阅读说明：本技术 Ha-1特异性t细胞受体及其用途 (HA-1 specific T cell receptor and uses thereof ) 是由 克里斯蒂安·埃林格尔 丹尼尔·萨默迈耶 阿利安·布拉赫尔 于 2019-08-06 设计创作，主要内容包括：本发明涵盖对次要组织相容性抗原1(HA-1)的一种等位基因变体、特别是等位基因变体HA-1～H具有特异性的分离的T细胞受体(TCR)。还描述了包含TCR的功能部分的分离的多肽。此外,限定了与TCR相关的多价TCR复合物、核酸分子、载体、细胞、抗体以及医学用途。(The invention encompasses an allelic variant of minor histocompatibility antigen 1(HA-1), in particular the allelic variant HA-1 H An isolated T Cell Receptor (TCR) with specificity. Isolated polypeptides comprising a functional portion of a TCR are also described. In addition, multivalent TCR complexes, nucleic acid molecules, vectors, cells, antibodies and medical uses related to TCRs are defined.)

1. An isolated T Cell Receptor (TCR) specific for an allelic variant of minor histocompatibility antigen 1 (HA-1).

2. The isolated TCR of claim 1, wherein the allelic variant of HA-1 is HA-1^H。

3. The isolated TCR of any of the preceding claims, wherein the TCR specifically recognizes the amino acid sequence SEQ ID NO 2 or a fragment thereof.

4. The isolated TCR of any of the preceding claims, wherein the TCR does not recognize the amino acid sequence SEQ ID NO 4 or a fragment thereof.

5. The isolated TCR of any of the preceding claims, wherein the recognition motif of the TCR comprises at least the sequence set forth in SEQ ID NO 127.

6. The isolated TCR of any of the preceding embodiments, wherein the TCR comprises

a) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 5, a CDR2 having the amino acid sequence of SEQ ID NO. 6 and a CDR3 having the sequence of SEQ ID NO. 7,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 8, CDR2 having the amino acid sequence of SEQ ID No. 9 and CDR3 having the sequence of SEQ ID No. 10; or

b) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 15, a CDR2 having the amino acid sequence of SEQ ID NO. 16 and a CDR3 having the sequence of SEQ ID NO. 17,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 18, CDR2 having the amino acid sequence of SEQ ID No. 19 and CDR3 having the sequence of SEQ ID No. 20; or

c) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 25, a CDR2 having the amino acid sequence of SEQ ID NO. 26 and a CDR3 having the sequence of SEQ ID NO. 27,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 28, CDR2 having the amino acid sequence of SEQ ID No. 29 and CDR3 having the sequence of SEQ ID No. 30;

d) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO 35, a CDR2 having the amino acid sequence of SEQ ID NO 36 and a CDR3 having the sequence of SEQ ID NO 37,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO:38, CDR2 having the amino acid sequence of SEQ ID NO:39 and CDR3 having the sequence of SEQ ID NO: 40; or

e) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO 45, a CDR2 having the amino acid sequence of SEQ ID NO 46 and a CDR3 having the sequence of SEQ ID NO 47,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 48, CDR2 having the amino acid sequence of SEQ ID No. 49 and CDR3 having the sequence of SEQ ID No. 50; or

f) A TCR alpha chain comprising CDR1 having the amino acid sequence of SEQ ID NO. 55, CDR2 having the amino acid sequence of SEQ ID NO. 56 and CDR3 having the sequence of SEQ ID NO. 57,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO:58, CDR2 having the amino acid sequence of SEQ ID NO:59 and CDR3 having the sequence of SEQ ID NO: 60.

7. The isolated TCR of any of the preceding claims, wherein the TCR comprises

a) A variable TCR α region having the amino acid sequence of SEQ ID NO. 11 and a variable TCR β region having the amino acid sequence of SEQ ID NO. 12; or

b) A variable TCR α region having the amino acid sequence of SEQ ID NO 21 and a variable TCR β region having the amino acid sequence of SEQ ID NO 22; or

c) A variable TCR α region having the amino acid sequence of SEQ ID NO. 31 and a variable TCR β region having the amino acid sequence of SEQ ID NO. 32; or

d) A variable TCR alpha region having the amino acid sequence of SEQ ID NO 41 and a variable TCR beta region having the amino acid sequence of SEQ ID NO 42; or

e) A variable TCR α region having the amino acid sequence of SEQ ID NO 51 and a variable TCR β region having the amino acid sequence of SEQ ID NO 52; or

f) A variable TCR alpha region having the amino acid sequence of SEQ ID NO 61 and a variable TCR beta region having the amino acid sequence of SEQ ID NO 62.

8. An isolated polypeptide comprising a functional portion of a TCR as claimed in any one of claims 1 to 7, wherein the functional portion comprises at least one of the following amino acid sequences: SEQ ID NO 7, SEQ ID NO 17, SEQ ID NO 27, SEQ ID NO 37, SEQ ID NO 47, SEQ ID NO 57, SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 30, SEQ ID NO 40, SEQ ID NO 50 and SEQ ID NO 60.

9. A multivalent TCR complex comprising at least two TCRs as claimed in any one of claims 1 to 7.

10. A nucleic acid encoding a TCR according to any one of claims 1 to 7 or encoding a polypeptide according to claim 8.

11. A vector comprising the nucleic acid of claim 10.

12. A cell expressing a TCR according to claims 1 to 7.

13. An antibody or antigen-binding fragment thereof that specifically binds to a portion of a TCR according to claims 1 to 7 that mediates specificity for one allelic variant of HA-1.

14. A TCR according to claims 1 to 7, a polypeptide according to claim 8, a multivalent TCR complex according to any one of claims 9, a nucleic acid according to claim 10, a vector according to claim 11, a cell according to claim 12, or an antibody according to claim 13, for use as a medicament.

15. The TCR of claims 1 to 7, the polypeptide of claim 8, the multivalent TCR complex of claim 9, the nucleic acid of claim 10, the vector of claim 11, or the cell of claim 12, for use in treating a cancer, wherein the cancer is preferably a hematological cancer.

Technical Field

The present invention relates to isolated T Cell Receptors (TCRs) that specifically bind to one allelic form of minor histocompatibility antigen 1(HA-1), and polypeptides comprising a functional portion of the TCRs. Also relates to multivalent TCR complexes, nucleic acids encoding TCRs, cells expressing TCRs, and pharmaceutical compositions comprising TCRs. The invention also relates to TCRs for use as a medicament, in particular to TCRs for use in the treatment of cancer.

Background

Bone Marrow Transplantation (BMT) and Hematopoietic Stem Cell Transplantation (HSCT) have been successfully used as first line treatments for a number of malignant and non-malignant diseases, such as severe aplastic anemia and leukemia, over the last 50 years. During BMT or HSCT, patients receive hematopoietic cells (i.e., hematopoietic stem cells) from healthy donors after the patient's own hematopoietic cell system has been destroyed by sublethal systemic irradiation or high dose cytotoxic drugs. The donor's stem cells may be derived from the donor's bone marrow or part of a stem cell transplant of donor mobilized hematopoietic stem cells. This approach is called allogeneic because the transplanted cells are derived from non-autologous sources, i.e., healthy, genetically non-identical donors.

One mode of failure of allogeneic BMT or HSCT is due to host rejection of the transplant, also known as host versus graft disease (HvGD), in which patient residual immune cells that escape destruction in a pre-transplant regimen of sublethal radiation or cytotoxic drugs identify the transplanted donor cells as "foreign" and subsequently elicit an immune response, resulting in rejection of the transplanted bone marrow or hematopoietic stem cells.

Another even more serious form of failure of BMT/HSCT is the so-called "graft versus host disease" (GvHD), which is actually the response of transplanted immune cells derived from bone marrow or hematopoietic stem cells transplanted from a donor to the patient's (recipient) somatic cells. GvHD is mainly attributed to differences in MHC molecules expressed by all nucleated somatic cells and platelets. MHC stands for major histocompatibility complex molecules, of which two major classes are known. MHC class I molecules presenting or displaying peptide fragments of cytoplasmic proteins play a major role in GvHD. In humans, MHC is also referred to as HLA complex (human leukocyte antigen complex) and is located on chromosome 6.

HLA complexes are polygenic and based on highly polymorphic genes, which means that there are many different alleles (alternative forms of the same gene) in different individuals of a population. Each human cell expresses six different MHC class I alleles (one HLA-A, HLA-B and HLA-C allele from each parent) and six to eight additional MHC class II alleles. In the case of BMT and HSCT, the MHC molecule itself may act as an antigen, i.e. it may be identified as "foreign" by the immune cells of the donor. Thus, the GvHD response is based primarily on HLA/MHC molecule expression mismatches between the donor and recipient. Therefore, it is most important to select donors with the least amount of mismatch in MHC class I and class II phenotypes to minimize the risk of GvHD.

Even though the donor and patient are matched as closely as possible in their MHC I/II genotypes, more than 50% of all patients suffer from GvHD. One possible way to prevent the patient from developing GvHD is to remove T cells from the transplant using, for example, an anti-CD 3 monoclonal antibody or other antibody specific for mature T cells, because donor T cells induce GvHD by recognizing HLA molecules of patients different from the donor pool. Although BMT and HSCT are currently performed in an HLA-matched environment, GvHD still occurs in a large number of patients, creating a need to overcome this problem.

This is likely due to a mismatch in the so-called minor histocompatibility antigen (MiHA), which is inherited independently of HLA genes. Over the last few years, a large number of MiHA (Frontiers in Immunology, vol 7, chapter 100, month 2016, 3) have been identified, which can be encoded by male-specific Y chromosomes (H-Y antigen) or other chromosomes (autosomal MiHA). In autosomal MiHA, the antigens designated HA-1, HA-2, HA-3, HA-4 and HA-5 are the earliest found antigens and all of these antigens are presented in the context of HLA-A1(HA-3) or in the context of HLA-A2(HA-1, HA-2, HA-4 and HA-5). It is also known that SNP variants (single nucleotide polymorphisms) of MiHA defining HA-1 are expressed in 69% of patients expressing HLA-A2, whereas HA-2 can be detected in 95%, HA-4 in 16% and HA-5 in about 7% of the population. In contrast, HA-3 presented in the context of HLA-A1 was found in 88% of patients expressing HLA-A1 (N Engl J Med.1996, 2 months 1; 334(5): 281-5).

Because HA-1 is almost exclusively present in hematopoietic cells and cancersExpressed on cells, it therefore represents a promising tumor-specific target for adoptive T cell therapy (ACT). Previously, HLA-A2-restricted HA-1-specific cytotoxic T lymphocytes (T cells) have been successfully isolated or generated (Haematologica.2005, 10 months; 90(10): 1415-21). HA-1 comprises an epitope in the form of nonapeptide having amino acid sequence VLHDDLLEA (SEQ ID NO:2) (science.1998, 13.2; 279(5353):1054-7) and is derived from an allele (SNP variant) of the HMHA1(KIAA0223) gene (rs number: rs 1801284). The HMHA1 locus contains two alleles HA-1H and HA-1R, which differ at two nucleotides, resulting in a single amino acid substitution. The second nonapeptide epitope, VLRDDLLEA (SEQ ID NO: 4; Science1998, 2.13: 279, 5353, 1054-1057), is inefficiently presented by HLA-A02: 01. Due to allelic polymorphism and Mendelian segregation pattern, an individual may be homozygous HA-1^H/HHeterozygote HA-1^H/ROr homozygote HA-1^R/R。

Disclosure of Invention

It is an object of the present invention to provide T cell receptors that specifically bind to only one allelic variant of MiHA HA-1.

The present invention relates to the treatment of only one allelic form of HA-1 (i.e., HA-1)^H) T Cell Receptors (TCR) with specificity. In a specific embodiment of the invention, the disclosure is directed to HA-1 presented by HLA-A2 molecule or in the context of HLA-A2 molecule^HT cell receptors with binding specificity. The invention also relates to TCRs for use as a medicament or for use in the treatment of malignant tumours. The TCRs may be used, for example, where the BMT/HSCT patient's HA-1H allele (presenting peptide VLHDDLLEA on an appropriate MHC/HLA molecule) is homozygous (i.e., the patient is HA-1)^H/H) Or are hybrid (i.e., HA-1)^H/R) And wherein the R allele of the donor is homozygous (i.e., HA-1)^R/R) And a portion of the donor T cells have been isolated and transduced with one of the TCRs of the invention and thus expressed against HA-1^HThe allele has a specific TCR, and wherein the transduced donor T cells are infused into the patient, and wherein the patient and donor are otherwise HLA identical or HLA matched. Expression of infused HA-1^HRecombinant donor T cell detection and depletion (by cytotoxic activity) of specific TCR expression of HA-1 in the context of appropriate MHC/HLA molecules^HAny residual hematopoietic tumor cells in the patient. It is also envisaged to preserve such HA-1^HSpecific donor T cells for future infusions to be able to deplete patient tumor cells that reappear during tumor recurrence. Another aspect of the invention is the use of a TCR of the invention or a recombinant T cell expressing a TCR of the invention for pretreatment therapy in a patient scheduled for BMT/HSCT. By using the TCR of the invention in this way or expressing this HA-1^HRecombinant T cells of specific TCRs which may replace or support sublethal radiation by infusion of such recombinant T cells are alloreactive to hematopoietic cells of a patient due to recombinant expression of the TCRs of the invention, and thus may eradicate presentation of HA-1 in the context of HLA-A2^HThe immune cell of (1). The T cells may be derived from a donor of BMT/HSCT.

TCRs according to the invention are isolated and/or purified and may be soluble or membrane bound.

In some embodiments, the amino acid sequence of the TCR can comprise one or more phenotypically silent substitutions. In addition, the TCRs of the invention may be labeled. Useful labels are known in the art and can be optionally coupled to a TCR or TCR variant via linkers of various lengths using conventional methods. The term "label" or "labeling group" refers to any detectable label. Additionally or alternatively, the amino acid sequence may be modified to include a therapeutic agent or a pharmacokinetic modifying moiety. The therapeutic agent may be selected from the group consisting of an immune effector molecule, a cytotoxic agent, and a radionuclide. The immune effector molecule may for example be a cytokine. The pharmacokinetic modifying moiety may be at least one polyethylene glycol repeat unit, at least one glycol group, at least one sialic acid group, or a combination thereof.

The TCR according to the invention, in particular the soluble form, may be modified by the attachment of further functional moieties, e.g. for reducing immunogenicity, increasing hydrodynamic size (size in solution), solubility and/or stability (e.g. by enhanced protection against proteolytic degradation) and/or increasing serum half-life. Other useful functional moieties and modifications include "suicide" or "safety switches" which can be used to turn off or on effector host cells carrying the TCRs of the invention in a patient.

TCRs having altered glycosylation patterns are also contemplated herein.

It is also contemplated to add a drug or therapeutic entity (e.g., a small molecule compound) to the TCR, particularly the soluble form of the inventive TCR.

TCRs, particularly soluble forms of the inventive TCRs, may additionally be modified to introduce additional domains that facilitate the identification, tracking, purification, and/or isolation of individual molecules (tags).

In some embodiments, the TCR is of the single chain type, wherein the TCR α chain and the TCR β chain are linked by a linker sequence.

Another aspect of the invention relates to a polypeptide comprising a functional portion of a TCR as described herein, wherein the functional portion comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: SEQ ID NO 7, SEQ ID NO 17, SEQ ID NO 27, SEQ ID NO 37, SEQ ID NO 47, SEQ ID NO 57, SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 30, SEQ ID NO 40, SEQ ID NO 50 and SEQ ID NO 60.

In particular embodiments, the functional moiety comprises a TCR α variable chain and/or a TCR β variable chain.

Particular embodiments relate to multivalent TCR complexes comprising at least two TCRs as described herein. In a more specific embodiment, at least one of the TCRs is associated with a therapeutic agent.

Some embodiments relate to a TCR of the invention expressed as a functional or functional multivalent polypeptide on effector cells, in particular on immune effector cells, wherein IFN- γ secretion is induced in the above TCR-expressing effector cells after binding to the amino acid sequence SEQ ID NO:2 presented by the HLA-a02:01 encoding molecule.

Wherein presentation by HLA-A02:01 encoding molecules means that the peptides, in particular epitopes, are bound to the binding groove of MHC molecules.

The MHC molecule presenting an epitope may be encoded by one of the HLA-a02 alleles, for example by the HLA-a02:01 or HLA-a02: 06 alleles, preferably by the HLA-a02:01 alleles.

At 10^-7[M]The secretion of IFN- γ induced by binding of a TCR of the invention expressed on effector cells to the amino acid sequence SEQ ID NO:2 presented by the HLA-a02:01 encoding molecule may be at least 3 times higher, e.g. 10 times higher, 20 times higher, 100 times higher, than the secretion of IFN- γ induced by binding to SEQ ID NO:4 presented by the HLA-a02:01 encoding molecule at the HA-1 peptide concentration of (a).

Thus, for all HA-1^HTCR transgenic T cells, for the identification of HA-1^RVariants, requiring conjugation to HA-1^HCompared to a peptide concentration at least 1,000-fold higher, preferably at least 5,000-fold higher, more preferably at least 8000-fold higher, most preferably at least 10,0000-fold higher.

In particular embodiments, for example, when the ratio of TCR transgenic T cells to T2 cells is 2:1, at 10^-7[M]Can be greater than 500pg/ml, such as greater than 1000pg/ml, more preferably greater than 2000pg/ml, most preferably greater than 3000pg/ml, by binding of a TCR of the invention expressed on effector cells to the amino acid sequence presented by the HLA-a02:01 encoding molecule, SEQ ID NO: 2.

Cytokine and chemokine release (e.g., IFN- γ secretion) can be measured as follows: the use of T-cell antibody-immobilized magnetic beads by in vitro assay, in which K562 cells (Greiner et al 2006, blood.2006Dec 15; 108(13):4109-17) transduced to express the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, respectively, were compared to CD8 expressing the TCR to be studied⁺Enriched and/or non-CD 8⁺Enriched PBMC were incubated together, or T2 cells externally loaded with VLHDDLLEA peptide (SEQ ID NO:2) or peptide VLRDDLLEA (e.g., peptide SEQ ID NO:4) were used in an in vitro assay and subsequently incubated with CD8 expressing the TCR to be studied⁺Enriched and/or non-CD 8⁺Enrichment of PBMC for co-incubation.

Another aspect of the invention relates to a nucleic acid encoding a TCR as described herein or encoding a polypeptide as described above.

Another aspect of the invention relates to a plasmid or vector comprising a nucleic acid of the present application as described above. Preferably, the vector is an expression vector or a vector suitable for transduction or transfection of cells, in particular eukaryotic cells. The vector may be, for example, a retroviral vector (e.g., a gamma-retrovirus) or a lentiviral vector.

Another aspect of the invention relates to a cell expressing a TCR as described herein. The cells may be isolated or non-naturally occurring.

Another aspect of the invention relates to a cell comprising a nucleic acid as described above or a plasmid or vector as described above. More specifically, the cell may comprise:

a) an expression vector comprising at least one nucleic acid as described above, or

b) A first expression vector comprising a nucleic acid encoding an alpha chain of a TCR as described herein, and a second expression vector comprising a nucleic acid encoding a beta chain of a TCR as described herein.

The cells may be Peripheral Blood Lymphocytes (PBLs) or Peripheral Blood Mononuclear Cells (PBMCs). Typically, the cells are immune effector cells, particularly T cells. Other suitable cell types include gamma-delta T cells, NK cells and NK-like T cells.

Another aspect relates to an antibody or antigen-binding fragment thereof that specifically binds to a portion of a TCR as described herein, said portion mediating binding to HA-1^HThe specificity of (A). In one embodiment, mediation of TCR HA-1^HSpecific portions comprise the alpha chain CDR3 of SEQ ID NO 7, SEQ ID NO 17, SEQ ID NO 27, SEQ ID NO 37, SEQ ID NO 47, SEQ ID NO 57 and/or the beta chain CDR3 of SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 30, SEQ ID NO 40, SEQ ID NO 50 and SEQ ID NO 60.

Another aspect of the invention relates to a pharmaceutical composition comprising a TCR as described herein, a polypeptide as described herein, a multivalent TCR complex as described herein, a nucleic acid as described herein, a vector as described herein, a cell as described herein, or an antibody as described herein.

Typically, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

Another aspect of the invention relates to the use of a composition as described hereinA TCR, a polypeptide as described herein, a multivalent TCR complex as described herein, a nucleic acid as described herein, a vector as described herein, a cell as described herein, or an antibody as described herein for use as a medicament, in particular for use in the treatment of cancer (in particular hematological cancer in the case of treatment of HLA matched bone marrow and/or stem cell transplantation), wherein the patient HAs previously received an allogeneic bone marrow and/or stem cell transplantation from an HLA matched donor, wherein the donor is HA-1^HNegative (and thus HA-1)^RHomozygous and/or HLA-A2 negative), and wherein the patient is homozygous HA-1^HOr hybrid HA-1^H/RAnd HLA-a2 positive) has recurrence or recurrence of hematologic cancer cells, or as a preventative measure (treatment of patients with no evidence of recurrence/recurrence following transplantation). By isolating CD8 from a patient or donor after transplantation⁺T cells and ex vivo transduction of such isolated CD8 with a TCR according to the invention⁺T cells to treat patients with a recurrence of hematologic cancer cells. The hematologic cancer may be selected from the group consisting of: non-hodgkin's lymphoma (NHL), Hodgkin's Lymphoma (HL), multiple myeloma, Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL), Mixed Phenotype Acute Leukemia (MPAL), Chronic Myeloid Leukemia (CML), B-cell pleomorphic lymphoma, hairy cell leukemia, Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), central nervous system lymphoma, CD37+ dendritic cell lymphoma, lymphoblastic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, extraosseous plasmacytoma, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT tissue), nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, precursor B lymphoblastic lymphoma, Acute Myelogenous Leukemia (AML), mixed phenotype Acute Lymphoblastic Leukemia (ALL), Mixed Phenotype Acute Leukemia (MPAL), mixed phenotype acute leukemia (mmal), Chronic Myelogenous Leukemia (CML), B cell pleomorphic lymphoma, hairy cell leukemia, Chronic Lymphocytic Leukemia (CLL), small, Immunoblastic large cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt's lymphoma/leukemia, B cell proliferation of unknown malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disease. Other hematologic cancers or diseases include myeloproliferationAnd (6) abnormal.

Drawings

FIG. 1 shows the use of a different HA-1^HReactive TCR transduced CD8⁺HA-1 of T cells^H-MHC-multimer binding. CD 8T cells were isolated from PBMCs of healthy donors and used with six different HA-1' s^HTCR and a non-recognition HA-1^HThe control TCR of (a) was transduced. Transduced CD 8T cells were enriched by FACS using murine constant β regions as transduction markers. After these cells were expanded, they were treated with HA-1^HMHC multimers and antibodies against CD8 and murine constant β region (mCb) and analyzed by flow cytometry. In live CD8⁺/mCb⁺The population was gated on the cells and staining of multimers/CD 8 was shown.

FIG. 2 shows HA-1^HTCR-transgenic T cells recognize HA-1 presented on HLA-A2^H-a peptide. The transgenic T cells and the outside are loaded with HA-1^HT2 cells of the peptide or already used to encode HA-1^H-partial transduction of the HMHA1 gene for epitopes K562/HLA-A2 cell co-culture. As negative controls, T2 cells loaded with control peptide and untransduced K562/HLA-A2 cells were used, respectively. The recognition of target cells was analyzed by measuring the IFN- γ concentration in the co-culture supernatants by standard ELISA.

FIGS. 3A and 3B show HA-1^HTCR-functional avidity of transgenic T cells. (A) Transgenic T cells were loaded with graded concentrations of HA-1 from the outside^H-a peptide (10)^-11M-10^-5M) or HA-1^R-a peptide (10)^-8M-10^-5M) of T2 cells. (B) Mixing HA-1^HTCR transgenic T cells against HA-1^RFunctional affinity of the peptide was compared to T cells transduced with HA-1 specific TCR (TCR2) as described in WO2018058002a 1. The data for TCRs 1-6(TCR _1, TCR _2, TCR _3, TCR _4, TCR _5, TCR _6) according to the invention are the same in FIGS. 3A and 3B.

FIGS. 4A and 4B show HA-1^HTCR transgenic T cells recognize physiological levels of HA-1 on unmodified target cells^HThe ability of the cell to perform. Co-culturing transgenic T-cells with different Lymphoblastoid Cell Lines (LCL), said cellsIs HA-1^HPositive (LCL 1-5) or HA-1^HNegative (LCL 6-7). (A) Cytokine release was analyzed by measuring IFN- γ concentration in co-culture supernatants by standard ELISA. T cells expressing the control TCR were used as a negative control. (B) By photographing the co-culture every two hoursZOOM (Essen Bioscience) measures cytotoxicity against LCL stably transduced with fluorescent markers. Untransduced CD 8T cells were used as negative control.

FIGS. 5A and 5B show HA-1^HTCR transgenic T cells recognize HA-1 present on tumor cell lines^HThe ability of the cell to perform. Combining transgenic T cells with different HA-1^HPositive tumor cell lines were co-cultured. (A) Cytokine release was analyzed by measuring IFN- γ concentration in co-culture supernatants by standard ELISA. (B) By taking pictures every two hoursZOOM measures cytotoxicity against tumor cell lines stably transduced with fluorescent markers.

FIGS. 6A and 6B show that T cells expressing TCR _3 or TCR _4 recognize and lyse (kill) HA-1 as compared to TCR2 as described in WO2018058002A1^HThe ability to positive tumor cell lines. (A) Combining transgenic T cells with different HA-1^HPositive tumor cell lines were co-cultured. Cytokine release was analyzed by measuring IFN- γ concentration in co-culture supernatants by standard ELISA. (B) Analysis by flow cytometry against HA-1^HCytotoxicity of positive tumor cell lines. Mixing HA-1^HHA-1 of negative/GFP-positive K562 cells and tumor cell lines^Hpositive/mCherry positive cells were mixed at a ratio of 1:1 and co-cultured with TCR transgenic T cells. mCherry positivity (HA-1) was calculated after 45 hours^HPositive) and GFP positive (HA-1)^HNegative) ratio of cells, and normalized to the ratio of control TCR measurements. The smaller numbers indicate HA-1 due to cytotoxicity of T cells^HPositive tumor cells were lysed.

Figure 7 shows an analysis of the recognition motifs of TCR _3 and TCR _4 compared to TCR2 as described in WO2018058002a 1. The transgenic T cells and the outside are loaded with HA-1^HPeptide T2 cells were co-cultured, transgenic T cells with the peptide or control peptide having each individual amino acid residue replaced sequentially with serine.

FIGS. 8A and 8B show a comparison of different constant TCR regions. TCR _3 was cloned using constant TCR regions of murine, minimally murine or human origin. (A) Combining transgenic T cells with different HA-1^HPositive tumor cell lines were co-cultured. Cytokine release was analyzed by measuring IFN- γ concentration in co-culture supernatants using standard ELISA. Analysis by flow cytometry against HA-1^HCytotoxicity of positive tumor cell lines. Mixing HA-1^HHA-1 of negative/GFP-positive K562 cells and tumor cell lines^Hpositive/mCherry positive cells were mixed at a ratio of 1:1 and co-cultured with TCR transgenic T cells. After 45 hours, mCherry positivity (HA-1) was calculated^HPositive) cells and GFP-positive (HA-1)^HNegative) ratio of cells, and normalized to the ratio of control TCR measurements. The smaller number represents HA-1^HLysis of positive tumor cells. (B) Combining transgenic T cells with different HA-1^HNegatives (OMW, BER) and HA-1^HPositive (FH22, SLE005, SPO-010, BM16, BSM) LCL (lymphoblastoid cell line) coculture. Cytokine release was analyzed by measuring IFN- γ concentration in co-culture supernatants by standard ELISA.

Detailed Description

Before describing the invention in detail with respect to certain preferred embodiments, the following general definitions are provided.

The invention illustratively described below suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims.

When the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising. If a group is defined below as comprising at least a certain number of embodiments, it is also to be understood that a group preferably consisting of only these embodiments is disclosed.

For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtainable". If in the following, for example, an antibody is defined as obtainable from a particular source, it is also to be understood that antibodies obtained from that source are disclosed.

When an indefinite or definite article is used when referring to a singular noun e.g. "a", "an" or "the", this word includes a plural of that noun unless something else is specifically stated. In the context of the present invention, the term "about" or "approximately" denotes a range of precision that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

Technical terms are used in their common sense or meaning to those skilled in the art. If a specific meaning is conveyed to a certain term, the definition of that term will be given in the context in which it is used below.

TCR background

The TCR is composed of two distinct and separate protein chains, namely the TCR alpha (α) and TCR beta (β) chains. The TCR alpha chain comprises a variable region (V), a connecting region (J) and a constant region (C). The TCR β chain comprises a variable region (V), a variable region (D), a connecting region (J) and a constant region (C). The rearranged v (d) J regions of both the TCR α and TCR β chains comprise hypervariable regions (CDRs, complementarity determining regions), of which the CDR3 region determines specific epitope recognition. In the C-terminal region, both TCR α and TCR β chains comprise a hydrophobic transmembrane domain and terminate in a short cytoplasmic tail.

Generally, TCRs are heterodimers of one α chain and one β chain. The heterodimer may be bound to an MHC molecule presenting a peptide.

In the context of the present invention, the term "variable TCR α region" or "TCR α variable chain" or "variable domain" refers to the variable region of the TCR α chain. In the context of the present invention, the term "variable TCR β region" or "TCR β variable chain" refers to the variable region of the TCR β chain.

Using the International Immunogenetics (IMGT) TCR nomenclature (IMGT database, www.IMGT.org; Giudilli, V., et al, IMGT/LIGM-DB, immunoglobulin and T cell receptor nucleotide sequencesIntegrated database, nuclear. acids res, 34, D781-D784(2006). PMID: 16381979; t cell Receptor facebook, LeFranc and LeFranc, Academic Press ISBN 0-12-441352-8) names the TCR loci and genes.

Target

A first aspect of the invention relates to an isolated TCR specific for an allelic variant of HA-1. The allelic variant is a type of single nucleotide polymorphism.

In particular, the TCR specifically recognizes the amino acid sequence of SEQ ID NO. 2 (VLHDDLLEA).

Generally, TCRs recognize peptide fragments of antigens presented by Major Histocompatibility Complex (MHC) molecules.

The Human Leukocyte Antigen (HLA) system or complex is a complex of genes encoding Major Histocompatibility Complex (MHC) proteins in humans. HLA-a02 is a specific class I Major Histocompatibility Complex (MHC) allele at the HLA-a locus. HLA-A02:01 is a specific HLA-A02 sub-allele.

Thus, in a specific embodiment, the TCR specifically recognizes the amino acid sequence of SEQ ID No. 2, which is presented by an MHC molecule encoded by the HLA-a02 allele, preferably by a molecule encoded by HLA-a02:01 or by a molecule encoded by HLA-a02: 06, more preferably by a molecule encoded by HLA-a02: 01.

TCR vs HA-1^HHAs high specificity and is resistant to other peptides, particularly allelic variants HA-1^RThere is substantially no cross-reaction. This means that it does not recognize the HA-1 allelic variation. In other words, it does not recognize the amino acid sequence of SEQ ID NO. 4. Cross-reactivity can be measured by IFN- γ secretion as described herein.

In the context of the present invention, the term "specific for" refers to the specific binding of a TCR to a target.

The terms "allelic variation" and "allelic form" are used herein the same.

In a preferred embodiment, the recognition motif of the TCR comprises at least the sequence shown in SEQ ID NO: 127. In a specific embodiment, the recognition motif of the TCR consists of the sequence shown in SEQ ID NO: 127. Wherein the recognition motif can be determined by serine substitution.

The recognition motif defines the amino acids of the epitope that influence TCR activation, as determined, for example, in IFN- γ secretion assays. In particular, the effect of amino acid residues in a binding epitope can be determined by amino acid substitution scans, such as serine scans.

In the serine scan, epitope peptides were used in which each individual amino acid residue was successively replaced by serine. If the peptide results in a significant reduction in TCR activation, as determined by IFN- γ secretion, this indicates that the position being replaced belongs to the recognition motif. The significant reduction may be at least a 3-fold, preferably at least a 5-fold, more preferably at least a 10-fold reduction in IFN- γ secretion compared to the unsubstituted peptide sequence (i.e. the unsubstituted epitope).

TCR specific sequences

The TCR α chain CDR3 of the TCR may have an amino acid sequence selected from the group consisting of: SEQ ID NO 7, SEQ ID NO 17, SEQ ID NO 27, SEQ ID NO 37, SEQ ID NO 47 and SEQ ID NO 57.

The TCR β chain CDR3 of the TCR may have an amino acid sequence selected from the group consisting of: 10, 20, 30, 40, 50 and 60.

Some embodiments relate to isolated TCRs comprising a TCR α chain and a TCR β chain, wherein

a) The TCR alpha chain comprises the complementarity determining region 3(CDR3) having the sequence of SEQ ID NO. 7,

-the TCR β chain comprises CDR3 having the amino acid sequence of SEQ ID NO: 10; or

b) The TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO 17,

-the TCR β chain comprises CDR3 having the amino acid sequence of SEQ ID NO: 20; or

c) The TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO 27,

-the TCR β chain comprises CDR3 having the amino acid sequence of SEQ ID NO: 30; or

d) The TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO 37,

-the TCR β chain comprises CDR3 having the amino acid sequence of SEQ ID NO: 40; or

e) The TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO 47,

-the TCR β chain comprises CDR3 having the amino acid sequence of SEQ ID NO: 50; or

f) The TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO. 57,

the TCR β chain comprises a CDR3 having the amino acid sequence of SEQ ID NO 60.

More specific embodiments relate to an isolated TCR, wherein the TCR comprises:

a) a TCR alpha chain comprising CDR1 having the amino acid sequence of SEQ ID NO. 5, CDR2 having the amino acid sequence of SEQ ID NO. 6 and CDR3 having the sequence of SEQ ID NO. 7,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 8, CDR2 having the amino acid sequence of SEQ ID No. 9 and CDR3 having the sequence of SEQ ID No. 10; or

b) A TCR alpha chain comprising CDR1 having the amino acid sequence of SEQ ID NO. 15, CDR2 having the amino acid sequence of SEQ ID NO. 16 and CDR3 having the sequence of SEQ ID NO. 17,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 18, CDR2 having the amino acid sequence of SEQ ID No. 19 and CDR3 having the sequence of SEQ ID No. 20; or

c) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 25, a CDR2 having the amino acid sequence of SEQ ID NO. 26 and a CDR3 having the sequence of SEQ ID NO. 27,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 28, CDR2 having the amino acid sequence of SEQ ID No. 29 and CDR3 having the sequence of SEQ ID No. 30;

d) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 35, a CDR2 having the amino acid sequence of SEQ ID NO. 36 and a CDR3 having the sequence of SEQ ID NO. 37,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 38, CDR2 having the amino acid sequence of SEQ ID No. 39 and CDR3 having the sequence of SEQ ID No. 40; or

e) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 45, a CDR2 having the amino acid sequence of SEQ ID NO. 46 and a CDR3 having the sequence of SEQ ID NO. 47,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 48, CDR2 having the amino acid sequence of SEQ ID No. 49 and CDR3 having the sequence of SEQ ID No. 50; or

f) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 55, a CDR2 having the amino acid sequence of SEQ ID NO. 56 and a CDR3 having the sequence of SEQ ID NO. 57,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO:58, CDR2 having the amino acid sequence of SEQ ID NO:59 and CDR3 having the sequence of SEQ ID NO: 60.

A preferred embodiment relates to an isolated TCR defined by CDRs, in particular CDR3 of the TCR α and TCR β chains as described above, wherein the recombinant TCR sequence is modified to comprise a murine-derived C α region and a C β region.

Some embodiments relate to an isolated TCR, wherein the TCR comprises

a) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 11 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 12; or

b) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 21 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 22; or

c) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 31 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 32; or

d) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 41 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 42; or

e) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 51 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 52; or

f) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 61 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 62.

As used herein, "at least 80% identical," in particular, "having an amino acid sequence that is at least 80% identical" includes amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences shown.

In some embodiments, the TCR comprises a TCR α chain and a TCR β chain, wherein

a) The variable TCR alpha region has an amino acid sequence which is at least 80% identical to SEQ ID NO. 11 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID NO. 7,

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 12 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 10; or

b) The variable TCR alpha region has an amino acid sequence which is at least 80% identical to SEQ ID NO 21 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID NO 17,

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 22 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 20; or

c) -the variable TCR α region has an amino acid sequence at least 80% identical to SEQ ID No. 31 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 27;

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 32 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID No. 30; or

d) -the variable TCR α region has an amino acid sequence at least 80% identical to SEQ ID No. 41 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 37;

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 42 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID No. 40; or

e) -the variable TCR α region has an amino acid sequence at least 80% identical to SEQ ID No. 51 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 47;

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 52 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 50; or

f) -the variable TCR α region has an amino acid sequence at least 80% identical to SEQ ID No. 61 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 57;

the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 62 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID No. 60.

Exemplary embodiments relate to isolated TCRs, wherein the TCRs comprise

a) A variable TCR α region having the amino acid sequence of SEQ ID NO. 11 and a variable TCR β region having the amino acid sequence of SEQ ID NO. 12; or

b) A variable TCR α region having the amino acid sequence of SEQ ID NO 21 and a variable TCR β region having the amino acid sequence of SEQ ID NO 22; or

c) A variable TCR α region having the amino acid sequence of SEQ ID NO. 31 and a variable TCR β region having the amino acid sequence of SEQ ID NO. 32; or

d) A variable TCR alpha region having the amino acid sequence of SEQ ID NO 41 and a variable TCR beta region having the amino acid sequence of SEQ ID NO 42; or

e) A variable TCR α region having the amino acid sequence of SEQ ID NO 51 and a variable TCR β region having the amino acid sequence of SEQ ID NO 52; or

f) A variable TCR alpha region having the amino acid sequence of SEQ ID NO 61 and a variable TCR beta region having the amino acid sequence of SEQ ID NO 62.

The following table shows a summary of exemplary TCRs of the invention.

Table 1: summary of exemplary TCRs of the invention

TCR

CDR1_α

CDR2_α

CDR3_α

TRAV

TRAJ

CDR1_β

CDR2_β

CDR3_β

TRBV

TRBJ

1

DSASNY

IRSNVGE

CAASDLNF

13-1

41

SEHNR

FQNEAQ

CASSLVSRVDGYTF

7-9

1-2

2

DSASNY

IRSNVGE

CAAHTPGYSTLTF

13-1

11

SEHNR

FQNEAQ

CASSPRAGGETQYF

7-9

2-5

3

TSINN

IRSNERE

CATGDQTGANNLFF

17

36

SEHNR

FQNEAQ

CASSLTRTEKLFF

7-9

1-4

4

DSASNY

IRSNVGE

CAGRGKLTF

13-1

48

SEHNR

FQNEAQ

CASSLVRDEKLFF

7-9

1-4

5

SVFSS

VVTGGEV

CAGAGNNDMRF

27

43

SEHNR

FQNEAQ

CASSLVRGIEAFF

7-9

1-1

6

DSSSTY

IFSNMDM

CAEKWIIF

5

30

SEHNR

FQNEAQ

CASSLTTPDGYTF

7-9

1-2

As can be seen from the examples, the TCR according to the invention is directed to HA-1^HSpecific and show only very low cross-reactivity to other epitopes or antigens.

Preference is given to using Vector NTI Advance^TMAlignX application of the program 10 (Invitrogen Corporation, Carlsbad CA, USA) accomplishes the determination of percent identity between multiple sequences. The program used a modified Clustal W algorithm (Thompson et al, 1994.Nucl Acids Res.22: pp. 4673-4680; Invitrogen Corporation; Vector NTI Advance^TMUser's Manual,2004, pp 389-662). The determination of percent identity is performed using the standard parameters of the AlignX application.

The TCR according to the invention is isolated or purified. In the context of the present invention, "isolated" means that the TCR is not present in the environment in which it naturally occurs in nature. In the context of the present invention, "purified" means, for example, that the TCR is free or substantially free of other proteinaceous and non-proteinaceous parts of the cell from which it was originally derived.

In some embodiments, the amino acid sequence of the TCR can comprise one or more phenotypically silent substitutions.

"phenotypically silent substitutions" are also referred to as "conservative amino acid substitutions". The concept of "conservative amino acid substitutions" is understood by those skilled in the art, and preferably means that codons encoding positively charged residues (H, K and R) are replaced with codons encoding positively charged residues, codons encoding negatively charged residues (D and E) are replaced with codons encoding negatively charged residues, codons encoding neutral polar residues (C, G, N, Q, S, T and Y) are replaced with codons encoding neutral polar residues, and codons encoding neutral non-polar residues (A, F, I, L, M, P, V and W) are replaced with codons encoding neutral non-polar residues. These variations may occur spontaneously, be introduced by random mutagenesis, or may be introduced by site-directed mutagenesis. These changes can be made without destroying the essential characteristics of these polypeptides. One of ordinary skill can readily and routinely screen amino acid variants and/or nucleic acids encoding them by methods known in the art to determine whether these variants substantially reduce or disrupt ligand binding capability.

The skilled artisan will appreciate that modifications may also be made to the nucleic acid encoding the TCR. Useful modifications of the entire nucleic acid sequence include codon optimization of the sequence. Changes can be made that result in conservative substitutions within the expressed amino acid sequence. These changes, which do not affect function, can be made in the complementarity determining regions and non-complementarity determining regions of the amino acid sequences of the TCR chains. Generally, additions and deletions should not be made in the CDR3 region.

According to some embodiments of the invention, the amino acid sequence of the TCR is modified to comprise a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety.

Non-limiting examples of detectable labels are radioactive labels, fluorescent labels, nucleic acid probes, enzymes, and contrast agents. Therapeutic agents that may be associated with the TCR include radioactive compounds, immunomodulators, enzymes or chemotherapeutic agents. The therapeutic agent may be encapsulated with liposomes linked to the TCR, such that the compound may be slowly released at the target site. This will avoid damage during in vivo transport and ensure that the therapeutic agent (e.g. toxin) has the greatest effect after binding of the TCR to the relevant antigen presenting cell. Other examples of therapeutic agents are:

peptidoglycan, i.e., a protein or peptide having the ability to kill mammalian cells, such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase, and RNase. Small molecule cytotoxic agents (i.e., compounds that have the ability to kill mammalian cells) have a molecular weight of less than 700 daltons. Such compounds may contain toxic metals capable of having a cytotoxic effect. Furthermore, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that degrade or convert under physiological conditions to release the cytotoxic agent. Such agents may include, for example, docetaxel, gemcitabine, cisplatin, maytansine derivatives, rachelmycin, calicheamicin (calicheamicin), etoposide, ifosfamide, irinotecan, porfimer sodium photosensitizer ii (porfimer sodium phorofrin ii), temozolomide, topotecan, trimetrexate gluconate, mitoxantrone, auristatin e (auristatin e), vincristine, and doxorubicin; radionuclides such as iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225, and astatine 213. The association of the radionuclide with the TCR or derivative thereof may be performed, for example, by a chelator; immunostimulators (also known as immunostimulants), i.e. immune effector molecules that stimulate an immune response. Exemplary immunostimulants are cytokines such as IL-2 and IFN- γ, antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD 3, anti-CD 28, or anti-CD 16); a surrogate protein scaffold having antibody-like binding properties; superantigens, i.e., antigens that cause non-specific activation of T cells, leading to polyclonal T cell activation and release of large amounts of cytokines, and mutants thereof; chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating protein, and the like; a complement activator; heterologous protein domains, allogeneic protein domains, viral/bacterial peptides.

Antigen receptor molecules on human T lymphocytes (T cell receptor molecules) associate non-covalently with complexes of CD3(T3) molecules on the cell surface. Perturbation of this complex with an anti-CD 3 monoclonal antibody induces T cell activation. Thus, some embodiments relate to a TCR as described herein associated (typically by fusion to the N-or C-terminus of the alpha or beta chain) with an anti-CD 3 antibody or a functional fragment or variant of said anti-CD 3 antibody. Antibody fragments and variants/analogs suitable for use in the compositions and methods described herein include minibodies (minibodies), Fab fragments, F (ab)<'>)2 fragments, dsFv and scFv fragments, Nanobodies^TM(ablynx (belgium), molecules comprising synthetic single immunoglobulin variable heavy domains derived from camelidae (e.g. camel or llama) Antibodies) and Domain Antibodies (Domain Antibodies) (comprising affinity matured single immunoglobulin variable heavy domains or immunoglobulin variable light domains (domainis (belgium)) or alternative protein scaffolds exhibiting antibody-like binding properties, such as Affibodies (comprising an engineered protein a scaffold Affibody (sweden)) or Antibodies (comprising engineered Anticalins spieries (germany)).

The therapeutic agent may preferably be selected from the group consisting of immune effector molecules, cytotoxic agents and radionuclides. Preferably, the immune effector molecule is a cytokine.

The pharmacokinetic modifying moiety may be, for example, at least one polyethylene glycol repeat unit, at least one diol group, at least one sialic acid group, or a combination thereof. The association of the at least one polyethylene glycol repeating unit, the at least one diol group, the at least one sialic acid group may be caused by a variety of means known to those skilled in the art. In a preferred embodiment, these units are covalently linked to the TCR. The TCR according to the present invention may be modified by one or several pharmacokinetic modifying moieties. In particular, the soluble form of the TCR is modified by one or several pharmacokinetic modifying moieties. The pharmacokinetic modifying moiety may effect beneficial changes in the pharmacokinetic properties of the therapeutic agent, such as improved plasma half-life, reduced or enhanced immunogenicity, and improved solubility.

TCRs according to the invention may be soluble or membrane bound. The term "soluble" refers to a TCR in soluble form (i.e., without a transmembrane or cytoplasmic domain), e.g., for use as a targeting agent for delivery of a therapeutic agent to an antigen presenting cell. For stability, the soluble α β heterodimeric TCR preferably has an introduced disulfide bond between residues of the respective constant domains, as described for example in WO 03/020763. One or both of the constant domains present in the α β heterodimers of the invention may be truncated at the C-terminus or C-terminus, e.g., up to 15, up to 10, or up to 8 or fewer amino acids. For use in adoptive therapy, α β heterodimeric TCRs can be, for example, transfected as full-length chains with both cytoplasmic and transmembrane domains. The TCR may comprise a disulfide bond corresponding to that between the corresponding alpha and beta constant domains found in nature, in addition or alternatively, a non-native disulfide bond may be present.

Thus, TCRs, in particular soluble forms of TCRs according to the invention, may be modified by the attachment of additional functional moieties, e.g. for reducing immunogenicity, increasing hydrodynamic size (size in solution), solubility and/or stability (e.g. by enhanced protection against proteolytic degradation) and/or increasing serum half-life.

Other useful functional moieties and modifications include "suicide" or "safety switches" which can be used to turn off effector host cells carrying the TCRs of the invention in a patient. One example is Gargett and Brown Front pharmacol.2014; 5:235, inducible Caspase 9(iCasp9) "safety switch". Briefly, effector host cells are modified by well-known methods to express Caspase 9 domains whose dimerization is dependent on small molecule dimerizer drugs, such as AP1903/CIP, and results in rapid induction of apoptosis in the modified effector cells. This system is described, for example, in EP2173869(a 2). Examples of other "suicide" safety switches "are known in the art, such as the expression of herpes simplex virus thymidine kinase (HSV-TK), CD20 and subsequent depletion using anti-CD 20 antibodies, expression of truncated EGFR and subsequent depletion using anti-EGFR antibodies (Wang et al, Blood, 8.2011.4; 118(5):1255-63), or myc-tag (Kieback et al, Proc Natl Acad Sci USA.2008.1.15; 105(2): 623-8).

TCRs with altered glycosylation patterns are also contemplated herein. As known in the art, the glycosylation pattern can depend on the amino acid sequence (e.g., the presence or absence of the particular glycosylated amino acid residue discussed below) and/or the host cell or organism producing the protein. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The addition of an N-linked glycosylation site to the binding molecule is conveniently achieved by altering the amino acid sequence to comprise one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine (where X is any amino group except proline). An O-linked glycosylation site can be introduced by adding or replacing one or more serine or threonine residues to the starting sequence.

Another method of glycosylation of TCRs is by chemically or enzymatically coupling a glycoside to a protein. Depending on the coupling means used, one or more sugars may be attached to: (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. Similarly, deglycosylation (i.e., removal of the carbohydrate moieties present on the binding molecule) can be achieved chemically, for example by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically by employing endoglycosidases and exoglycosidases.

It is also contemplated to add a drug, such as a small molecule compound, to the TCR, particularly the soluble form of the TCR of the invention. Attachment may be achieved by covalent bonds or non-covalent interactions, such as by electrostatic forces. To form a drug conjugate, various linkers known in the art may be used.

TCRs, particularly soluble forms of the inventive TCRs, may additionally be modified to introduce additional domains that facilitate the identification, tracking, purification, and/or isolation of individual molecules (tags). Thus, in some embodiments, the TCR α chain or TCR β chain may be modified to comprise an epitope tag.

Epitope tags are useful examples of tags that can be incorporated into the TCRs of the invention. Epitope tags are short amino acid fragments that allow binding of specific antibodies and thus enable identification and tracking of binding and movement of soluble TCR or host cells or cultured (host) cells in a patient. Detection of the epitope tag and thus the tagged TCR can be achieved using a variety of different techniques.

The tags can be further used to stimulate and expand host cells carrying the TCRs of the invention by culturing the cells in the presence of a binding molecule (antibody) specific for the tag.

Generally, in some cases, the TCR may be modified to have a variety of mutations that alter the affinity and dissociation rate of the TCR with the target antigen. In particular, mutations may increase avidity and/or decrease off-rate. Thus, the TCR may be mutated in at least one CDR and its variable domain framework region.

However, in a preferred embodiment, the CDR regions of the TCR are not modified or affinity matured in vitro, for example for the TCR receptor in the examples. This means that the CDR regions have naturally occurring sequences. This may be advantageous because affinity maturation in vitro may lead to immunogenicity on the TCR molecule. This may lead to the production of anti-drug antibodies that reduce or inactivate therapeutic efficacy and treatment and/or cause adverse reactions.

The mutation may be one or more substitutions, deletions or insertions. These mutations can be introduced by any suitable method known in the art, such as polymerase chain reaction, restriction enzyme-based Cloning, ligation independent Cloning procedures, described in, for example, Sambrook, Molecular Cloning-4 th edition (2012) Cold Spring Harbor Laboratory Press.

Theoretically, unpredictable TCR specificities with the risk of cross-reactivity may occur due to mismatches between endogenous and exogenous TCR chains. To avoid TCR sequence mismatches, recombinant TCR sequences can be modified to include a murinized C α region and a C β region, a technique that has been shown to be effective in enhancing the correct pairing of a variety of differently transduced TCR chains. Murine humanization of TCRs (i.e., the exchange of human constant regions in the α and β chains by their murine counterparts) is a commonly used technique for improving cell surface expression of TCRs in host cells. Without wishing to be bound by a particular theory, it is believed that the murinized TCR associates more efficiently with the CD3 co-receptor; and/or preferentially pair with each other and are less likely to form mixed TCRs on ex vivo genetically modified human T cells to express a TCR with the desired antigen specificity, but still retain and express its "original" (i.e., endogenous) TCR. Nine amino acids responsible for improving the expression of the murine TCR have been identified (Sommermeyer and Uckert, J Immunol.2010, 6.1; 184(11):6223-31) and at least one or all of the amino acid residues in the constant region of the TCR alpha and/or beta chains are envisaged to be replaced by their murine counterparts. This technique is also referred to as "minimurization" and offers the advantage of enhancing cell surface expression while at the same time reducing the number of "foreign" amino acid residues in the amino acid sequence and thus the risk of immunogenicity. Thus, in some embodiments, the TCR sequences may be modified to include a minimally murine ca region and a C β region.

Some embodiments relate to an isolated TCR as described herein, wherein the TCR is of the single chain type, wherein the TCR α chain and the TCR β chain are linked by a linker sequence.

Suitable single chain TCR formats comprise a first segment consisting of an amino acid sequence corresponding to the variable TCR α region, a second segment consisting of an amino acid sequence corresponding to the variable TCR β region fused to the N-terminus of the amino acid sequence corresponding to the extracellular sequence of the TCR β chain constant region, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment. Alternatively, the first segment may be comprised of an amino acid sequence corresponding to the TCR β chain variable region and the second segment may be comprised of an amino acid sequence corresponding to the TCR α chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to the TCR α chain constant region extracellular sequence. The single chain TCR described above may further comprise a disulphide bond between the first and second chains, and wherein the length of the linker sequence and the position of the disulphide bond are such that the variable domain sequences of the first and second segments are mutually orientated substantially as in a native TCR. More specifically, the first segment may be composed of an amino acid sequence corresponding to a TCR α chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a TCR α chain constant region, the second segment may be composed of an amino acid sequence corresponding to a TCR β variable region fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a TCR β chain constant region, and a disulfide bond may be provided between the first and second chains. The linker sequence may be any sequence which does not impair TCR function.

In the context of the present invention, a "functional" TCR α and/or β chain fusion protein shall mean a TCR or TCR variant which retains at least essential biological activity, e.g., is modified by addition, deletion or substitution of amino acids. For the α and/or β chains of the TCR this means that both chains are still able to form a T cell receptor (with an unmodified α and/or β chain or with another fusion protein of the invention α and/or β chain) which performs its biological function, in particular binding to a specific peptide-MHC complex of the TCR, and/or functional signal transduction following specific peptide: MHC interaction.

In particular embodiments, the TCR may be modified to a functional TCR alpha and/or beta chain fusion protein, wherein the epitope tag is 6 to 15 amino acids, preferably 9 to 11 amino acids in length. In another embodiment, the TCR may be modified to a functional TCR a and/or β chain fusion protein, wherein the TCR a and/or β chain fusion protein comprises two or more epitope tags, either spaced apart or directly in tandem. Embodiments of the fusion protein may comprise 2, 3, 4, 5, or even more epitope tags, as long as the fusion protein retains its biological activity/activities ("functionality").

Preferred are functional TCR alpha and/or beta chain fusion proteins according to the present invention, wherein the epitope tag is selected from, but not limited to, the CD20 or Her2/neu tag or other conventional tags such as myc tag, FLAG tag, T7 tag, HA (hemagglutinin) tag, His tag, S tag, GST tag or GFP tag. Myc, T7, GST, GFP tags are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, e.g., U.S. patent nos. 4,703,004 and 4,851,341). Myc tags may be preferred because high quality reagents are available for their detection. In addition to being recognized by an antibody, an epitope tag may of course have one or more additional functions. The sequences of these tags are described in the literature and are well known to those skilled in the art.

TCR fragments and variants

Another aspect of the invention relates to a polypeptide comprising a functional portion of a TCR as described herein. The functional portion may comprise at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: SEQ ID NO 7, SEQ ID NO 17, SEQ ID NO 27, SEQ ID NO 37, SEQ ID NO 47, SEQ ID NO 57, SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 30, SEQ ID NO 40, SEQ ID NO 50 and SEQ ID NO 60.

In particular embodiments, the polypeptide may be a functional part of the TCR alone, for example in soluble form. Alternatively, the polypeptide may be conjugated to other domains.

The functional moiety may mediate binding of the TCR to an antigen, particularly to an antigen-MHC complex.

In one embodiment, the functional moiety comprises a TCR α variable chain and/or a TCR β variable chain as described herein.

TCR variant molecules (i.e., molecules that combine a polypeptide comprising a functional portion of a TCR and other domains) may have the binding properties of a TCR receptor, but may be combined with the signaling domain of effector cells (other than T cells), particularly with the signaling domain of NK cells. Thus, some embodiments relate to proteins comprising a functional portion of a TCR as described herein in combination with a signaling domain of an effector cell (e.g., NK cell).

"binding" refers to the ability to specifically and non-covalently associate, associate or bind with a target.

Another aspect of the invention relates to a multivalent TCR complex comprising at least two TCRs as described herein. In one embodiment of this aspect, at least two TCR molecules are linked by a linker moiety to form a multivalent complex. Preferably, the complex is water soluble and therefore the linker moiety should be chosen accordingly. Preferably, the linker moiety is capable of attaching to a defined position on the TCR molecule, thereby minimizing the structural diversity of the complex formed. One embodiment of this aspect is provided by a TCR complex of the invention, wherein the polymer chain or peptide linker sequence extends between amino acid residues of each TCR which are not located in the variable region sequence of the TCR. Since the complexes of the invention are useful in medicine, the choice of linker moiety should be appropriately based on its pharmaceutical suitability, e.g. its immunogenicity. Examples of linker moieties that meet the desired criteria described above are known in the art, for example, in the art of linking antibody fragments.

Examples of linkers are hydrophilic polymers and peptide linkers. An example of a hydrophilic polymer is a polyalkylene glycol. The most common of these are based on polyethylene glycol or PEG. However, others are based on other suitable optionally substituted polyalkylene glycols, including polypropylene glycol, and copolymers of ethylene glycol and propylene glycol. The peptide linker comprises a chain of amino acids and serves to create a simple linker or multimerization domain to which the TCR molecule can be linked.

One embodiment relates to a multivalent TCR complex, wherein at least one of the TCRs is associated with a therapeutic agent.

Cytokine and chemokine release

Some embodiments relate to an isolated TCR as described herein, a polypeptide as described herein, a multivalent TCR complex as described herein, wherein IFN- γ secretion is induced by binding of a TCR of the invention expressed on effector cells to the amino acid sequence of SEQ ID NO:2 presented by HLA-a02:01 encoding molecules.

At 10^-7[M]When the binding to the amino acid sequence of SEQ ID NO:2 presented by the HLA a02:01 encoding molecule is compared to the binding of SEQ ID NO:4 presented by the HLA a02:01 encoding molecule, the secretion of IFN- γ induced by the binding of the TCR of the invention expressed on effector cells to the amino acid sequence of SEQ ID NO:2 presented by the HLA a02:01 encoding molecule may be at least 3-fold higher, preferably at least 10-fold higher, more preferably at least 20-fold higher, even more preferably at least 50-fold higher, most preferably at least 100-fold higher at the HA-1 peptide concentration of (a).

At 10^-6[M]When the binding to the amino acid sequence of SEQ ID NO:2 presented by the HLA a02:01 encoding molecule is compared to the binding of SEQ ID NO:4 presented by the HLA a02:01 encoding molecule, the secretion of IFN- γ induced by the binding of the TCR of the invention expressed on effector cells to the amino acid sequence of SEQ ID NO:2 presented by the HLA a02:01 encoding molecule may be at least 3-fold higher, preferably at least 10-fold higher, more preferably at least 20-fold higher, even more preferably at least 50-fold higher, most preferably at least 100-fold higher at the HA-1 peptide concentration of (a).

At 10^-5[M]When the binding to the amino acid sequence of SEQ ID NO:2 presented by the HLA-A02:01 encoding molecule is compared to the binding to SEQ ID NO:4 presented by the HLA-A02:01 encoding molecule, the secretion of IFN- γ induced by the binding of a TCR of the invention expressed on effector cells to the amino acid sequence of SEQ ID NO:2 presented by the HLA-A02:01 encoding molecule may be at least 3 times higher, such as at least 10 times higher, preferably at least 10 times higher, such as at least 20 times higher, at the HA-1 peptide concentration of (b)High, at least 50 times higher, at least 100 times higher.

Thus, for all HA-1^HTCR transgenic T cells, for the identification of HA-1^RVariants, requiring conjugation to HA-1^HCompared to a peptide concentration at least 1,000-fold higher, preferably at least 5,000-fold higher, more preferably at least 8000-fold higher, most preferably at least 10,0000-fold higher.

In particular embodiments, for example, when the ratio of TCR transgenic T cells to T2 cells is 2:1, at 10^-7[M]The secretion of IFN-. gamma.induced by binding of a TCR of the invention expressed on effector cells to the amino acid sequence of SEQ ID NO:2 presented by HLA-A02:01 encoding molecules may be greater than 500pg/ml, for example greater than 1000pg/ml at HA-1 peptide concentrations of

pg/ml, more preferably greater than 2000pg/ml, most preferably greater than 3000 pg/ml.

The "effector cells" may be Peripheral Blood Lymphocytes (PBLs) or Peripheral Blood Mononuclear Cells (PBMCs). Typically, the effector cell is an immune effector cell, particularly a T cell. Other suitable cell types include gamma-delta T cells and NK-like T cells.

The invention also relates to a method for identifying a TCR or a fragment thereof that binds to the target amino acid sequence SEQ ID No. 2 or an HLA-a 2-binding form thereof, preferably SEQ ID No. 2 presented by an HLA-a02: 01-encoding molecule or an HLA-a02: 06-encoding molecule, preferably presented by an HLA-a02: 01-encoding molecule or an HLA-a02: 06-encoding molecule, wherein the method comprises contacting a candidate TCR or a fragment thereof with the amino acid sequence SEQ ID No. 2 or an HLA-a 02-binding form thereof, preferably with an HLA-a02: 01-encoding molecule or an HLA-a02: 06-encoding molecule, preferably presented by an HLA-a02: 01-encoding molecule, and determining whether the candidate TCR or fragment thereof binds to the target and/or mediates an immune response.

Whether a candidate TCR, or fragment thereof, mediates an immune response can be determined, for example, by measuring cytokine secretion (e.g., IFN- γ secretion). As described above, cytokine secretion can be measured by an in vitro assay in which K562 cells (or other APCs) transfected with the ivtRNA encoding the amino acid sequence SEQ ID NO:2 are incubated with CD8+ -enriched PBMC expressing the TCR or a molecule comprising the TCR fragment to be studied.

Nucleic acid, vector

Another aspect of the invention relates to a nucleic acid encoding a TCR as described herein or encoding a polynucleotide encoding a TCR as described herein.

The following table lists the nucleotide sequences encoding each peptide sequence:

"nucleic acid molecule" generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthetic or obtained (e.g., isolated and/or purified) from a natural source, which may comprise natural, non-natural or altered nucleotides, and which may comprise natural, non-natural or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, rather than phosphodiesters between nucleotides of unmodified oligonucleotides. Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to a molecule that is either (i) constructed outside a living cell by linking natural or synthetic nucleic acid fragments to a nucleic acid molecule that is replicable in the living cell, or (ii) obtained from the replication of those described in (i) above. For the purposes herein, replication may be in vitro or in vivo. Nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using methods known in the art or commercially available methods (e.g., from Genscript, Thermo Fisher, and the like). Nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides), see, e.g., Sambrook et al. The nucleic acid may comprise any nucleotide sequence encoding any recombinant TCR, polypeptide, or protein, or a functional portion or functional variant thereof.

The present disclosure also provides variants of the isolated or purified nucleic acid, wherein the nucleic acid variant comprises a nucleotide sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence encoding a TCR described herein. Such variant nucleotide sequences encode sequences that specifically recognize HA-1^HFunctional TCR of an antigen.

The present disclosure also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to a nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of any of the nucleic acids described herein.

Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. By "high stringency conditions" is meant that the nucleotide sequence hybridizes specifically to the target sequence (the nucleotide sequence of any of the nucleic acids described herein) in a more detectable amount than does non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides having exactly complementary sequences or polynucleotides that contain only few discrete mismatches from random sequences that happen to have few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are easier to melt than full-length complementarity of 14-17 bases or more, and high stringency hybridization makes them easy to distinguish. Relatively high stringency conditions include, for example, low salt and/or high temperature conditions, such as those provided by about 0.02 to 0.1M NaCl, or equivalent, at a temperature of about 50 to 70 ℃. Such high stringency conditions allow for few, if any, mismatches between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any of the TCRs described herein. It is generally believed that conditions can be made more stringent by adding increased amounts of formamide.

The nucleic acid encoding the TCR may be modified as already described elsewhere herein. Useful modifications in the overall nucleic acid sequence may be codon optimization. Changes can be made that result in conservative substitutions within the expressed amino acid sequence. These changes, which do not affect function, can be made in the complementarity determining regions and non-complementarity determining regions of the amino acid sequences of the TCR chains. Generally, additions and deletions should not be made in the CDR3 region.

Another embodiment relates to a vector comprising a nucleic acid encoding a TCR as described herein.

The vector is preferably a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, adenoviral vector or a particle and/or vector for gene therapy.

A "vector" is any molecule or composition having the ability to carry a nucleic acid sequence to a suitable host cell in which synthesis of the encoded polypeptide can occur. Typically and preferably, the vector is a nucleic acid that has been engineered to introduce a desired nucleic acid sequence (e.g., a nucleic acid of the invention) using recombinant DNA techniques known in the art. The vector may comprise DNA or RNA and/or comprise liposomes. The vector may be a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, lentiviral vector, adenoviral vector or a particle and/or vector for gene therapy. A vector may comprise a nucleic acid sequence, such as an origin of replication, which allows it to replicate in a host cell. The vector may also comprise one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. The vector is preferably an expression vector comprising a nucleic acid according to the invention operably linked to a sequence allowing the expression of said nucleic acid.

Preferably, the vector is an expression vector. More preferably, the vector is a retrovirus, more particularly a gamma-retrovirus or lentivirus vector.

Cell, cell line

Another aspect of the invention relates to a cell expressing a TCR as described herein.

In some embodiments, the cell is isolated or non-naturally occurring.

In particular embodiments, the cell may comprise a nucleic acid encoding a TCR as described herein or a vector comprising the nucleic acid.

In a cell, the above-described vector comprising a nucleic acid sequence encoding the above-described TCR can be introduced, or the ivtRNA encoding the TCR can be introduced. The cells may be peripheral blood lymphocytes, such as T cells. Cloning and exogenous expression methods for TCRs are described, for example, in Engels et al (Relay or organization of cancer is predicted by peptide-major histocompatibility complex affinity. cancer Cell,23(4), 516-26.2013). Transduction of primary human T cells with lentiviral vectors is described, for example, in Cribbs "simplified production and differentiation of viral vectors to achievehigh transduction in primary human T cells" BMC Biotechnol.2013; 13: 98.

The terms "transfection" and "transduction" are interchangeable and refer to the process of introducing an exogenous nucleic acid sequence into a host cell (e.g., a eukaryotic host cell). Note that introduction or transfer of nucleic acid sequences is not limited to the mentioned methods, but may be achieved by any number of means, including electroporation, microinjection, gene gun delivery, lipofection, repeat transfection (superfection), and infection by a retrovirus or other virus suitable for transduction or transfection as mentioned.

Some embodiments relate to a cell comprising:

a) an expression vector comprising at least one nucleic acid as described herein, or

b) A first expression vector comprising a nucleic acid encoding an alpha chain of a TCR as described herein, and a second expression vector comprising a nucleic acid encoding a beta chain of a TCR as described herein.

In some embodiments, the cell is a Peripheral Blood Lymphocyte (PBL) or a Peripheral Blood Mononuclear Cell (PBMC). The cell may be a natural killer cell or a T cell. Preferably, the cell is a T cell. The T cells may be CD4+ or CD8+ T cells.

In some embodiments, the cell is a stem cell-like memory T cell.

Stem cell-like memory T cells (TSCMs) are a less differentiated subset of CD8+ T cells, characterized by the ability to self-renew and persist for a long period of time. Once these cells encounter their antigens in vivo, they further differentiate into central memory T Cells (TCM), effector memory T cells (TEM), and terminally differentiated effector memory T cells (TEMRA), while some TSCMs remain quiescent (Flynn et al, Clinical & Translational Immunology (2014)). These remaining TSCM cells show the ability to establish a persistent immunological memory in vivo and are therefore considered to be an important T cell subset for adoptive T cell therapy (Lugli et al, Nature Protocols 8, 33-42 (2013) gattinini et al, nat. med.2011 10 months; 17(10): 1290-. Immuno-magnetic selection can be used to limit the T cell bank to stem cell memory T cell subtypes (Riddell et al 2014, Cancer Journal 20(2): 141-44).

Antibodies targeting TCR

Another aspect of the invention relates to an antibody or antigen-binding fragment thereof that specifically binds to a portion of a TCR described herein, which portion mediates binding to HA-1^HThe specificity of (A). In one embodiment, mediation of TCR HA-1^HSpecific portions comprise the alpha chain CDR3 of SEQ ID NO 7, SEQ ID NO 17, SEQ ID NO 27, SEQ ID NO 37, SEQ ID NO 47 and SEQ ID NO 57 and/or the beta chain CDR3 of SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 30, SEQ ID NO 40, SEQ ID NO 50 and SEQ ID NO 60.

The antibody or antigen binding fragment thereof can modulate the activity of the TCR. Which may or may not block TCR from HA-1^HIn combination with (1). Which can be used to modulate the therapeutic activity of the TCR or for diagnostic purposes.

Pharmaceutical compositions, drug treatments and kits

Another aspect of the invention relates to a pharmaceutical composition comprising: a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex as described herein, a nucleic acid encoding a TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or an antibody that specifically binds to a portion of a TCR as described herein.

Those active ingredients of the invention are preferably used in such pharmaceutical compositions in a dosage which is in admixture with an acceptable carrier or carrier material, whereby the disease can be treated or at least alleviated. Such compositions may contain, in addition to the active ingredient and carrier, filler materials, salts, buffers, stabilizers, solubilizers and other materials, which are known in the art.

The term "pharmaceutically acceptable" defines non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. The choice of carrier depends on the application.

The pharmaceutical composition may comprise additional ingredients that enhance the activity of the active ingredient or supplement therapy. Such additional ingredients and/or factors may be part of the pharmaceutical composition to achieve a synergistic effect or to minimize adverse or undesirable effects.

Techniques for the formulation or preparation and application/administration of the active ingredients of the present invention are disclosed in the latest edition of "Remington's Pharmaceutical Sciences", Mack Publishing co. Suitable administration is parenteral administration, for example intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injections. Intravenous injection is the preferred treatment for patients.

According to a preferred embodiment, the pharmaceutical composition is an infusion or injection.

The injectable composition is a pharmaceutically acceptable fluid composition comprising at least one active ingredient, such as an expanded T cell population that expresses a TCR (e.g., autologous or allogeneic to the patient to be treated). The active ingredient is typically dissolved or suspended in a physiologically acceptable carrier, and the composition may additionally contain minor amounts of one or more non-toxic auxiliary substances, such as emulsifiers, preservatives, pH buffers and the like. Such injectable compositions useful for use with the fusion proteins of the present disclosure are conventional; suitable formulations are well known to those of ordinary skill in the art.

Typically, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

Thus, another aspect of the invention relates to a TCR as described herein, a polypeptide comprising a functional portion of said TCR, a multivalent TCR complex according to the invention, a nucleic acid encoding said TCR, a vector comprising said nucleic acid, a cell comprising said TCR, or an antibody specifically binding to a portion of a TCR as described herein, for use as a medicament.

Some embodiments relate to a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex according to the invention, a nucleic acid encoding the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, for use in treating cancer.

In one embodiment, the cancer is a hematologic cancer.

Hematologic cancers, also known as blood cancers, do not form solid tumors and are therefore mainly dispersed in the body.

The hematologic cancer may be selected from the group consisting of: non-hodgkin's lymphoma (NHL), Hodgkin's Lymphoma (HL), multiple myeloma, Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL), Mixed Phenotype Acute Leukemia (MPAL), Chronic Myeloid Leukemia (CML), B-cell pleomorphic lymphoma, hairy cell leukemia, Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), central nervous system lymphoma, CD37+ dendritic cell lymphoma, lymphoblastic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, extraosseous plasmacytoma, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT tissue), nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, precursor B lymphoblastic lymphoma, Acute Myelogenous Leukemia (AML), mixed phenotype Acute Lymphoblastic Leukemia (ALL), Mixed Phenotype Acute Leukemia (MPAL), mixed phenotype acute leukemia (mmal), Chronic Myelogenous Leukemia (CML), B cell pleomorphic lymphoma, hairy cell leukemia, Chronic Lymphocytic Leukemia (CLL), small, Immunoblastic large cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt's lymphoma/leukemia, B cell proliferation of unknown malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disease. Other hematologic cancers or diseases include myelodysplasia.

Also contemplated herein are pharmaceutical compositions and kits comprising one or more of the following: (i) an isolated TCR as described herein; (ii) a viral particle comprising a nucleic acid encoding a recombinant TCR; (iii) an immune cell, e.g., a T cell or NK cell, modified to express a recombinant TCR as described herein; (iv) a nucleic acid encoding a recombinant TCR as described herein. In some embodiments, the disclosure provides compositions comprising lentiviral vector particles (or T cells that have been modified to express recombinant TCRs using vector particles described herein) comprising nucleotide sequences encoding recombinant TCRs described herein. Such compositions may be administered to a subject in the methods of the present disclosure as further described herein.

Compositions comprising modified T cells described herein may be used in methods and compositions for adoptive immunotherapy according to known techniques or variations thereof that will be apparent to those of skill in the art based on the present disclosure.

In some embodiments, the cells are formulated as follows: the cells are first harvested from their culture medium and then washed and concentrated in a therapeutically effective amount in a medium or container system ("pharmaceutically acceptable" carrier) suitable for administration. A suitable infusion medium may be any isotonic medium preparation, typically physiological saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

The number of cells in the composition for effective treatment is typically greater than 10 cells, and up to 10⁶Up to and including 10⁸Or 10⁹And can be greater than 10¹⁰And (4) cells. The number of cells will depend on the intended end use of the composition, as well as the type of cells contained therein. For example, if cells specific for a particular antigen are desired, the population will contain greater than 70%, typically greater than 80%, 85% and 90-95% of such cells. For the uses provided herein, the cells are typically in a volume of one liter or less, and may be 500ml or less, even 250ml or 100ml or less. Thus, the desired density of cells is typically greater than 10⁶Individual cells/ml, and usually greater than 10⁷Individual cells/ml, usually greater than 10⁸Individual cells/ml or greater. Clinically relevant numbers of immune cells can be distributed to multiple infusionsA cumulative value of 10 or more⁹、10¹⁰Or 10¹¹And (4) cells. The pharmaceutical compositions provided herein can be in a variety of forms, such as solid, liquid, powder, aqueous, or lyophilized forms. Examples of suitable pharmaceutical carriers are known in the art. Such carriers and/or additives may be formulated by conventional methods and may be administered to a subject in a suitable dosage. Stabilizers such as lipids, nuclease inhibitors, polymers and chelating agents can protect the composition from degradation in vivo. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, and isotonic agents may be included.

The recombinant TCRs described herein or viral vector particles comprising nucleotide sequences encoding the recombinant TCRs provided herein can be packaged into kits. Kits may optionally include one or more components, such as instructions for use, devices, and other reagents, as well as components, such as tubes, containers, and syringes for performing the methods. An exemplary kit can comprise a nucleic acid encoding a recombinant TCR, a recombinant TCR polypeptide, or a virus provided herein, and can optionally comprise instructions for use, a device for detecting the virus in a subject, a device for administering the composition to the subject, and a device for administering the composition to the subject.

Also contemplated herein are kits comprising a polynucleotide encoding a gene of interest (e.g., a recombinant TCR). Also contemplated herein are kits comprising a viral vector encoding a sequence of interest (e.g., a recombinant TCR) and optionally a polynucleotide sequence encoding an immune checkpoint inhibitor.

Kits contemplated herein also include kits for carrying out a method of detecting the presence of a polynucleotide encoding any one or more of the TCRs disclosed herein. In particular, such diagnostic kits may comprise suitable amplification and detection primer sets and other related reagents for performing deep sequencing to detect polynucleotides encoding the TCRs disclosed herein. In other embodiments, the kits herein may comprise reagents, such as antibodies or other binding molecules, for detecting the TCRs disclosed herein. The diagnostic kit can further comprise instructions for determining the presence of a polynucleotide encoding a TCR disclosed herein or for determining the presence of a TCR disclosed herein. The kit may further comprise instructions. The specification generally includes tangible expressions that describe: the components contained in the kit, as well as methods of administration, including methods for determining the appropriate state, appropriate dosage, and appropriate method of administration for a subject. The instructions may also include instructions for monitoring the subject during the treatment period.

The kits provided herein can also include a device for administering a composition described herein to a subject. Any of a variety of devices known in the art for administering drugs or vaccines can be included in the kits provided herein. Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers, such as droppers (eyedroppers). Typically, the device used to administer the virus of the kit will be compatible with the virus of the kit; for example, needleless injection devices such as high pressure injection devices may be included in kits that contain viruses that are not destroyed by high pressure injection, but are generally not included in kits that contain viruses that are destroyed by high pressure injection.

The kits provided herein may also include a device for administering a compound (e.g., a T cell activator or stimulator, or a TLR agonist such as a TLR4 agonist) to a subject. Any of a variety of devices known in the art for administering a drug to a subject can be included in the kits provided herein. Exemplary devices include hypodermic needles, intravenous needles, catheters, needleless injections, but are not limited to hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers, such as droppers. Generally, the device used to administer the compounds of the kit will be compatible with the desired method of administering the compounds.

Experiment of

Example (b):

example 1: HA-1^HTCR transgenic T cells binding HA-1^HMultimer

Isolation of HA-1 Using in vitro priming method^HReactive T cellsAnd (4) cloning. Priming System mature dendritic cells (mDCs) from HLA-A02:01 Positive donors were used as antigen presenting cells and autologous CD8⁺The enriched T cells act as responder cells. In vitro transcribed RNA (ivtRNA) encoding 31 amino acids (ARFAEGLEKLKECVLHDDLLEARRPRAHECL; SEQ ID NO:126) of the human HMHA1 gene was used as a source of specific antigen. After electroporation into mdcs, HMHA1 encodes ivtRNA that is translated into protein, subsequently processed and presented as a peptide by HLA-a02:01 molecules on mdcs. In vitro co-culture of T cells with mdcs transfected with ivtRNA from the same donor resulted in de novo induction of antigen-specific T cells, which served as the source of the corresponding TCR. Antigen-specific T cells can be enriched by a variety of methods and cloned by limiting dilution or FACS-based single cell sorting. Sequencing HA-1 by Next Generation^HThe sequences of the TCR α and TCR β chains of the reactive T cell clones were identified and cloned into the retroviral vector pes.12-6 after the constant TCR region was exchanged by its murine counterpart. PBMCs from healthy donors were isolated by ficoll gradient centrifugation. CD 8T cells were enriched by negative magnetic selection (Miltenyi) and stimulated with anti-CD 3 and anti-CD 28mAh (BD Pharmingen, Heidelberg, Germany) in non-tissue culture 24-well plates. Amphotropic retroviral particles were generated by transfecting HEK293T cells with the corresponding TCR-encoding retroviral plasmid and the two expression plasmids. The day two post-stimulation, CD 8T cells were transduced and on day twelve transduced CD8 was enriched by FACS using murine constant β regions as transduction markers⁺The cells were then expanded by a rapid expansion protocol (Riddell SR, Science,1992Jul 10; 257(5067): 238-41).

As a result:

with a secondary amine of HA-1^HSix different TCRs isolated from reactive T cell clones and one that did not recognize HA-1^HTo transduce CD 8T cells. Using the Gene optimizer^TMThe algorithm (ThermoFisher) codon-optimizes the nucleotide sequence of the isolated TCR and murinizes the constant region. Codon-optimized and murine TCRs were used for all other experiments described herein. Applying it to HA-1^HMHC multimer (immunAware) and antibody staining against CD8 and murine constant beta region. All but one HA-1^HThe TCR transgenic T cell populations all bind HA-1 very efficiently^H-MHC multimers (>90%); TCR _6 alone shows a lower percentage of HA-1^HMHC multimer positive cells (37%). No HA-1 observed for the control TCR^H-MHC multimer staining. These results show that HA-1 was obtained from^HTCRs isolated from reactive T cell clones can be expressed transgenically in T cells from healthy donors. (FIG. 1)

Example 2: HA-1^HRecognition of HA-1 by TCR transgenic T cells^HPositive target cell

Confirmation of HA-1 of TCR transgenic T cells according to the following protocol^HSpecificity:

to T2 cells (HLA-A02: 01) as target cells^{Yang (Yang)}) Load saturation capacity (10)^-5M) HA-1^HPeptide (SEQ ID NO:2) or control peptide Astn 1P 1268L (KLYGLDWAEL). In addition, HLA-A02:01 and the coding HA-1 are used^HThe part of the HMHA1 gene of the epitope transduced K562 cells. K562 cells transduced with HLA-a02:01 only were used as controls. Each target cell line and TCR transgenic T cells were co-cultured using 20,000T cells and 10,000 target cells in a 2:1 ratio. After 20-24 hours, co-culture supernatants were analyzed for IFN- γ concentration by standard sandwich ELISA (BD human IFN- γ ELISA device).

As a result:

HA-1^Hrecognition of TCR transgenic T cells loaded with HA-1^HT2 and HMHA1 transduced K562 cells, but did not recognize control target cells. T cells expressing the control TCR only recognized T2 cells loaded with the control peptide. These results show that HA-1 was obtained from^HTCR isolated from reactive T cell clones is functional after transfer to T cells of a healthy donor. (FIG. 2)

Example 3: HA-1^HThe inability of TCR transgenic T cells to recognize HA-1 loaded cells^RTarget cells for peptides

For analysis, two variants of HA-1 were identified^HAnd HA-1^RThe difference in (A) was analyzed for HA-1^HFunctional avidity of TCR transgenic T cells for both variants loaded on HLA-a02: 01:

t2 cells were loaded externally with different concentrations (10)^-11M-10^-5M) HA-1^H-peptides and (10)^-8M-10^-5M) HA-1^RPeptides and coculture with TCR transgenic T cells using 20,000T cells and 10,000T 2 cells in a 2:1 ratio. After 20-24 hours, co-culture supernatants were analyzed for IFN- γ concentration by standard sandwich ELISA (BD human IFN- γ ELISA device).

As a result:

TCR _3 and TCR _4 show HA-1 loading on HLA-A02:01 encoding molecules^H-the highest functional affinity of the peptide, and TCR _2 shows the lowest functional affinity. At non-physiologically high peptide concentrations (10)^-5M) some HA-1 was observed^HTCR transgenic T cell Pair HA-1^RIs only very rarely identified and for all HA-1 s^HTCR transgenic T cells, for the identification of HA-1^RVariants, requiring comparison with HA-1^HAt least 10,0000 times higher peptide concentration. These results show that HA-1^HTCR for HA-1^HVariants are highly specific. In addition, for HLA-A02:01 encoding molecules loaded HA-1^RThe functional affinity of the peptide was compared with the TCR (TCR2) described in WO2018058002A 1. In contrast to the TCRs described herein, the TCR is also at 10^-6Recognition of HA-1 at M concentration^R-a peptide, and at 10^-5M release significantly more IFN- γ at concentrations indicating that all TCRs described herein have significantly higher specificity than the TCR described in WO2018058002a 1. (FIGS. 3A and 3B)

Example 4: HA-1^HRecognition of HA-1 by TCR transgenic T cells^HPositive LCL

To analyze HA-1^HTCR transgenic T cells recognize physiological levels of HA-1 on unmodified target cells^HCo-culture of T cells with different Lymphoblast Cell Lines (LCLs), and analysis of cytokine release and cytotoxicity of TCR transgenic T cells:

seven LCL DNAExtract solutions (Illumina) were isolated and the portion of HMHA1 gene encoding HA-1 was amplified by PCR. PCR products were separated from agarose gels for sequencing (Eu)rofins Genomics). Assignment of LCL to HA-1 based on sequencing results^HPositive (HA-1)^H/HAnd HA-1^H/R) Or HA-1^HNegative (HA-1)^R/R) And (4) grouping. To analyze cytokine release, TCR transgenic T cells and LCLs were co-cultured at a 2:1 ratio using 20,000T cells and 10,000T 2 cells. After 20-24 hours, co-culture supernatants were analyzed for IFN- γ concentration by standard sandwich ELISA (BD human IFN- γ ELISA device). For cytotoxicity assays, co-cultures were established at an effector to target ratio of 5:1 using 100,000 TCR transgenic T cells and 20,000 LCLs that had been transduced with fluorescent marker genes. Monitoring using living cells (ZOOM) the decrease of fluorescent target cells (total integrated intensity in RCU. times. mu.m) was measured every two hours over a total time period of 20 hours²Image, RCU ═ red calibration unit).

As a result:

all HA-1^HTCR transgenic T cells in HA-1^HPositive LCL (LCL 1-5) releases IFN-. gamma.upon co-culture, but upon co-culture with HA-1^HNegative LCL (LCL 6-7) did not follow co-cultivation. T cells expressing the control TCR did not recognize any LCL. Further, HA-1^HPositive LCL quilt HA-1^HTCR transgenic T cell lysis. Untransduced CD 8T cells were used as a negative control and LCLs were not lysed. These results show that HA-1^HThe TCR can recognize physiological level of HLA-A02:01 encoding molecule loaded with HA-1^H. (FIGS. 4A and 4B)

Example 5: HA-1H-TCR transgenic T cell recognition HA-1H positive tumor cell line

To analyze HA-1^HTCR transgenic T cells recognize HA-1 presented on tumor cell lines^HAbility of T cells to associate different HA-1 with^HPositive tumor cell lines were co-cultured.

DNA from tumor cell lines was isolated (Quick Extract DNA Extract Solution, Illumina) and the portion of the HMHA1 gene encoding HA-1 was amplified by PCR. PCR products were separated from agarose gels and extracted from the gels for useFollowed by sequencing (Eurofins Genomics). Mixing HA-1^HPositive (HA-1)^H/HAnd HA-1^H/R) Tumor cell lines were used for the experiments. To analyze cytokine release, TCR transgenic T cells and tumor cell lines were co-cultured at a 2:1 ratio using 20,000T cells and 10,000T 2 cells. After 20-24 hours, co-culture supernatants were analyzed for IFN- γ concentration by standard sandwich ELISA (BD human IFN- γ ELISA device). For cytotoxicity assays, co-cultures were established with 100,000 TCR transgenic T cells and 20,000 tumor cells that had been transduced with fluorescent marker genes at an effector to target ratio of about 5: 1. Monitoring using living cells (ZOOM) the reduction of fluorescent target cells (total integrated intensity in RCU x μm) was measured every two hours over a total time period of 20 hours²Image, RCU ═ red calibration unit).

As a result:

all HA-1 except TCR _2 when co-cultured with tumor cell lines^HTCR transgenic T cells all release IFN- γ. T cells expressing the control TCR did not recognize tumor cell lines. HA-1^HTCR transgenic T cells also show cytotoxic activity against tumor cell lines. Consistent with the cytokine release data, TCR _2 lyses target cells with lower efficiency. Untransduced CD 8T cells were used as negative control. (FIGS. 5A and 5B)

Example 6: t cells expressing TCR _3 and TCR _4 recognize HA-1 at levels comparable to the TCR previously described^HPositive tumor cell line

To add HA-1^HTCR transgenic T cell Pair HA-1^HIdentification of positive tumor cell lines in comparison to TCR 2-expressing T cells described in WO2018058002a1, 20,000T cells and 10,000 tumor cells were co-cultured. After 20 hours, the co-culture supernatants were analyzed for IFN- γ concentration by standard sandwich ELISA (BD human IFN- γ ELISA device). For cytotoxicity assays, 10,000 HA-1 s were used^Hnegative/GFP positive K562 cells and 10,000 cells of the mCherry expressing tumor cell line were co-cultured with 20,000 TCR transgenic T cells. 45After hours, the samples were analyzed by flow cytometry and mCherry positivity (HA-1) was calculated^HPositive) and GFP positive (HA-1)^HNegative) proportion of cells and normalized to the proportion measured for the control TCR.

As a result:

in the presence of HA-1^HAfter co-culture of positive tumor cell lines, T cells expressing TCR _3 and TCR _4 released IFN- γ in similar amounts to T cells expressing TCR2 described in WO2018058002a1 (fig. 6A). T cells expressing TCR _3 and TCR _4 also showed cytotoxic activity against different tumor cell lines comparable to TCR2 (fig. 6B).

Example 7: TCR recognition motifs

To analyze the specific recognition motif of the TCR, a serine substitution scan was performed. By single substitution of the original amino acids of the epitope with serine, positions within the peptide essential for TCR-mediated recognition can be identified. To define the recognition motif, effector T cells expressing different HA-1 TCRs (TCR _3, TCR _4 and TCR2 described in WO2018058002A1) were co-cultured with T2 cells, the T2 cells being loaded with the HA-1H peptide (SEQ ID NO:2) with each individual amino acid residue successively replaced by serine, or with a control peptide. T2 cells were loaded individually with saturating concentrations (10)^-5M), washed, and co-cultured with effector cells at 1: 1E: T. After approximately 20 hours of co-incubation, the supernatants were harvested and analyzed for secreted IFN- γ by ELISA. T cells expressing the control TCR were used as a negative control.

As a result:

compared to TCR2(WO2018058002a1), TCR _3 and TCR _4 show a more specific recognition pattern at serine, e.g. recognizing less peptide.

Comparison of different constant TCR regions

To compare the effects of different constant TCR regions, TCR _3 was cloned with murine, minimally murine (Sommermeyer and Uckert,2010, j. Testing of HA-1 with different constant TCR regions by Co-culturing 20,000T cells and 10,000 tumor cells^HTCR transgenic T cell Pair HA-1^HIdentification of TCR-positive tumor cell lines or LCLs. After 20-24 hours, pass the standardSandwich ELISA (BD human IFN- γ ELISA device) assay IFN- γ concentration in co-culture supernatants. For cytotoxicity assays, 10,000 HA-1 s were used^Hnegative/GFP positive K562 cells and 10,000 cells of the mCherry expressing tumor cell line were co-cultured with 20,000 TCR transgenic T cells. After 45 hours, the samples were analyzed by flow cytometry and mCherry positivity (HA-1) was calculated^HPositive) and GFP positive (HA-1)^HNegative) proportion of cells and normalized to the proportion measured for the control TCR.

As a result:

as expected, HA-1 when the murine-derived constant region was replaced with the minimally murine-derived or human constant TCR region^HSlightly reduced reactivity of TCR transgenic T cells (fig. 8A and B). However, even with human constant TCR regions, the peptide HAs been found to interact with HA-1^HAfter co-culture of positive target cells, T cells still specifically released IFN-gamma and showed cytotoxic effects.

The description also includes the following embodiments:

embodiment 1. isolated T Cell Receptor (TCR) specific for an allelic variant of minor histocompatibility antigen 1 (HA-1).

Embodiment 2. the isolated TCR of embodiment 1, wherein the allelic variant of HA-1 is HA-1^H。

Embodiment 3 the isolated TCR of any of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence SEQ ID NO 2 or a fragment thereof.

Embodiment 4 the isolated TCR of any of the preceding embodiments, wherein the TCR does not recognize the amino acid sequence SEQ ID NO 4 or a fragment thereof.

Embodiment 5. the isolated TCR of any of the preceding embodiments, wherein the recognition motif of the TCR comprises at least the sequence set forth in SEQ ID NO: 127.

Embodiment 6 the isolated TCR according to any of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO. 2 in HLA-A2-bound form.

Embodiment 7. the isolated TCR of any of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO:2 presented by HLA-a02:01 encoding molecules.

Embodiment 8 the isolated TCR of any of the preceding embodiments, wherein the TCR comprises a TCR a chain comprising complementarity determining region 3(CDR3), the CDR3 having an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO 7, SEQ ID NO 17, SEQ ID NO 27, SEQ ID NO 37, SEQ ID NO 47 and SEQ ID NO 57.

Embodiment 9 the isolated TCR of any one of the preceding embodiments, wherein the TCR comprises a TCR β chain comprising CDR3, the CDR3 having an amino acid sequence selected from the group consisting of: 10, 20, 30, 40, 50 and 60.

Embodiment 10 the isolated TCR of any one of the preceding embodiments, wherein the TCR comprises

a) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 5, a CDR2 having the amino acid sequence of SEQ ID NO. 6 and a CDR3 having the sequence of SEQ ID NO. 7,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 8, CDR2 having the amino acid sequence of SEQ ID No. 9 and CDR3 having the sequence of SEQ ID No. 10; or

b) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 15, a CDR2 having the amino acid sequence of SEQ ID NO. 16 and a CDR3 having the sequence of SEQ ID NO. 17,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 18, CDR2 having the amino acid sequence of SEQ ID No. 19 and CDR3 having the sequence of SEQ ID No. 20; or

c) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 25, a CDR2 having the amino acid sequence of SEQ ID NO. 26 and a CDR3 having the sequence of SEQ ID NO. 27,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 28, CDR2 having the amino acid sequence of SEQ ID No. 29 and CDR3 having the sequence of SEQ ID No. 30;

d) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO 35, a CDR2 having the amino acid sequence of SEQ ID NO 36 and a CDR3 having the sequence of SEQ ID NO 37,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO:38, CDR2 having the amino acid sequence of SEQ ID NO:39 and CDR3 having the sequence of SEQ ID NO: 40; or

e) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO 45, a CDR2 having the amino acid sequence of SEQ ID NO 46 and a CDR3 having the sequence of SEQ ID NO 47,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 48, CDR2 having the amino acid sequence of SEQ ID No. 49 and CDR3 having the sequence of SEQ ID No. 50; or

f) A TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 55, a CDR2 having the amino acid sequence of SEQ ID NO. 56 and a CDR3 having the sequence of SEQ ID NO. 57,

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO:58, CDR2 having the amino acid sequence of SEQ ID NO:59 and CDR3 having the sequence of SEQ ID NO: 60.

Embodiment 11 the isolated TCR of any of the preceding embodiments, wherein the TCR comprises a TCR a chain and a TCR β chain, wherein

a) The variable TCR alpha region has an amino acid sequence which is at least 80% identical to SEQ ID NO. 11 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID NO. 7,

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 12 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 10; or

b) The variable TCR alpha region has an amino acid sequence which is at least 80% identical to SEQ ID NO 21 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID NO 17,

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 22 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 20; or

c) -the variable TCR α region has an amino acid sequence at least 80% identical to SEQ ID No. 31 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 27;

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 32 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID No. 30; or

d) -the variable TCR α region has an amino acid sequence at least 80% identical to SEQ ID No. 41 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 37;

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 42 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID No. 40; or

e) -the variable TCR α region has an amino acid sequence at least 80% identical to SEQ ID No. 51 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 47;

-the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 52 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 50; or

f) -the variable TCR α region has an amino acid sequence at least 80% identical to SEQ ID No. 61 and comprises a CDR3 region having the amino acid sequence set forth in SEQ ID No. 57;

the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 62 and comprises a CDR3 region having the amino acid sequence shown in SEQ ID No. 60.

Embodiment 12 the isolated TCR of any one of the preceding embodiments, wherein the TCR comprises

a) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 11 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 12; or

b) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 21 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 22; or

c) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 31 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 32; or

d) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 41 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 42; or

e) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 51 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 52; or

f) A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 61 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 62.

Embodiment 13: the isolated TCR of any of the preceding embodiments, wherein the TCR comprises

a) A variable TCR α region having the amino acid sequence of SEQ ID NO. 11 and a variable TCR β region having the amino acid sequence of SEQ ID NO. 12; or

b) A variable TCR α region having the amino acid sequence of SEQ ID NO 21 and a variable TCR β region having the amino acid sequence of SEQ ID NO 22; or

c) A variable TCR α region having the amino acid sequence of SEQ ID NO. 31 and a variable TCR β region having the amino acid sequence of SEQ ID NO. 32; or

d) A variable TCR alpha region having the amino acid sequence of SEQ ID NO 41 and a variable TCR beta region having the amino acid sequence of SEQ ID NO 42; or

e) A variable TCR α region having the amino acid sequence of SEQ ID NO 51 and a variable TCR β region having the amino acid sequence of SEQ ID NO 52; or

f) A variable TCR alpha region having the amino acid sequence of SEQ ID NO 61 and a variable TCR beta region having the amino acid sequence of SEQ ID NO 62.

Embodiment 14 the isolated TCR of any one of the preceding embodiments, wherein the TCR is purified.

Embodiment 15 the isolated TCR of any one of the preceding embodiments, wherein the amino acid sequence of the TCR comprises one or more phenotypically silent substitutions.

Embodiment 16 the isolated TCR of any one of the preceding embodiments, wherein the amino acid sequence of the TCR is modified to comprise a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety.

Embodiment 17 the isolated TCR of embodiment 16, wherein the therapeutic agent is selected from the group consisting of an immune effector molecule, a cytotoxic agent, and a radionuclide.

Embodiment 18 the isolated TCR of embodiment 17, wherein the immune effector molecule is a cytokine.

Embodiment 19 the isolated TCR of any of the preceding embodiments, wherein the TCR is soluble or membrane-bound.

Embodiment 20: the isolated TCR of embodiment 16, wherein the pharmacokinetic modifying moiety is at least one polyethylene glycol repeat unit, at least one diol group, at least one sialic acid group, or a combination thereof.

Embodiment 21 the isolated TCR of any of the preceding embodiments, wherein the TCR is single-chain, wherein the TCR a chain is linked to the TCR β chain by a linker sequence.

Embodiment 22 the isolated TCR of any of the preceding embodiments, wherein the TCR a chain or the TCR β chain is modified to comprise an epitope tag.

Embodiment 23 the isolated TCR of any of the preceding embodiments, wherein the recombinant TCR sequence can be modified to comprise a minimally murine ca region and a C β region.

Embodiment 24. an isolated polypeptide comprising a functional portion of a TCR of any one of embodiments 1 to 21, wherein the functional portion comprises at least one of the following amino acid sequences: SEQ ID NO 7, SEQ ID NO 17, SEQ ID NO 27, SEQ ID NO 37, SEQ ID NO 47, SEQ ID NO 57, SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 30, SEQ ID NO 40, SEQ ID NO 50 and SEQ ID NO 60.

Embodiment 25 the isolated polypeptide of embodiment 24, wherein the functional moiety comprises a TCR a variable chain and/or a TCR β variable chain.

Embodiment 26. a multivalent TCR complex comprising at least two TCRs as embodied in any one of embodiments 1 to 23.

Embodiment 27. multivalent TCR complexes wherein at least one of the TCRs is associated with a therapeutic agent.

Embodiment 28 the isolated TCRs according to embodiments 1 to 23, the polypeptides according to embodiments 24 and 25, the multivalent TCR complexes according to embodiments 25 and 26, wherein IFN- γ secretion is induced by binding to the amino acid sequence of SEQ ID NO:2 presented by HLA-a02:01 encoding molecules.

Embodiment 29. a nucleic acid encoding a TCR according to any one of embodiments 1 to 22 or encoding a polypeptide according to embodiments 24 to 25.

Embodiment 30. the nucleic acid of embodiment 29, wherein the nucleic acid is codon optimized.

Embodiment 31. a vector comprising the nucleic acid of embodiment 29 or 30.

Embodiment 32. the vector of embodiment 31, wherein the vector is an expression vector.

Embodiment 33. the vector according to embodiment 31 or 32, wherein the vector is a retroviral vector.

Embodiment 34 the vector of embodiment 31 or 32, wherein the vector is a lentiviral vector.

Embodiment 35. cells expressing a TCR according to embodiments 1 to 23.

Embodiment 36. the cell of embodiment 34, wherein the cell is isolated or non-naturally occurring.

Embodiment 37. a cell comprising a nucleic acid according to embodiment 29 or 30 or a vector according to embodiments 31 to 35.

Embodiment 38 the cell of embodiments 35-37, wherein the cell comprises:

a) an expression vector comprising at least one nucleic acid as embodied in embodiment 29 or 30,

b) a first expression vector comprising a nucleic acid encoding an alpha chain of a TCR as embodied in any one of embodiments 1 to 23, and a second expression vector comprising a nucleic acid encoding a beta chain of a TCR as embodied in any one of embodiments 1 to 23.

Embodiment 39 the cell of any one of embodiments 35 to 38, wherein the cell is a Peripheral Blood Lymphocyte (PBL) or a Peripheral Blood Mononuclear Cell (PBMC).

Embodiment 40 the cell of any one of embodiments 35 to 38, wherein the cell is a T cell.

Embodiment 41. an antibody or antigen-binding fragment thereof that specifically binds to a portion of a TCR according to embodiments 1 to 23, which portion mediates specificity for one allelic variant of HA-1.

Embodiment 42 the antibody or antigen binding fragment thereof of embodiment 40, wherein the portion of the TCR that mediates specificity for one allelic variant of HA-1 comprises the alpha chain CDR3 of SEQ ID NO 7, SEQ ID NO 17, SEQ ID NO 27, SEQ ID NO 37, SEQ ID NO 47, SEQ ID NO 57, and/or the beta chain CDR3 of SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 30, SEQ ID NO 40, SEQ ID NO 50, and SEQ ID NO 60.

Embodiment 43 the antibody or antigen binding fragment thereof of embodiment 41 or 42, wherein the allelic variant of HA-1 is HA-1^H。

Embodiment 44. a pharmaceutical composition comprising a TCR according to embodiments 1 to 22, a polypeptide according to embodiments 24 to 25, a multivalent TCR complex according to any one of embodiments 27 to 28, a nucleic acid according to embodiment 29 or 30, a vector according to embodiments 31 to 34, a cell according to any one of embodiments 35 to 40, or an antibody according to embodiments 41 to 42.

Embodiment 45: the pharmaceutical composition according to embodiment 44, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

Embodiment 46: a TCR according to embodiment 1 to 23, a polypeptide according to embodiment 24 to 25, a multivalent TCR complex according to any one of embodiments 27 to 28, a nucleic acid according to embodiment 29 or 30, a vector according to embodiment 31 to 34, a cell according to any one of embodiments 34 to 39, or an antibody according to embodiment 41 to 43, for use as a medicament.

Embodiment 47: a TCR according to embodiment 1 to 23, a polypeptide according to embodiment 24 to 25, a multivalent TCR complex according to any one of embodiments 26 to 27, a nucleic acid according to embodiment 29 or 30, or a cell according to any one of embodiments 35 to 40, for use in treating cancer.

Embodiment 48 the TCR, the polypeptide, the multivalent TCR complex, the nucleic acid, or the cell for use according to embodiment 45, wherein the cancer is a hematological cancer.

Embodiment 49: the TCR, the polypeptide, the multivalent TCR complex, the nucleic acid, or the cell for use according to embodiment 45, wherein the hematologic cancer is selected from the group consisting of: non-hodgkin's lymphoma (NHL), Hodgkin's Lymphoma (HL), multiple myeloma, Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL), Mixed Phenotype Acute Leukemia (MPAL), Chronic Myeloid Leukemia (CML), B-cell pleomorphic lymphoma, hairy cell leukemia, Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), central nervous system lymphoma, CD37+ dendritic cell lymphoma, lymphoblastic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, extraosseous plasmacytoma, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT tissue), nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, precursor B lymphoblastic lymphoma, Acute Myelogenous Leukemia (AML), mixed phenotype Acute Lymphoblastic Leukemia (ALL), Mixed Phenotype Acute Leukemia (MPAL), mixed phenotype acute leukemia (mmal), Chronic Myelogenous Leukemia (CML), B cell pleomorphic lymphoma, hairy cell leukemia, Chronic Lymphocytic Leukemia (CLL), small, Immunoblastic large cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt's lymphoma/leukemia, B cell proliferation of unknown malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, and myelodysplastic disorders.

Sequence listing

<110> Gene medical immunotherapy Limited liability company

<120> HA-1 specific T cell receptor and uses thereof

<130> M11350WO

<150> EP18187539.4

<151> 2018-08-06

<160> 127

<170> PatentIn version 3.5

<210> 1

<211> 1136

<212> PRT

<213> Intelligent people

<400> 1

Met Phe Ser Arg Lys Lys Arg Glu Leu Met Lys Thr Pro Ser Ile Ser

1 5 10 15

Lys Lys Asn Arg Ala Gly Ser Pro Ser Pro Gln Pro Ser Gly Glu Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Gly Ala Ala Ser Trp Thr Leu Gly Arg Ser His Arg Ser Pro Leu Thr

85 90 95

Ala Ala Ser Pro Gly Glu Leu Pro Thr Glu Gly Ala Gly Pro Asp Val

100 105 110

Val Glu Asp Ile Ser His Leu Leu Ala Asp Val Ala Arg Phe Ala Glu

115 120 125

Gly Leu Glu Lys Leu Lys Glu Cys Val Leu His Asp Asp Leu Leu Glu

130 135 140

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

145 150 155 160

Met His Gln Ile Ile Ser Lys Tyr Pro Leu Leu Asn Thr Val Glu Thr

165 170 175

Leu Thr Ala Ala Gly Thr Leu Ile Ala Lys Val Lys Ala Phe His Tyr

180 185 190

Glu Ser Asn Asn Asp Leu Glu Lys Gln Glu Phe Glu Lys Ala Leu Glu

195 200 205

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

210 215 220

Glu Val Asp Ser Ser Thr Leu Leu Ala Val Pro Pro Gly Asp Ser Ser

225 230 235 240

Gln Ser Met Glu Ser Leu Tyr Gly Pro Gly Ser Glu Gly Thr Pro Pro

245 250 255

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

260 265 270

Val Leu Leu Gln Arg Cys Glu Gly Gly Val Asp Ala Ala Leu Leu Tyr

275 280 285

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

290 295 300

Lys Arg Thr Thr Leu Glu Met Glu Phe Ala Lys Gly Leu Gln Lys Ile

305 310 315 320

Ala His Asn Cys Arg Gln Ser Val Met Gln Glu Pro His Met Pro Leu

325 330 335

Leu Ser Ile Tyr Ser Leu Ala Leu Glu Gln Asp Leu Glu Phe Gly His

340 345 350

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

355 360 365

Pro Leu Thr Leu Arg Arg Leu Glu His Glu Lys Arg Arg Lys Glu Ile

370 375 380

Lys Glu Ala Trp His Arg Ala Gln Arg Lys Leu Gln Glu Ala Glu Ser

385 390 395 400

Asn Leu Arg Lys Ala Lys Gln Gly Tyr Val Gln Arg Cys Glu Asp His

405 410 415

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

420 425 430

Ser Ala Pro Gly Ala Gly Ser Thr Ala Thr Lys Thr Leu Asp Lys Arg

435 440 445

Arg Arg Leu Glu Glu Glu Ala Lys Asn Lys Ala Glu Glu Ala Met Ala

450 455 460

Thr Tyr Arg Thr Cys Val Ala Asp Ala Lys Thr Gln Lys Gln Glu Leu

465 470 475 480

Glu Asp Thr Lys Val Thr Ala Leu Arg Gln Ile Gln Glu Val Ile Arg

485 490 495

Gln Ser Asp Gln Thr Ile Lys Ser Ala Thr Ile Ser Tyr Tyr Gln Met

500 505 510

Met His Met Gln Thr Ala Pro Leu Pro Val His Phe Gln Met Leu Cys

515 520 525

Glu Ser Ser Lys Leu Tyr Asp Pro Gly Gln Gln Tyr Ala Ser His Val

530 535 540

Arg Gln Leu Gln Arg Asp Gln Glu Pro Asp Val His Tyr Asp Phe Glu

545 550 555 560

Pro His Val Ser Ala Asn Ala Trp Ser Pro Val Met Arg Ala Arg Lys

565 570 575

Ser Ser Phe Asn Val Ser Asp Val Ala Arg Pro Glu Ala Ala Gly Ser

580 585 590

Pro Pro Glu Glu Gly Gly Cys Thr Glu Gly Thr Pro Ala Lys Asp His

595 600 605

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

610 615 620

Ser Asp Ser Asp Ser Gly Leu Asp Pro Gly Pro Gly Ala Gly Asp Phe

625 630 635 640

Lys Lys Phe Glu Arg Thr Ser Ser Ser Gly Thr Met Ser Ser Thr Glu

645 650 655

Glu Leu Val Asp Pro Asp Gly Gly Ala Gly Ala Ser Ala Phe Glu Gln

660 665 670

Ala Asp Leu Asn Gly Met Thr Pro Glu Leu Pro Val Ala Val Pro Ser

675 680 685

Gly Pro Phe Arg His Glu Gly Leu Ser Lys Ala Ala Arg Thr His Arg

690 695 700

Leu Arg Lys Leu Arg Thr Pro Ala Lys Cys Arg Glu Cys Asn Ser Tyr

705 710 715 720

Val Tyr Phe Gln Gly Ala Glu Cys Glu Glu Cys Cys Leu Ala Cys His

725 730 735

Lys Lys Cys Leu Glu Thr Leu Ala Ile Gln Cys Gly His Lys Lys Leu

740 745 750

Gln Gly Arg Leu Gln Leu Phe Gly Gln Asp Phe Ser His Ala Ala Arg

755 760 765

Ser Ala Pro Asp Gly Val Pro Phe Ile Val Lys Lys Cys Val Cys Glu

770 775 780

Ile Glu Arg Arg Ala Leu Arg Thr Lys Gly Ile Tyr Arg Val Asn Gly

785 790 795 800

Val Lys Thr Arg Val Glu Lys Leu Cys Gln Ala Phe Glu Asn Gly Lys

805 810 815

Glu Leu Val Glu Leu Ser Gln Ala Ser Pro His Asp Ile Ser Asn Val

820 825 830

Leu Lys Leu Tyr Leu Arg Gln Leu Pro Glu Pro Leu Ile Ser Phe Arg

835 840 845

Leu Tyr His Glu Leu Val Gly Leu Ala Lys Asp Ser Leu Lys Ala Glu

850 855 860

Ala Glu Ala Lys Ala Ala Ser Arg Gly Arg Gln Asp Gly Ser Glu Ser

865 870 875 880

Glu Ala Val Ala Val Ala Leu Ala Gly Arg Leu Arg Glu Leu Leu Arg

885 890 895

Asp Leu Pro Pro Glu Asn Arg Ala Ser Leu Gln Tyr Leu Leu Arg His

900 905 910

Leu Arg Arg Ile Val Glu Val Glu Gln Asp Asn Lys Met Thr Pro Gly

915 920 925

Asn Leu Gly Ile Val Phe Gly Pro Thr Leu Leu Arg Pro Arg Pro Thr

930 935 940

Glu Ala Thr Val Ser Leu Ser Ser Leu Val Asp Tyr Pro His Gln Ala

945 950 955 960

Arg Val Ile Glu Thr Leu Ile Val His Tyr Gly Leu Val Phe Glu Glu

965 970 975

Glu Pro Glu Glu Thr Pro Gly Gly Gln Asp Glu Ser Ser Asn Gln Arg

980 985 990

Ala Glu Val Val Val Gln Val Pro Tyr Leu Glu Ala Gly Glu Ala Val

995 1000 1005

Val Tyr Pro Leu Gln Glu Ala Ala Ala Asp Gly Cys Arg Glu Ser

1010 1015 1020

Arg Val Val Ser Asn Asp Ser Asp Ser Asp Leu Glu Glu Ala Ser

1025 1030 1035

Glu Leu Leu Ser Ser Ser Glu Ala Ser Ala Leu Gly His Leu Ser

1040 1045 1050

Phe Leu Glu Gln Gln Gln Ser Glu Ala Ser Leu Glu Val Ala Ser

1055 1060 1065

Gly Ser His Ser Gly Ser Glu Glu Gln Leu Glu Ala Thr Ala Arg

1070 1075 1080

Glu Asp Gly Asp Gly Asp Glu Asp Gly Pro Ala Gln Gln Leu Ser

1085 1090 1095

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

1100 1105 1110

Pro Pro Met Arg Leu Arg Gly Gly Arg Met Thr Leu Gly Ser Cys

1115 1120 1125

Arg Glu Arg Gln Pro Glu Phe Val

1130 1135

<210> 2

<211> 9

<212> PRT

<213> Intelligent people

<400> 2

Val Leu His Asp Asp Leu Leu Glu Ala

1 5

<210> 3

<211> 1136

<212> PRT

<213> Intelligent people

<400> 3

Met Phe Ser Arg Lys Lys Arg Glu Leu Met Lys Thr Pro Ser Ile Ser

1 5 10 15

Lys Lys Asn Arg Ala Gly Ser Pro Ser Pro Gln Pro Ser Gly Glu Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Gly Ala Ala Ser Trp Thr Leu Gly Arg Ser His Arg Ser Pro Leu Thr

85 90 95

Ala Ala Ser Pro Gly Glu Leu Pro Thr Glu Gly Ala Gly Pro Asp Val

100 105 110

Val Glu Asp Ile Ser His Leu Leu Ala Asp Val Ala Arg Phe Ala Glu

115 120 125

Gly Leu Glu Lys Leu Lys Glu Cys Val Leu Arg Asp Asp Leu Leu Glu

130 135 140

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

145 150 155 160

Met His Gln Ile Ile Ser Lys Tyr Pro Leu Leu Asn Thr Val Glu Thr

165 170 175

Leu Thr Ala Ala Gly Thr Leu Ile Ala Lys Val Lys Ala Phe His Tyr

180 185 190

Glu Ser Asn Asn Asp Leu Glu Lys Gln Glu Phe Glu Lys Ala Leu Glu

195 200 205

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

210 215 220

Glu Val Asp Ser Ser Thr Leu Leu Ala Val Pro Pro Gly Asp Ser Ser

225 230 235 240

Gln Ser Met Glu Ser Leu Tyr Gly Pro Gly Ser Glu Gly Thr Pro Pro

245 250 255

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

260 265 270

Val Leu Leu Gln Arg Cys Glu Gly Gly Val Asp Ala Ala Leu Leu Tyr

275 280 285

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

290 295 300

Lys Arg Thr Thr Leu Glu Met Glu Phe Ala Lys Gly Leu Gln Lys Ile

305 310 315 320

Ala His Asn Cys Arg Gln Ser Val Met Gln Glu Pro His Met Pro Leu

325 330 335

Leu Ser Ile Tyr Ser Leu Ala Leu Glu Gln Asp Leu Glu Phe Gly His

340 345 350

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

355 360 365

Pro Leu Thr Leu Arg Arg Leu Glu His Glu Lys Arg Arg Lys Glu Ile

370 375 380

Lys Glu Ala Trp His Arg Ala Gln Arg Lys Leu Gln Glu Ala Glu Ser

385 390 395 400

Asn Leu Arg Lys Ala Lys Gln Gly Tyr Val Gln Arg Cys Glu Asp His

405 410 415

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

420 425 430

Ser Ala Pro Gly Ala Gly Ser Thr Ala Thr Lys Thr Leu Asp Lys Arg

435 440 445

Arg Arg Leu Glu Glu Glu Ala Lys Asn Lys Ala Glu Glu Ala Met Ala

450 455 460

Thr Tyr Arg Thr Cys Val Ala Asp Ala Lys Thr Gln Lys Gln Glu Leu

465 470 475 480

Glu Asp Thr Lys Val Thr Ala Leu Arg Gln Ile Gln Glu Val Ile Arg

485 490 495

Gln Ser Asp Gln Thr Ile Lys Ser Ala Thr Ile Ser Tyr Tyr Gln Met

500 505 510

Met His Met Gln Thr Ala Pro Leu Pro Val His Phe Gln Met Leu Cys

515 520 525

Glu Ser Ser Lys Leu Tyr Asp Pro Gly Gln Gln Tyr Ala Ser His Val

530 535 540

Arg Gln Leu Gln Arg Asp Gln Glu Pro Asp Val His Tyr Asp Phe Glu

545 550 555 560

Pro His Val Ser Ala Asn Ala Trp Ser Pro Val Met Arg Ala Arg Lys

565 570 575

Ser Ser Phe Asn Val Ser Asp Val Ala Arg Pro Glu Ala Ala Gly Ser

580 585 590

Pro Pro Glu Glu Gly Gly Cys Thr Glu Gly Thr Pro Ala Lys Asp His

595 600 605

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

610 615 620

Ser Asp Ser Asp Ser Gly Leu Asp Pro Gly Pro Gly Ala Gly Asp Phe

625 630 635 640

Lys Lys Phe Glu Arg Thr Ser Ser Ser Gly Thr Met Ser Ser Thr Glu

645 650 655

Glu Leu Val Asp Pro Asp Gly Gly Ala Gly Ala Ser Ala Phe Glu Gln

660 665 670

Ala Asp Leu Asn Gly Met Thr Pro Glu Leu Pro Val Ala Val Pro Ser

675 680 685

Gly Pro Phe Arg His Glu Gly Leu Ser Lys Ala Ala Arg Thr His Arg

690 695 700

Leu Arg Lys Leu Arg Thr Pro Ala Lys Cys Arg Glu Cys Asn Ser Tyr

705 710 715 720

Val Tyr Phe Gln Gly Ala Glu Cys Glu Glu Cys Cys Leu Ala Cys His

725 730 735

Lys Lys Cys Leu Glu Thr Leu Ala Ile Gln Cys Gly His Lys Lys Leu

740 745 750

Gln Gly Arg Leu Gln Leu Phe Gly Gln Asp Phe Ser His Ala Ala Arg

755 760 765

Ser Ala Pro Asp Gly Val Pro Phe Ile Val Lys Lys Cys Val Cys Glu

770 775 780

Ile Glu Arg Arg Ala Leu Arg Thr Lys Gly Ile Tyr Arg Val Asn Gly

785 790 795 800

Val Lys Thr Arg Val Glu Lys Leu Cys Gln Ala Phe Glu Asn Gly Lys

805 810 815

Glu Leu Val Glu Leu Ser Gln Ala Ser Pro His Asp Ile Ser Asn Val

820 825 830

Leu Lys Leu Tyr Leu Arg Gln Leu Pro Glu Pro Leu Ile Ser Phe Arg

835 840 845

Leu Tyr His Glu Leu Val Gly Leu Ala Lys Asp Ser Leu Lys Ala Glu

850 855 860

Ala Glu Ala Lys Ala Ala Ser Arg Gly Arg Gln Asp Gly Ser Glu Ser

865 870 875 880

Glu Ala Val Ala Val Ala Leu Ala Gly Arg Leu Arg Glu Leu Leu Arg

885 890 895

Asp Leu Pro Pro Glu Asn Arg Ala Ser Leu Gln Tyr Leu Leu Arg His

900 905 910

Leu Arg Arg Ile Val Glu Val Glu Gln Asp Asn Lys Met Thr Pro Gly

915 920 925

Asn Leu Gly Ile Val Phe Gly Pro Thr Leu Leu Arg Pro Arg Pro Thr

930 935 940

Glu Ala Thr Val Ser Leu Ser Ser Leu Val Asp Tyr Pro His Gln Ala

945 950 955 960

Arg Val Ile Glu Thr Leu Ile Val His Tyr Gly Leu Val Phe Glu Glu

965 970 975

Glu Pro Glu Glu Thr Pro Gly Gly Gln Asp Glu Ser Ser Asn Gln Arg

980 985 990

Ala Glu Val Val Val Gln Val Pro Tyr Leu Glu Ala Gly Glu Ala Val

995 1000 1005

Val Tyr Pro Leu Gln Glu Ala Ala Ala Asp Gly Cys Arg Glu Ser

1010 1015 1020

Arg Val Val Ser Asn Asp Ser Asp Ser Asp Leu Glu Glu Ala Ser

1025 1030 1035

Glu Leu Leu Ser Ser Ser Glu Ala Ser Ala Leu Gly His Leu Ser

1040 1045 1050

Phe Leu Glu Gln Gln Gln Ser Glu Ala Ser Leu Glu Val Ala Ser

1055 1060 1065

Gly Ser His Ser Gly Ser Glu Glu Gln Leu Glu Ala Thr Ala Arg

1070 1075 1080

Glu Asp Gly Asp Gly Asp Glu Asp Gly Pro Ala Gln Gln Leu Ser

1085 1090 1095

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

1100 1105 1110

Pro Pro Met Arg Leu Arg Gly Gly Arg Met Thr Leu Gly Ser Cys

1115 1120 1125

Arg Glu Arg Gln Pro Glu Phe Val

1130 1135

<210> 4

<211> 9

<212> PRT

<213> Intelligent people

<400> 4

Val Leu Arg Asp Asp Leu Leu Glu Ala

1 5

<210> 5

<211> 6

<212> PRT

<213> Intelligent people

<400> 5

Asp Ser Ala Ser Asn Tyr

1 5

<210> 6

<211> 7

<212> PRT

<213> Intelligent people

<400> 6

Ile Arg Ser Asn Val Gly Glu

1 5

<210> 7

<211> 8

<212> PRT

<213> Intelligent people

<400> 7

Cys Ala Ala Ser Asp Leu Asn Phe

1 5

<210> 8

<211> 5

<212> PRT

<213> Intelligent people

<400> 8

Ser Glu His Asn Arg

1 5

<210> 9

<211> 6

<212> PRT

<213> Intelligent people

<400> 9

Phe Gln Asn Glu Ala Gln

1 5

<210> 10

<211> 14

<212> PRT

<213> Intelligent people

<400> 10

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

1 5 10

<210> 11

<211> 127

<212> PRT

<213> Intelligent people

<400> 11

Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp

1 5 10 15

Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val

20 25 30

Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala

35 40 45

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

50 55 60

Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg

65 70 75 80

Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile

85 90 95

Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser

100 105 110

Asp Leu Asn Phe Gly Lys Gly Thr Ser Leu Leu Val Thr Pro His

115 120 125

<210> 12

<211> 134

<212> PRT

<213> Intelligent people

<400> 12

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Leu Val Ser Arg Val Asp Gly Tyr Thr Phe Gly Ser Gly Thr

115 120 125

Arg Leu Thr Val Val Glu

130

<210> 13

<211> 263

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 13

Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp

1 5 10 15

Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val

20 25 30

Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala

35 40 45

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

50 55 60

Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg

65 70 75 80

Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile

85 90 95

Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser

100 105 110

Asp Leu Asn Phe Gly Lys Gly Thr Ser Leu Leu Val Thr Pro His Ile

115 120 125

Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln

130 135 140

Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val

145 150 155 160

Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu

165 170 175

Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser

180 185 190

Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala

195 200 205

Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys

210 215 220

Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met

225 230 235 240

Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met

245 250 255

Thr Leu Arg Leu Trp Ser Ser

260

<210> 14

<211> 306

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 14

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Leu Val Ser Arg Val Asp Gly Tyr Thr Phe Gly Ser Gly Thr

115 120 125

Arg Leu Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val

130 135 140

Thr Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala

145 150 155 160

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

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg

195 200 205

Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg

210 215 220

Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu

225 230 235 240

Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly

245 250 255

Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu

260 265 270

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

275 280 285

Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys

290 295 300

Asn Ser

305

<210> 15

<211> 6

<212> PRT

<213> Intelligent people

<400> 15

Asp Ser Ala Ser Asn Tyr

1 5

<210> 16

<211> 7

<212> PRT

<213> Intelligent people

<400> 16

Ile Arg Ser Asn Val Gly Glu

1 5

<210> 17

<211> 13

<212> PRT

<213> Intelligent people

<400> 17

Cys Ala Ala His Thr Pro Gly Tyr Ser Thr Leu Thr Phe

1 5 10

<210> 18

<211> 5

<212> PRT

<213> Intelligent people

<400> 18

Ser Glu His Asn Arg

1 5

<210> 19

<211> 6

<212> PRT

<213> Intelligent people

<400> 19

Phe Gln Asn Glu Ala Gln

1 5

<210> 20

<211> 14

<212> PRT

<213> Intelligent people

<400> 20

Cys Ala Ser Ser Pro Arg Ala Gly Gly Glu Thr Gln Tyr Phe

1 5 10

<210> 21

<211> 132

<212> PRT

<213> Intelligent people

<400> 21

Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp

1 5 10 15

Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val

20 25 30

Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala

35 40 45

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

50 55 60

Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg

65 70 75 80

Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile

85 90 95

Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala His

100 105 110

Thr Pro Gly Tyr Ser Thr Leu Thr Phe Gly Lys Gly Thr Met Leu Leu

115 120 125

Val Ser Pro Asp

130

<210> 22

<211> 134

<212> PRT

<213> Intelligent people

<400> 22

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Pro Arg Ala Gly Gly Glu Thr Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Leu Val Leu Glu

130

<210> 23

<211> 268

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 23

Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp

1 5 10 15

Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val

20 25 30

Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala

35 40 45

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

50 55 60

Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg

65 70 75 80

Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile

85 90 95

Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala His

100 105 110

Thr Pro Gly Tyr Ser Thr Leu Thr Phe Gly Lys Gly Thr Met Leu Leu

115 120 125

Val Ser Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys

130 135 140

Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp

145 150 155 160

Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr

165 170 175

Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly

180 185 190

Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe

195 200 205

Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala

210 215 220

Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln

225 230 235 240

Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly

245 250 255

Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 24

<211> 306

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 24

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Pro Arg Ala Gly Gly Glu Thr Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Leu Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val

130 135 140

Thr Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala

145 150 155 160

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

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg

195 200 205

Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg

210 215 220

Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu

225 230 235 240

Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly

245 250 255

Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu

260 265 270

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

275 280 285

Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys

290 295 300

Asn Ser

305

<210> 25

<211> 5

<212> PRT

<213> Intelligent people

<400> 25

Thr Ser Ile Asn Asn

1 5

<210> 26

<211> 7

<212> PRT

<213> Intelligent people

<400> 26

Ile Arg Ser Asn Glu Arg Glu

1 5

<210> 27

<211> 14

<212> PRT

<213> Intelligent people

<400> 27

Cys Ala Thr Gly Asp Gln Thr Gly Ala Asn Asn Leu Phe Phe

1 5 10

<210> 28

<211> 5

<212> PRT

<213> Intelligent people

<400> 28

Ser Glu His Asn Arg

1 5

<210> 29

<211> 6

<212> PRT

<213> Intelligent people

<400> 29

Phe Gln Asn Glu Ala Gln

1 5

<210> 30

<211> 13

<212> PRT

<213> Intelligent people

<400> 30

Cys Ala Ser Ser Leu Thr Arg Thr Glu Lys Leu Phe Phe

1 5 10

<210> 31

<211> 133

<212> PRT

<213> Intelligent people

<400> 31

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

1 5 10 15

Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser

20 25 30

Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser

35 40 45

Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val

50 55 60

His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg

65 70 75 80

Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile

85 90 95

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

100 105 110

Asp Gln Thr Gly Ala Asn Asn Leu Phe Phe Gly Thr Gly Thr Arg Leu

115 120 125

Thr Val Ile Pro Tyr

130

<210> 32

<211> 133

<212> PRT

<213> Intelligent people

<400> 32

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Leu Thr Arg Thr Glu Lys Leu Phe Phe Gly Ser Gly Thr Gln

115 120 125

Leu Ser Val Leu Glu

130

<210> 33

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 33

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

1 5 10 15

Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser

20 25 30

Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser

35 40 45

Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val

50 55 60

His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg

65 70 75 80

Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile

85 90 95

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

100 105 110

Asp Gln Thr Gly Ala Asn Asn Leu Phe Phe Gly Thr Gly Thr Arg Leu

115 120 125

Thr Val Ile Pro Tyr Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu

130 135 140

Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe

145 150 155 160

Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile

165 170 175

Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn

180 185 190

Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile

195 200 205

Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp

210 215 220

Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe

225 230 235 240

Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala

245 250 255

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 34

<211> 305

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 34

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Leu Thr Arg Thr Glu Lys Leu Phe Phe Gly Ser Gly Thr Gln

115 120 125

Leu Ser Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Thr

130 135 140

Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

180 185 190

Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu

195 200 205

Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys

210 215 220

Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly

225 230 235 240

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

245 250 255

Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser

260 265 270

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

275 280 285

Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn

290 295 300

Ser

305

<210> 35

<211> 6

<212> PRT

<213> Intelligent people

<400> 35

Asp Ser Ala Ser Asn Tyr

1 5

<210> 36

<211> 7

<212> PRT

<213> Intelligent people

<400> 36

Ile Arg Ser Asn Val Gly Glu

1 5

<210> 37

<211> 9

<212> PRT

<213> Intelligent people

<400> 37

Cys Ala Gly Arg Gly Lys Leu Thr Phe

1 5

<210> 38

<211> 5

<212> PRT

<213> Intelligent people

<400> 38

Ser Glu His Asn Arg

1 5

<210> 39

<211> 6

<212> PRT

<213> Intelligent people

<400> 39

Phe Gln Asn Glu Ala Gln

1 5

<210> 40

<211> 13

<212> PRT

<213> Intelligent people

<400> 40

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

1 5 10

<210> 41

<211> 128

<212> PRT

<213> Intelligent people

<400> 41

Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp

1 5 10 15

Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val

20 25 30

Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala

35 40 45

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

50 55 60

Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg

65 70 75 80

Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile

85 90 95

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

100 105 110

Gly Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu Thr Ile Ile Pro Asn

115 120 125

<210> 42

<211> 133

<212> PRT

<213> Intelligent people

<400> 42

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

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

115 120 125

Leu Ser Val Leu Glu

130

<210> 43

<211> 264

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 43

Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp

1 5 10 15

Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val

20 25 30

Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala

35 40 45

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

50 55 60

Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg

65 70 75 80

Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile

85 90 95

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

100 105 110

Gly Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu Thr Ile Ile Pro Asn

115 120 125

Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser

130 135 140

Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn

145 150 155 160

Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val

165 170 175

Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp

180 185 190

Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn

195 200 205

Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu

210 215 220

Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val

225 230 235 240

Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

245 250 255

Met Thr Leu Arg Leu Trp Ser Ser

260

<210> 44

<211> 305

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 44

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

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

115 120 125

Leu Ser Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Thr

130 135 140

Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

180 185 190

Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu

195 200 205

Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys

210 215 220

Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly

225 230 235 240

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

245 250 255

Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser

260 265 270

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

275 280 285

Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn

290 295 300

Ser

305

<210> 45

<211> 5

<212> PRT

<213> Intelligent people

<400> 45

Ser Val Phe Ser Ser

1 5

<210> 46

<211> 7

<212> PRT

<213> Intelligent people

<400> 46

Val Val Thr Gly Gly Glu Val

1 5

<210> 47

<211> 11

<212> PRT

<213> Intelligent people

<400> 47

Cys Ala Gly Ala Gly Asn Asn Asp Met Arg Phe

1 5 10

<210> 48

<211> 5

<212> PRT

<213> Intelligent people

<400> 48

Ser Glu His Asn Arg

1 5

<210> 49

<211> 6

<212> PRT

<213> Intelligent people

<400> 49

Phe Gln Asn Glu Ala Gln

1 5

<210> 50

<211> 13

<212> PRT

<213> Intelligent people

<400> 50

Cys Ala Ser Ser Leu Val Arg Gly Ile Glu Ala Phe Phe

1 5 10

<210> 51

<211> 128

<212> PRT

<213> Intelligent people

<400> 51

Met Val Leu Lys Phe Ser Val Ser Ile Leu Trp Ile Gln Leu Ala Trp

1 5 10 15

Val Ser Thr Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser Ile Gln

20 25 30

Glu Gly Glu Asn Leu Thr Val Tyr Cys Asn Ser Ser Ser Val Phe Ser

35 40 45

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

50 55 60

Val Thr Val Val Thr Gly Gly Glu Val Lys Lys Leu Lys Arg Leu Thr

65 70 75 80

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

85 90 95

Ala Gln Thr Gly Asp Thr Gly Leu Tyr Leu Cys Ala Gly Ala Gly Asn

100 105 110

Asn Asp Met Arg Phe Gly Ala Gly Thr Arg Leu Thr Val Lys Pro Asn

115 120 125

<210> 52

<211> 133

<212> PRT

<213> Intelligent people

<400> 52

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

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

115 120 125

Leu Thr Val Val Glu

130

<210> 53

<211> 264

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 53

Met Val Leu Lys Phe Ser Val Ser Ile Leu Trp Ile Gln Leu Ala Trp

1 5 10 15

Val Ser Thr Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser Ile Gln

20 25 30

Glu Gly Glu Asn Leu Thr Val Tyr Cys Asn Ser Ser Ser Val Phe Ser

35 40 45

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

50 55 60

Val Thr Val Val Thr Gly Gly Glu Val Lys Lys Leu Lys Arg Leu Thr

65 70 75 80

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

85 90 95

Ala Gln Thr Gly Asp Thr Gly Leu Tyr Leu Cys Ala Gly Ala Gly Asn

100 105 110

Asn Asp Met Arg Phe Gly Ala Gly Thr Arg Leu Thr Val Lys Pro Asn

115 120 125

Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser

130 135 140

Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn

145 150 155 160

Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val

165 170 175

Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp

180 185 190

Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn

195 200 205

Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu

210 215 220

Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val

225 230 235 240

Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

245 250 255

Met Thr Leu Arg Leu Trp Ser Ser

260

<210> 54

<211> 305

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 54

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

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

115 120 125

Leu Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Thr

130 135 140

Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

180 185 190

Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu

195 200 205

Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys

210 215 220

Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly

225 230 235 240

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

245 250 255

Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser

260 265 270

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

275 280 285

Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn

290 295 300

Ser

305

<210> 55

<211> 6

<212> PRT

<213> Intelligent people

<400> 55

Asp Ser Ser Ser Thr Tyr

1 5

<210> 56

<211> 7

<212> PRT

<213> Intelligent people

<400> 56

Ile Phe Ser Asn Met Asp Met

1 5

<210> 57

<211> 8

<212> PRT

<213> Intelligent people

<400> 57

Cys Ala Glu Lys Trp Ile Ile Phe

1 5

<210> 58

<211> 5

<212> PRT

<213> Intelligent people

<400> 58

Ser Glu His Asn Arg

1 5

<210> 59

<211> 6

<212> PRT

<213> Intelligent people

<400> 59

Phe Gln Asn Glu Ala Gln

1 5

<210> 60

<211> 13

<212> PRT

<213> Intelligent people

<400> 60

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

1 5 10

<210> 61

<211> 128

<212> PRT

<213> Intelligent people

<400> 61

Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu

1 5 10 15

Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser

20 25 30

Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser

35 40 45

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

50 55 60

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

65 70 75 80

Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg

85 90 95

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

100 105 110

Lys Trp Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Asn

115 120 125

<210> 62

<211> 133

<212> PRT

<213> Intelligent people

<400> 62

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Leu Thr Thr Pro Asp Gly Tyr Thr Phe Gly Ser Gly Thr Arg

115 120 125

Leu Thr Val Val Glu

130

<210> 63

<211> 264

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 63

Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu

1 5 10 15

Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser

20 25 30

Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser

35 40 45

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

50 55 60

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

65 70 75 80

Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg

85 90 95

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

100 105 110

Lys Trp Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Asn

115 120 125

Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser

130 135 140

Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn

145 150 155 160

Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val

165 170 175

Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp

180 185 190

Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn

195 200 205

Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu

210 215 220

Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val

225 230 235 240

Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

245 250 255

Met Thr Leu Arg Leu Trp Ser Ser

260

<210> 64

<211> 305

<212> PRT

<213> Artificial sequence

<220>

<223> human with murine constant region

<400> 64

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Leu Thr Thr Pro Asp Gly Tyr Thr Phe Gly Ser Gly Thr Arg

115 120 125

Leu Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Thr

130 135 140

Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

180 185 190

Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu

195 200 205

Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys

210 215 220

Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly

225 230 235 240

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

245 250 255

Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser

260 265 270

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

275 280 285

Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn

290 295 300

Ser

305

<210> 65

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> control peptide Astn 1P 1268L

<400> 65

Lys Leu Tyr Gly Leu Asp Trp Ala Glu Leu

1 5 10

<210> 66

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 66

gacagcgcca gcaactac 18

<210> 67

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 67

atcagatcca acgtgggcga g 21

<210> 68

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 68

tgcgccgcca gcgacctgaa tttt 24

<210> 69

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 69

agcgagcaca accgg 15

<210> 70

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 70

ttccagaacg aggcccag 18

<210> 71

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 71

tgtgccagca gcctggtgtc cagagtggat ggctacacat tt 42

<210> 72

<211> 379

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 72

atgaccagca tccgggccgt gttcatcttc ctgtggctgc agctggacct ggtcaacggc 60

gagaatgtgg aacagcaccc cagcacactg agcgtgcaag agggcgattc tgccgtgatc 120

aagtgcacct acagcgacag cgccagcaac tacttcccct ggtacaagca agaactcggc 180

aagggccctc agctgatcat cgacatcaga tccaacgtgg gcgagaagaa ggaccagcgg 240

attgccgtga cactgaacaa gaccgccaag cacttcagcc tgcacatcac cgagacacag 300

cctgaggata gcgccgtgta cttttgcgcc gccagcgacc tgaattttgg caagggcaca 360

agcctgctgg tcacccctc 379

<210> 73

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 73

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgccagca gcctggtgtc cagagtggat 360

ggctacacat ttggcagcgg caccagactg acagtggtgg 400

<210> 74

<211> 792

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 74

atgaccagca tccgggccgt gttcatcttc ctgtggctgc agctggacct ggtcaacggc 60

gagaatgtgg aacagcaccc cagcacactg agcgtgcaag agggcgattc tgccgtgatc 120

aagtgcacct acagcgacag cgccagcaac tacttcccct ggtacaagca agaactcggc 180

aagggccctc agctgatcat cgacatcaga tccaacgtgg gcgagaagaa ggaccagcgg 240

attgccgtga cactgaacaa gaccgccaag cacttcagcc tgcacatcac cgagacacag 300

cctgaggata gcgccgtgta cttttgcgcc gccagcgacc tgaattttgg caagggcaca 360

agcctgctgg tcacccctca catccagaat ccggagcccg ccgtatacca gctgaaggac 420

cctagaagcc aggacagcac cctgtgcctg ttcaccgact tcgacagcca gatcaacgtg 480

cccaagacca tggaaagcgg caccttcatc accgacaaga cagtgctgga catgaaggcc 540

atggacagca agtccaacgg cgcaatcgcc tggtccaacc agaccagctt cacatgccag 600

gacatcttca aagagacaaa cgccacatac cccagcagcg acgtgccctg tgatgccacc 660

ctgacagaga agtccttcga gacagacatg aacctgaact tccagaatct gtccgtgatg 720

ggcctgagaa tcctgctgct gaaggtggcc ggcttcaatc tgctgatgac cctgcggctg 780

tggtccagct ga 792

<210> 75

<211> 921

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 75

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgccagca gcctggtgtc cagagtggat 360

ggctacacat ttggcagcgg caccagactg acagtggtgg aagatctccg gaacgtgacc 420

ccccctaaag tgaccctgtt cgaacccagc aaggccgaga tcgccaacaa gcagaaagcc 480

accctcgtgt gcctggccag aggcttcttc cccgaccatg tggaactgtc ttggtgggtc 540

aacggcaaag aggtgcacag cggagtgtcc accgaccctc aggcctacaa agagagcaac 600

tacagctact gcctgagcag cagactgcgg gtgtccgcca ccttctggca caacccccgg 660

aaccacttca gatgccaggt gcagtttcac ggcctgagcg aagaggacaa gtggcccgaa 720

ggctccccca agcccgtgac ccagaatatc tctgccgagg cctggggcag agccgactgt 780

ggaattacca gcgccagcta ccaccagggc gtgctgtctg ccaccatcct gtacgagatc 840

ctgctgggca aggccaccct gtacgccgtg ctggtgtctg gcctggtgct gatggccatg 900

gtcaagaaga agaacagctg a 921

<210> 76

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 76

gacagcgcca gcaactac 18

<210> 77

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 77

atcagatcca acgtgggcga g 21

<210> 78

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 78

tgcgccgctc acacacctgg ctacagcacc ctgacattt 39

<210> 79

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 79

agcgagcaca accgg 15

<210> 80

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 80

ttccagaacg aggcccag 18

<210> 81

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 81

tgtgctagct ctcctagagc cggcggagag acacagtatt tc 42

<210> 82

<211> 394

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 82

atgaccagca tccgggccgt gttcatcttc ctgtggctgc agctggacct ggtcaacggc 60

gagaatgtgg aacagcaccc cagcacactg agcgtgcaag agggcgattc tgccgtgatc 120

aagtgcacct acagcgacag cgccagcaac tacttcccct ggtacaagca agaactcggc 180

aagggccctc agctgatcat cgacatcaga tccaacgtgg gcgagaagaa ggaccagcgg 240

attgccgtga cactgaacaa gaccgccaag cacttcagcc tgcacatcac cgagacacag 300

cctgaggata gcgccgtgta cttctgcgcc gctcacacac ctggctacag caccctgaca 360

tttggcaagg gcaccatgct gctggtgtcc ccag 394

<210> 83

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 83

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtacctg tgtgctagct ctcctagagc cggcggagag 360

acacagtatt tcggccctgg aacacggctg ctggttctgg 400

<210> 84

<211> 807

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 84

atgaccagca tccgggccgt gttcatcttc ctgtggctgc agctggacct ggtcaacggc 60

gagaatgtgg aacagcaccc cagcacactg agcgtgcaag agggcgattc tgccgtgatc 120

aagtgcacct acagcgacag cgccagcaac tacttcccct ggtacaagca agaactcggc 180

aagggccctc agctgatcat cgacatcaga tccaacgtgg gcgagaagaa ggaccagcgg 240

attgccgtga cactgaacaa gaccgccaag cacttcagcc tgcacatcac cgagacacag 300

cctgaggata gcgccgtgta cttctgcgcc gctcacacac ctggctacag caccctgaca 360

tttggcaagg gcaccatgct gctggtgtcc ccagacatcc agaatccgga gcccgccgta 420

taccagctga aggaccctag aagccaggac agcaccctgt gcctgttcac cgacttcgac 480

agccagatca acgtgcccaa gaccatggaa agcggcacct tcatcaccga caagacagtg 540

ctggacatga aggccatgga cagcaagtcc aacggcgcaa tcgcctggtc caaccagacc 600

agcttcacat gccaggacat cttcaaagag acaaacgcca cataccccag cagcgacgtg 660

ccctgtgatg ccaccctgac agagaagtcc ttcgagacag acatgaacct gaacttccag 720

aatctgtccg tgatgggcct gagaatcctg ctgctgaagg tggccggctt caatctgctg 780

atgaccctgc ggctgtggtc cagctga 807

<210> 85

<211> 921

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 85

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtacctg tgtgctagct ctcctagagc cggcggagag 360

acacagtatt tcggccctgg aacacggctg ctggttctgg aagatctccg gaacgtgacc 420

ccccctaaag tgaccctgtt cgaacccagc aaggccgaga tcgccaacaa gcagaaagcc 480

accctcgtgt gcctggccag aggcttcttc cccgaccatg tggaactgtc ttggtgggtc 540

aacggcaaag aggtgcacag cggagtgtcc accgaccctc aggcctacaa agagagcaac 600

tacagctact gcctgagcag cagactgcgg gtgtccgcca ccttctggca caacccccgg 660

aaccacttca gatgccaggt gcagtttcac ggcctgagcg aagaggacaa gtggcccgaa 720

ggctccccca agcccgtgac ccagaatatc tctgccgagg cctggggcag agccgactgt 780

ggaattacca gcgccagcta ccaccagggc gtgctgtctg ccaccatcct gtacgagatc 840

ctgctgggca aggccaccct gtacgccgtg ctggtgtctg gcctggtgct gatggccatg 900

gtcaagaaga agaacagctg a 921

<210> 86

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 86

accagcatca acaac 15

<210> 87

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 87

atcagaagca acgagagaga g 21

<210> 88

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 88

tgcgccactg gcgatcagac cggcgccaac aatctgttct tt 42

<210> 89

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 89

agcgagcaca accgg 15

<210> 90

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 90

ttccagaacg aggcccag 18

<210> 91

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 91

tgtgccagca gcctgaccag aaccgagaag ctgtttttc 39

<210> 92

<211> 397

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 92

atggaaacac tgctgggcgt gtccctggtc atcctgtggc tgcaactggc cagagtgaac 60

agccagcagg gcgaagaaga tccccaggct ctgtctatcc aagagggcga gaacgccacc 120

atgaactgca gctacaagac cagcatcaac aacctgcagt ggtacagaca gaacagcggc 180

agaggactgg tgcacctgat cctgatcaga agcaacgaga gagagaagca ctccggcaga 240

ctgagagtga ccctggacac cagcaagaag tccagcagcc tgctgatcac cgcctctaga 300

gctgccgata ccgccagcta cttttgcgcc actggcgatc agaccggcgc caacaatctg 360

ttctttggca ccggaaccag gctgacagtg atccctt 397

<210> 93

<211> 397

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 93

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgccagca gcctgaccag aaccgagaag 360

ctgtttttcg gcagcggcac ccagctgtct gttctgg 397

<210> 94

<211> 810

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 94

atggaaacac tgctgggcgt gtccctggtc atcctgtggc tgcaactggc cagagtgaac 60

agccagcagg gcgaagaaga tccccaggct ctgtctatcc aagagggcga gaacgccacc 120

atgaactgca gctacaagac cagcatcaac aacctgcagt ggtacagaca gaacagcggc 180

agaggactgg tgcacctgat cctgatcaga agcaacgaga gagagaagca ctccggcaga 240

ctgagagtga ccctggacac cagcaagaag tccagcagcc tgctgatcac cgcctctaga 300

gctgccgata ccgccagcta cttttgcgcc actggcgatc agaccggcgc caacaatctg 360

ttctttggca ccggaaccag gctgacagtg atcccttaca tccagaatcc ggagcccgcc 420

gtataccagc tgaaggaccc tagaagccag gacagcaccc tgtgcctgtt caccgacttc 480

gacagccaga tcaacgtgcc caagaccatg gaaagcggca ccttcatcac cgacaagaca 540

gtgctggaca tgaaggccat ggacagcaag tccaacggcg caatcgcctg gtccaaccag 600

accagcttca catgccagga catcttcaaa gagacaaacg ccacataccc cagcagcgac 660

gtgccctgtg atgccaccct gacagagaag tccttcgaga cagacatgaa cctgaacttc 720

cagaatctgt ccgtgatggg cctgagaatc ctgctgctga aggtggccgg cttcaatctg 780

ctgatgaccc tgcggctgtg gtccagctga 810

<210> 95

<211> 918

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 95

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgccagca gcctgaccag aaccgagaag 360

ctgtttttcg gcagcggcac ccagctgtct gttctggaag atctccggaa cgtgaccccc 420

cctaaagtga ccctgttcga acccagcaag gccgagatcg ccaacaagca gaaagccacc 480

ctcgtgtgcc tggccagagg cttcttcccc gaccatgtgg aactgtcttg gtgggtcaac 540

ggcaaagagg tgcacagcgg agtgtccacc gaccctcagg cctacaaaga gagcaactac 600

agctactgcc tgagcagcag actgcgggtg tccgccacct tctggcacaa cccccggaac 660

cacttcagat gccaggtgca gtttcacggc ctgagcgaag aggacaagtg gcccgaaggc 720

tcccccaagc ccgtgaccca gaatatctct gccgaggcct ggggcagagc cgactgtgga 780

attaccagcg ccagctacca ccagggcgtg ctgtctgcca ccatcctgta cgagatcctg 840

ctgggcaagg ccaccctgta cgccgtgctg gtgtctggcc tggtgctgat ggccatggtc 900

aagaagaaga acagctga 918

<210> 96

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 96

gacagcgcca gcaactac 18

<210> 97

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 97

atcagatcca acgtgggcga g 21

<210> 98

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 98

tgtgccggca gaggcaagct gaccttt 27

<210> 99

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 99

agcgagcaca accgg 15

<210> 100

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 100

ttccagaacg aggcccag 18

<210> 101

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 101

tgtgccagtt ctctcgtgcg ggacgagaag ctgtttttc 39

<210> 102

<211> 382

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 102

atgaccagca tccgggccgt gttcatcttc ctgtggctgc agctggacct ggtcaacggc 60

gagaatgtgg aacagcaccc cagcacactg agcgtgcaag agggcgattc tgccgtgatc 120

aagtgcacct acagcgacag cgccagcaac tacttcccct ggtacaagca agaactcggc 180

aagggccctc agctgatcat cgacatcaga tccaacgtgg gcgagaagaa ggaccagcgg 240

attgccgtga cactgaacaa gaccgccaag cacttcagcc tgcacatcac cgagacacag 300

cctgaggata gcgccgtgta cttctgtgcc ggcagaggca agctgacctt tggcacaggc 360

acccggctga caatcatccc ta 382

<210> 103

<211> 397

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 103

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgccagtt ctctcgtgcg ggacgagaag 360

ctgtttttcg gcagcggcac acagctgagc gttctgg 397

<210> 104

<211> 795

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 104

atgaccagca tccgggccgt gttcatcttc ctgtggctgc agctggacct ggtcaacggc 60

gagaatgtgg aacagcaccc cagcacactg agcgtgcaag agggcgattc tgccgtgatc 120

aagtgcacct acagcgacag cgccagcaac tacttcccct ggtacaagca agaactcggc 180

aagggccctc agctgatcat cgacatcaga tccaacgtgg gcgagaagaa ggaccagcgg 240

attgccgtga cactgaacaa gaccgccaag cacttcagcc tgcacatcac cgagacacag 300

cctgaggata gcgccgtgta cttctgtgcc ggcagaggca agctgacctt tggcacaggc 360

acccggctga caatcatccc taacatccag aatccggagc ccgccgtata ccagctgaag 420

gaccctagaa gccaggacag caccctgtgc ctgttcaccg acttcgacag ccagatcaac 480

gtgcccaaga ccatggaaag cggcaccttc atcaccgaca agacagtgct ggacatgaag 540

gccatggaca gcaagtccaa cggcgcaatc gcctggtcca accagaccag cttcacatgc 600

caggacatct tcaaagagac aaacgccaca taccccagca gcgacgtgcc ctgtgatgcc 660

accctgacag agaagtcctt cgagacagac atgaacctga acttccagaa tctgtccgtg 720

atgggcctga gaatcctgct gctgaaggtg gccggcttca atctgctgat gaccctgcgg 780

ctgtggtcca gctga 795

<210> 105

<211> 918

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 105

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgccagtt ctctcgtgcg ggacgagaag 360

ctgtttttcg gcagcggcac acagctgagc gttctggaag atctccggaa cgtgaccccc 420

cctaaagtga ccctgttcga acccagcaag gccgagatcg ccaacaagca gaaagccacc 480

ctcgtgtgcc tggccagagg cttcttcccc gaccatgtgg aactgtcttg gtgggtcaac 540

ggcaaagagg tgcacagcgg agtgtccacc gaccctcagg cctacaaaga gagcaactac 600

agctactgcc tgagcagcag actgcgggtg tccgccacct tctggcacaa cccccggaac 660

cacttcagat gccaggtgca gtttcacggc ctgagcgaag aggacaagtg gcccgaaggc 720

tcccccaagc ccgtgaccca gaatatctct gccgaggcct ggggcagagc cgactgtgga 780

attaccagcg ccagctacca ccagggcgtg ctgtctgcca ccatcctgta cgagatcctg 840

ctgggcaagg ccaccctgta cgccgtgctg gtgtctggcc tggtgctgat ggccatggtc 900

aagaagaaga acagctga 918

<210> 106

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 106

agcgtgttca gcagc 15

<210> 107

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 107

gttgtgacag gcggcgaagt g 21

<210> 108

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 108

tgtgccggcg ctggcaacaa cgacatgaga ttt 33

<210> 109

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 109

agcgagcaca accgg 15

<210> 110

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 110

ttccagaacg aggcccag 18

<210> 111

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 111

tgtgcctctt ctctcgtgcg gggcatcgag gcctttttt 39

<210> 112

<211> 382

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 112

atggtgctga agttcagcgt gtccatcctg tggatccagc tggcctgggt ttccacacag 60

ctgctggaac agagccctca gttcctgagc atccaagagg gcgagaacct gaccgtgtac 120

tgcaacagca gcagcgtgtt cagcagcctg cagtggtaca gacaagagcc tggcgaagga 180

cctgtgctgc tggtcacagt tgtgacaggc ggcgaagtga agaagctgaa gcggctgacc 240

ttccagttcg gcgacgccag aaaggatagc tccctgcaca ttaccgccgc tcagacaggc 300

gataccggcc tgtatctttg tgccggcgct ggcaacaacg acatgagatt tggcgccgga 360

accagactga ccgtgaagcc ta 382

<210> 113

<211> 397

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 113

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgcctctt ctctcgtgcg gggcatcgag 360

gccttttttg gccaaggcac cagactgacc gtggtgg 397

<210> 114

<211> 795

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 114

atggtgctga agttcagcgt gtccatcctg tggatccagc tggcctgggt ttccacacag 60

ctgctggaac agagccctca gttcctgagc atccaagagg gcgagaacct gaccgtgtac 120

tgcaacagca gcagcgtgtt cagcagcctg cagtggtaca gacaagagcc tggcgaagga 180

cctgtgctgc tggtcacagt tgtgacaggc ggcgaagtga agaagctgaa gcggctgacc 240

ttccagttcg gcgacgccag aaaggatagc tccctgcaca ttaccgccgc tcagacaggc 300

gataccggcc tgtatctttg tgccggcgct ggcaacaacg acatgagatt tggcgccgga 360

accagactga ccgtgaagcc taacatccag aatccggagc ccgccgtata ccagctgaag 420

gaccctagaa gccaggacag caccctgtgc ctgttcaccg acttcgacag ccagatcaac 480

gtgcccaaga ccatggaaag cggcaccttc atcaccgaca agacagtgct ggacatgaag 540

gccatggaca gcaagtccaa cggcgcaatc gcctggtcca accagaccag cttcacatgc 600

caggacatct tcaaagagac aaacgccaca taccccagca gcgacgtgcc ctgtgatgcc 660

accctgacag agaagtcctt cgagacagac atgaacctga acttccagaa tctgtccgtg 720

atgggcctga gaatcctgct gctgaaggtg gccggcttca atctgctgat gaccctgcgg 780

ctgtggtcca gctga 795

<210> 115

<211> 918

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 115

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgcctctt ctctcgtgcg gggcatcgag 360

gccttttttg gccaaggcac cagactgacc gtggtggaag atctccggaa cgtgaccccc 420

cctaaagtga ccctgttcga acccagcaag gccgagatcg ccaacaagca gaaagccacc 480

ctcgtgtgcc tggccagagg cttcttcccc gaccatgtgg aactgtcttg gtgggtcaac 540

ggcaaagagg tgcacagcgg agtgtccacc gaccctcagg cctacaaaga gagcaactac 600

agctactgcc tgagcagcag actgcgggtg tccgccacct tctggcacaa cccccggaac 660

cacttcagat gccaggtgca gtttcacggc ctgagcgaag aggacaagtg gcccgaaggc 720

tcccccaagc ccgtgaccca gaatatctct gccgaggcct ggggcagagc cgactgtgga 780

attaccagcg ccagctacca ccagggcgtg ctgtctgcca ccatcctgta cgagatcctg 840

ctgggcaagg ccaccctgta cgccgtgctg gtgtctggcc tggtgctgat ggccatggtc 900

aagaagaaga acagctga 918

<210> 116

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 116

gacagctcct ccacctac 18

<210> 117

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 117

attttttcaa atatggacat g 21

<210> 118

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 118

tgtgcagaga aatggatcat cttt 24

<210> 119

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 119

agcgagcaca accgg 15

<210> 120

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 120

ttccagaacg aggcccag 18

<210> 121

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 121

tgtgccagca gcctgaccac acctgacggc tacacattt 39

<210> 122

<211> 382

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 122

atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60

agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120

ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180

ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240

agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300

cagactgggg actcagctat ctacttctgt gcagagaaat ggatcatctt tggaaaaggg 360

acacgacttc atattctccc ca 382

<210> 123

<211> 397

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized homo sapiens sequence

<400> 123

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgccagca gcctgaccac acctgacggc 360

tacacatttg gcagcggcac cagactgacc gtggtgg 397

<210> 124

<211> 795

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 124

atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60

agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120

ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180

ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240

agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300

cagactgggg actcagctat ctacttctgt gcagagaaat ggatcatctt tggaaaaggg 360

acacgacttc atattctccc caacatccag aatccggagc ccgccgtata ccagctgaag 420

gaccctagaa gccaggacag caccctgtgc ctgttcaccg acttcgacag ccagatcaac 480

gtgcccaaga ccatggaaag cggcaccttc atcaccgaca agacagtgct ggacatgaag 540

gccatggaca gcaagtccaa cggcgcaatc gcctggtcca accagaccag cttcacatgc 600

caggacatct tcaaagagac aaacgccaca taccccagca gcgacgtgcc ctgtgatgcc 660

accctgacag agaagtcctt cgagacagac atgaacctga acttccagaa tctgtccgtg 720

atgggcctga gaatcctgct gctgaaggtg gccggcttca atctgctgat gaccctgcgg 780

ctgtggtcca gctga 795

<210> 125

<211> 918

<212> DNA

<213> Artificial sequence

<220>

<223> human with murine constant region (codon optimization)

<400> 125

atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60

acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120

agatgcgacc ctatcagcga gcacaaccgg ctgtactggt acagacagac actcggccag 180

ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag cagactgctg 240

agcgacagat tcagcgccga aagacccaag ggcagcttca gcaccctgga aatccagaga 300

accgagcagg gcgacagcgc catgtatctg tgtgccagca gcctgaccac acctgacggc 360

tacacatttg gcagcggcac cagactgacc gtggtggaag atctccggaa cgtgaccccc 420

cctaaagtga ccctgttcga acccagcaag gccgagatcg ccaacaagca gaaagccacc 480

ctcgtgtgcc tggccagagg cttcttcccc gaccatgtgg aactgtcttg gtgggtcaac 540

ggcaaagagg tgcacagcgg agtgtccacc gaccctcagg cctacaaaga gagcaactac 600

agctactgcc tgagcagcag actgcgggtg tccgccacct tctggcacaa cccccggaac 660

cacttcagat gccaggtgca gtttcacggc ctgagcgaag aggacaagtg gcccgaaggc 720

tcccccaagc ccgtgaccca gaatatctct gccgaggcct ggggcagagc cgactgtgga 780

attaccagcg ccagctacca ccagggcgtg ctgtctgcca ccatcctgta cgagatcctg 840

ctgggcaagg ccaccctgta cgccgtgctg gtgtctggcc tggtgctgat ggccatggtc 900

aagaagaaga acagctga 918

<210> 126

<211> 31

<212> PRT

<213> Intelligent people

<400> 126

Ala Arg Phe Ala Glu Gly Leu Glu Lys Leu Lys Glu Cys Val Leu His

1 5 10 15

Asp Asp Leu Leu Glu Ala Arg Arg Pro Arg Ala His Glu Cys Leu

20 25 30

<210> 127

<211> 5

<212> PRT

<213> Intelligent people

<400> 127

Asp Asp Leu Leu Glu

1 5