HA-1 specific T cell receptor and its use

Abstract

The present invention encompasses an allelic variant of minor histocompatibility antigen 1 (HA-1), in particular an allelic variant HA-1 H A T cell receptor (TCR) having specificity is isolated. Also described are isolated polypeptides comprising a functional portion of a TCR. In addition, multivalent TCR complexes, nucleic acid molecules, vectors, cells, antibodies, and medical uses associated with a TCR are defined.

Description

Technical Field

[0001] The present invention relates to an isolated T cell receptor (TCR) that specifically binds to an allelic form of minor histocompatibility antigen 1 (HA-1), and a polypeptide comprising a functional portion of the TCR. It also relates to a multivalent TCR complex, a nucleic acid encoding the TCR, a cell expressing the TCR, and a pharmaceutical composition comprising the TCR. The present invention also relates to the use of the TCR as a drug, and in particular to the use of the TCR in the treatment of cancer. Background Art

[0002] Over the past 50 years, bone marrow transplantation (BMT) and hematopoietic stem cell transplantation (HSCT) have been successfully used as first-line treatments for many malignant and non-malignant diseases, such as severe aplastic anemia and leukemia. During BMT or HSCT, the patient receives hematopoietic cells (i.e., hematopoietic stem cells) from a healthy donor after the patient's own hematopoietic cell system has been destroyed by sublethal whole-body irradiation or high-dose cytotoxic drugs. The donor's stem cells can be derived from the donor's bone marrow, or they can be part of a stem cell transplant of donor-mobilized hematopoietic stem cells. This approach is called allogeneic because the transplanted cells are derived from a non-autologous source, i.e., a healthy, genetically non-identical donor.

[0003] One mode of failure of allogeneic BMT or HSCT is due to host rejection of the graft, also known as host-versus-graft disease (HvGD), in which residual immune cells of the patient that escaped destruction during a pre-transplant regimen of sublethal irradiation 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.

[0004] Another even more serious form of failure of BMT / HSCT is the so-called "graft versus host disease" (GvHD), which is actually a reaction of transplanted immune cells derived from the bone marrow or hematopoietic stem cells transplanted from the donor to the patient's (recipient) somatic cells. GvHD is mainly due to the differences in the MHC molecules expressed by all nucleated somatic cells and platelets. MHC stands for major histocompatibility complex molecules, of which two main classes are known. MHC class I molecules that present or display peptide fragments of cytoplasmic proteins play a major role in GvHD. In humans, MHC is also referred to as the HLA complex (human leukocyte antigen complex) and is located on chromosome 6.

[0005] HLA complex is 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 colony. Each human cell expresses six different MHC class I alleles (from one HLA-A, HLA-B and HLA-C allele of each parent) and six to eight other MHC class II alleles. In the case of BMT and HSCT, the MHC molecule itself may serve as an antigen, that is, it may be identified as "exogenous" by the immune cells of the donor. Therefore, GvHD reactions are mainly based on the mismatch of the HLA / MHC molecule expression of the donor and the recipient. Therefore, the most important thing is to select the donor to have a minimum mismatch in terms of MHC class I and class II phenotypes, so that the risk of GvHD is minimized.

[0006] Even if the donor and patient are matched as closely as possible in terms of their MHC I / II genotype, more than 50% of all patients still suffer from GvHD. One possible way to prevent patients from developing GvHD is to remove T cells from the graft using, for example, anti-CD3 monoclonal antibodies or other antibodies specific for mature T cells, because the donor's T cells induce GvHD by recognizing the patient's HLA molecules that are different from the donor's pool. Although BMT and HSCT are currently performed in an HLA-matched setting, GvHD still occurs in a large number of patients, creating a need to overcome this problem.

[0007] This is probably due to the mismatch in the so-called minor histocompatibility antigen (MiHA), which is inherited independently of the HLA gene. In the past few years, a large number of MiHA have been identified (Frontiers in Immunology, Vol. 7, Chapter 100, March 2016), which can be encoded by male-specific Y chromosomes (HY antigens) or other chromosomes (autosomal MiHA). In autosomal MiHA, the antigens referred to as HA-1, HA-2, HA-3, HA-4 and HA-5 are the earliest discovered 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 the SNP variants (single nucleotide polymorphisms) of the MiHA limiting HA-1 are expressed in 69% of patients expressing HLA-A2, while 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 Feb 1;334(5):281-5).

[0008] Because HA-1 is almost only expressed on hematopoietic cells and cancer cells, it represents a promising tumor-specific target for adoptive T cell therapy (ACT). Previously, HA-1 specific cytotoxic T lymphocytes (T cells) restricted by HLA-A2 have been successfully isolated or produced (Haematologica. October 2005; 90 (10): 1415-21). HA-1 includes an epitope in the form of a nine peptide with an amino acid sequence of VLHDDLLEA (SEQ ID NO: 2) (Science. February 13, 1998; 279 (5353): 1054-7), and is derived from an allele (SNP variant) of the HMHA1 (KIAA0223) gene (rs number: rs1801284). The HMHA1 locus includes two alleles, HA-1H and HA-1R, which are different at two nucleotides, resulting in a single amino acid replacement. The second nonapeptide epitope, VLRDDLLEA (SEQ ID NO: 4; Science Feb. 13, 1998: Vol. 279, No. 5353, pp. 1054-1057), is inefficiently presented by HLA-A*02:01. Due to allelic polymorphism and Mendelian segregation patterns, an individual can be homozygous for HA-1 H / H , heterozygous HA-1 H / R or homozygous HA-1 R / R . Summary of the invention

[0009] It is an object of the present invention to provide a T cell receptor that specifically binds to only one allelic variant of MiHA HA-1.

[0010] The present invention relates to the regulation of only one allelic form of HA-1 (i.e., HA-1 H ) has a specific T cell receptor (TCR). In a specific embodiment of the present invention, a method for HA-1 presented by HLA-A2 molecules or in the context of HLA-A2 molecules is disclosed. H T cell receptors with binding specificity. The present invention also relates to TCRs for use as drugs or for use in the treatment of malignant tumors. TCRs can be used, for example, in patients with BMT / HSCT whose HA-1H allele (presenting the peptide VLHDDLLEA on a suitable MHC / HLA molecule) is homozygous (i.e., the patient is HA-1 H / H ) or heterozygous (ie, HA-1 H / R ) and the donor is homozygous for the R allele (ie, 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 express HA-1 HThe transduced donor T cells are infused into the patient, and the patient and donor are otherwise HLA identical or HLA matched. H Recombinant donor T cells with specific TCR detect and eliminate (through cytotoxic activity) HA-1 expressed in the context of appropriate MHC / HLA molecules H It is also contemplated that preservation of such HA-1 H Another aspect of the invention is the use of the TCR of the invention or recombinant T cells expressing the TCR of the invention for pretreatment therapy in patients who are scheduled for BMT / HSCT. By using the TCR of the invention or recombinant T cells expressing such HA-1 H Recombinant T cells expressing specific TCRs can replace or support sublethal radiation by infusing such recombinant T cells, which are alloreactive to the patient's hematopoietic cells due to the recombinant expression of the TCRs of the present invention and can therefore eradicate HA-1 in the context of HLA-A2. H Immune cells. T cells can be derived from the donor of BMT / HSCT.

[0011] The TCR according to the invention is isolated and / or purified and may be soluble or membrane bound.

[0012] In some embodiments, the amino acid sequence of TCR may include one or more phenotypic silent replacements. In addition, the TCR of the present invention may be labeled. Useful labels are known in the art and can be optionally coupled to TCR or TCR variants via joints 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 modification portion. 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 be, for example, a cytokine. The pharmacokinetic modification portion may be at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group, or a combination thereof.

[0013] According to the TCR of the present invention, in particular the TCR in soluble form, can be modified by connecting additional functional parts, for example, to reduce immunogenicity, increase hydrodynamic size (size in solution), solubility and / or stability (e.g., by enhanced protection against proteolytic degradation) and / or extend serum half-life. Other useful functional parts and modifications include "suicide" or "safety switches" that can be used to turn off or on effector host cells carrying the TCR of the present invention in a patient.

[0014] Also contemplated herein are TCRs with altered glycosylation patterns.

[0015] It is also contemplated that drugs or therapeutic entities (eg, small molecule compounds) may be added to TCRs, particularly soluble forms of the TCRs of the invention.

[0016] The TCR, in particular the soluble form of the TCR of the invention, may additionally be modified to introduce additional domains that facilitate identification, tracking, purification and / or isolation of individual molecules (tags).

[0017] In some embodiments, the TCR is of the single-chain type, in which the TCR alpha chain and the TCR beta chain are connected by a linker sequence.

[0018] Another aspect of the present 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 the following 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.

[0019] In a specific embodiment, the functional portion comprises a TCR alpha variable chain and / or a TCR beta variable chain.

[0020] Specific 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.

[0021] Some embodiments relate to a TCR of the present invention expressed as a functional polypeptide or a functional multivalent polypeptide on effector cells, in particular on immune effector cells, wherein after binding to the amino acid sequence SEQ IDNO:2 presented by the HLA-A*02:01 encoded molecule, IFN-γ secretion is induced in the above-mentioned effector cells expressing the TCR.

[0022] Presentation by the HLA-A*02:01 encoded molecule refers to the binding of the peptide, especially the epitope, to the binding groove of the MHC molecule.

[0023] The MHC molecule presenting the epitope may be encoded by one of the HLA-A*02 alleles, such as the HLA-A*02:01 or HLA-A*02:06 allele, preferably the HLA-A*02:01 allele.

[0024] In 10-7 At an HA-1 peptide concentration of [M], IFN-γ secretion induced by binding of the TCR of the present invention expressed on effector cells to the amino acid sequence SEQ ID NO: 2 presented by the HLA-A*02:01 encoded molecule can be at least 3 times higher, for example, 10 times higher, 20 times higher, or 100 times higher than IFN-γ secretion induced by binding to SEQ ID NO: 4 presented by the HLA-A*02:01 encoded molecule.

[0025] Therefore, for all HA-1 H -TCR transgenic T cells, to recognize HA-1 R Variant, requires HA-1 H Compared to a peptide concentration that is at least 1,000 times higher, preferably at least 5,000 times higher, more preferably at least 8000 times higher, most preferably at least 10,0000 times higher.

[0026] In a specific embodiment, for example, when the ratio of TCR transgenic T cells to T2 cells is 2:1, at 10 -7 At an HA-1 peptide concentration of [M], the IFN-γ secretion induced by the binding of the TCR of the present invention expressed on effector cells to the amino acid sequence SEQ ID NO: 2 presented by the HLA-A*02:01 encoded molecule can be greater than 500 pg / ml, for example greater than 1000 pg / ml, more preferably greater than 2000 pg / ml, and most preferably greater than 3000 pg / ml.

[0027] Cytokine and chemokine release (e.g. IFN-γ secretion) can be measured by an in vitro assay using magnetic beads immobilized with T cell antibodies, wherein K562 cells transduced to express the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, respectively (Greiner et al. 2006, Blood. 2006 Dec 15; 108(13):4109-17) are incubated with CD8 T cells expressing the TCR to be investigated. + Enriched and / or non-CD8 + The enriched PBMCs are incubated together, or in an in vitro assay, T2 cells are used that are externally loaded with the VLHDDLLEA peptide (SEQ ID NO: 2) or the peptide VLRDDLLEA (e.g., the peptide SEQ ID NO: 4) and subsequently incubated with a CD8 T cell expressing the TCR to be investigated. + Enriched and / or non-CD8 + Co-incubation with enriched PBMCs.

[0028] Another aspect of the invention relates to a nucleic acid encoding a TCR as described herein or encoding a polypeptide as described above.

[0029] Another aspect of the present invention relates to a plasmid or vector comprising the 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, especially eukaryotic cells. The vector can be, for example, a retroviral vector (e.g., a γ-retrovirus) or a lentiviral vector.

[0030] Another aspect of the invention relates to a cell expressing a TCR as described herein. The cell may be isolated or non-naturally occurring.

[0031] Another aspect of the present 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:

[0032] a) an expression vector comprising at least one nucleic acid as described above, or

[0033] 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.

[0034] The cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). Typically, the cell is an immune effector cell, especially a T cell. Other suitable cell types include γ-δ T cells, NK cells and NK-like T cells.

[0035] Another aspect relates to an antibody or antigen-binding fragment thereof that specifically binds to a portion of a TCR as described herein that mediates the binding to HA-1 H In one embodiment, the TCR mediates HA-1 H The specific portion comprises the α 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 β 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.

[0036] 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.

[0037] Typically, pharmaceutical compositions comprise at least one pharmaceutically acceptable carrier.

[0038] Another aspect of the invention relates to 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 for use as a medicament, in particular for use in the treatment of cancer, in particular in the treatment of hematological cancers in the context of an HLA-matched bone marrow and / or stem cell transplant, wherein the patient has previously received an allogeneic bone marrow and / or stem cell transplant from an HLA-matched donor, wherein the donor is HA-1 H Negative (and therefore HA-1 R homozygous and / or HLA-A2 negative), and in which the patient (is homozygous HA-1 H or heterozygous HA-1 H / R and HLA-A2 positive) with relapse or recurrence of hematological cancer cells, or as a preventive measure (to treat the patient if there is no sign of relapse / recurrence after transplantation). + T cells and transducing the isolated CD8 T cells ex vivo with the TCR according to the invention + T cells to treat patients with relapse of hematological cancer cells. The hematological cancer can 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 polymorphic 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, 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. Other hematological cancers or diseases include myelodysplasia. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Shown with different HA-1 H -Reactive TCR-transduced CD8 + HA-1 in T cells H-MHC-multimer binding. CD8 T cells were isolated from PBMCs of healthy donors and expressed in vitro and in vitro with six different HA-1 H -TCR and a protein that does not recognize HA-1 H The transduced CD8 T cells were enriched by FACS using the mouse constant β region as a transduction marker. After expansion of these cells, they were treated with HA-1 H -MHC multimers and antibodies against CD8 and murine constant beta region (mCb) were stained and analyzed by flow cytometry. + / mCb + Populations were gated on cells and staining for multimer / CD8 is shown.

[0040] Figure 2 HA-1 H -TCR-transgenic T cells recognize HA-1 presented on HLA-A2 H -peptide. H -peptide T2 cells or cells that have been expressed with HA-1 H - epitope. T2 cells loaded with control peptide and non-transduced K562 / HLA-A2 cells were used as negative controls. Target cell recognition was analyzed by measuring IFN-γ concentration in the co-culture supernatant using standard ELISA.

[0041] Figure 3 A and 3B show HA-1 H - Functional affinity of TCR-transgenic T cells. (A) Transgenic T cells were loaded with HA-1 H -peptide (10 -11 M-10 -5 M) or HA-1 R -peptide (10 -8 M-10 -5 M) T2 cells were co-cultured. (B) HA-1 H -TCR transgenic T cells targeting HA-1 R -The functional affinity of the peptides was compared with T cells transduced with HA-1 specific TCR (TCR2) as described in WO2018058002A1. The data of TCR 1-6 (TCR_1, TCR_2, TCR_3, TCR_4, TCR_5, TCR_6) according to the present invention are Figure 3 Same as in A and 3B.

[0042] Figure 4A and 4BHA-1 H -TCR transgenic T cells recognize physiological levels of HA-1 on unmodified target cells H The transgenic T cells were co-cultured with different lymphoblastoid cell lines (LCL), which were HA-1 H Positive (LCL1-5) or HA-1 H Negative (LCL 6-7). (A) Cytokine release was analyzed by measuring IFN-γ concentrations in co-culture supernatants using a standard ELISA. T cells expressing a control TCR were used as a negative control. (B) Co-cultures were photographed every two hours to monitor cytokine release. ZOOM (Essen BioScience) measured cytotoxicity against LCL stably transduced with fluorescent markers. Untransduced CD8 T cells were used as negative control.

[0043] Figure 5A and 5B HA-1 H -TCR transgenic T cells recognize HA-1 present on tumor cell lines H The ability of transgenic T cells to express different HA-1 H Positive tumor cell lines were co-cultured. (A) Cytokine release was analyzed by measuring IFN-γ concentrations in the co-culture supernatant using a standard ELISA. (B) Cytokine release was analyzed by taking pictures every two hours. ZOOM measures cytotoxicity against tumor cell lines stably transduced with fluorescent markers.

[0044] Figure 6 A and 6B show that T cells expressing TCR_3 or TCR_4 recognize and lyse (kill) HA-1 compared to TCR2 as described in WO2018058002A1. H The ability of transgenic T cells to express HA-1 H Co-culture of positive tumor cell lines. Cytokine release was analyzed by measuring IFN-γ concentration in the co-culture supernatant using standard ELISA. (B) Analysis of IFN-γ against HA-1 by flow cytometry H Cytotoxicity of positive tumor cell lines. H HA-1 negative / GFP positive K562 cells and tumor cell lines H Positive / mCherry positive cells were mixed at a 1:1 ratio and co-cultured with TCR transgenic T cells. mCherry positive (HA-1 H positive) and GFP positive (HA-1 HThe ratio of negative (negative) cells was measured and normalized to the ratio of control TCR measurements. Smaller numbers indicate that HA-1 H Positive tumor cells were lysed.

[0045] Figure 7 Analysis of the recognition motifs of TCR_3 and TCR_4 compared to TCR2 as described in WO2018058002A1 is shown. Transgenic T cells were incubated with externally loaded HA-1 H T2 cells were co-cultured with peptides, transgenic T cells having peptides in which each individual amino acid residue was replaced sequentially with serine, or control peptides.

[0046] Figure 8 A and 8B show a comparison of different constant TCR regions. TCR-3 was cloned using murine, minimally humanized, or human constant TCR regions. (A) Transgenic T cells were co-cultured with different HA-1 H Positive tumor cell lines were co-cultured. Cytokine release was analyzed by measuring IFN-γ concentrations in the co-culture supernatants using standard ELISA. IFN-γ against HA-1 was analyzed by flow cytometry. H Cytotoxicity of positive tumor cell lines. H HA-1 negative / GFP positive K562 cells and tumor cell lines H Positive / mCherry positive cells were mixed at a 1:1 ratio and co-cultured with TCR transgenic T cells. After 45 hours, the number of mCherry positive (HA-1 H positive) cells and GFP positive (HA-1 H The ratios of HA-1 negative cells were normalized to the ratios measured with the control TCR. H Lysis of positive tumor cells. (B) Transgenic T cells were incubated with different HA-1 H Negative (OMW, BER) and HA-1 H Positive (FH22, SLE005, SPO-010, BM16, BSM) LCL (lymphoblastoid cell lines) co-cultures. Cytokine release was analyzed by measuring IFN-γ concentrations in the co-culture supernatants using a standard ELISA. DETAILED DESCRIPTION

[0047] Before describing the present invention in detail with respect to certain preferred embodiments, the following general definitions are provided.

[0048] The present invention as illustratively described below may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0049] 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.

[0050] When the term "comprises / comprising" is used in the present description and claims, it does not exclude other elements. For the purpose of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprises / comprising". If a group is defined hereinafter as comprising at least a certain number of embodiments, it is also understood that a group preferably consisting of only these embodiments is disclosed.

[0051] 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 being obtainable from a particular source, this is also to be understood as disclosing the antibody obtained from this source.

[0052] When an indefinite or definite article (e.g. "a", "an" or "the") is used in reference to a singular noun, the word includes the plural form of the noun unless otherwise specified. In the context of the present invention, the term "about" or "approximately" indicates a range of accuracy that a person skilled in the art will understand as still ensuring the technical effect of the feature in question. The term generally indicates a deviation of ±10%, preferably ±5%, from the indicated numerical value.

[0053] Technical terms are used with their common sense or meaning to those skilled in the art. If a specific meaning is conveyed to certain terms, the definition of the term will be given in the context of the term's use below.

[0054] TCR Background

[0055] TCR is composed of two different and separate protein chains, namely TCR alpha (α) and TCR beta (β) chains. The TCR α chain contains a variable region (V), a connecting region (J) and a constant region (C). The TCR β chain contains a variable region (V), a multivariable region (D), a connecting region (J) and a constant region (C). The rearranged V (D) J region of both the TCR α chain and the TCR β chain contains a hypervariable region (CDR, complementary determining region), of which the CDR3 region determines specific epitope recognition. In the C-terminal region, both the TCR α chain and the TCR β chain contain a hydrophobic transmembrane domain and terminate in a short cytoplasmic tail region.

[0056] Typically, TCR is a heterodimer of an α chain and a β chain that can bind to a peptide-presenting MHC molecule.

[0057] In the context of the present invention, the term "variable TCR alpha region" or "TCR alpha variable chain" or "variable domain" refers to the variable region of the TCR alpha chain. In the context of the present invention, the term "variable TCR beta region" or "TCR beta variable chain" refers to the variable region of the TCR beta chain.

[0058] The International Immunogenetics (IMGT) TCR nomenclature was used (IMGT Database, www.IMGT.org; Giudicelli, V., et al., IMGT / LIGM-DB, Immunoglobulin and T Cell Receptor Nucleotide Sequences, TCR loci and genes are named in the comprehensive database, Nucl. Acids Res., 34, D781-D784 (2006). PMID: 16381979; T cell Receptor Factsbook, LeFranc and LeFranc, Academic Press ISBN 0-12-441352-8).

[0059] Target

[0060] A first aspect of the present invention relates to an isolated TCR specific for an allelic variant of HA-1. The allelic variant is of the type of a single nucleotide polymorphism.

[0061] In particular, the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 2 (VLHDDLLEA).

[0062] Typically, TCRs recognize peptide fragments of antigens presented by major histocompatibility complex (MHC) molecules.

[0063] The human leukocyte antigen (HLA) system or complex is a complex of genes in the human body that encodes major histocompatibility complex (MHC) proteins. HLA-A*02 is a specific class I major histocompatibility complex (MHC) allele group at the HLA-A locus. HLA-A*02:01 is a specific HLA-A*02 suballele.

[0064] Therefore, 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-A*02 allele, preferably a molecule encoded by HLA-A*02:01 or a molecule encoded by HLA-A*02:06, and more preferably a molecule encoded by HLA-A02:01.

[0065] TCR for HA-1 H Highly specific and to other peptides, especially allelic variants of HA-1 R Essentially no cross-reactivity. This means that it does not recognize HA-1 allelic variations. 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.

[0066] In the context of the present invention, the term "specific for" refers to the specific binding of the TCR to the target.

[0067] As used herein, the terms "allelic variation" and "allelic form" are synonymous.

[0068] In a preferred embodiment, the recognition motif of TCR comprises at least the sequence shown in SEQ ID NO: 127. In a specific embodiment, the recognition motif of TCR consists of the sequence shown in SEQ ID NO: 127. The recognition motif can be determined by serine substitution.

[0069] The recognition motif defines the amino acids of the epitope that influence TCR activation, for example as determined in an IFN-γ secretion assay. In particular, the influence of amino acid residues in the binding epitope can be determined by amino acid substitution scanning, for example serine scanning.

[0070] In serine scanning, epitope peptides in which each individual amino acid residue is successively replaced by serine are used. If the peptide causes a significant reduction in TCR activation, such as determined by IFN-γ secretion, it is shown that the replaced position belongs to a recognition motif. Significant reduction can be at least 3 times, preferably at least 5 times, more preferably at least 10 times the reduction in IFN-γ secretion compared to an unsubstituted peptide sequence (i.e., an unsubstituted epitope).

[0071] TCR-specific sequences

[0072] The TCR alpha 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.

[0073] The TCR β chain CDR3 of the TCR may have an amino acid sequence selected from the group consisting 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.

[0074] Some embodiments relate to an isolated TCR comprising a TCR alpha chain and a TCR beta chain, wherein

[0075] a) - the TCR alpha chain comprises a complementarity determining region 3 (CDR3) having the sequence of SEQ ID NO: 7,

[0076] - the TCR β chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 10; or

[0077] b) the TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO: 17,

[0078] - the TCR β chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 20; or

[0079] c) the TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO: 27,

[0080] - the TCR β chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 30; or

[0081] d) the TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO: 37,

[0082] - the TCR β chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 40; or

[0083] e) the TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO: 47,

[0084] - the TCR β chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 50; or

[0085] f) the TCR alpha chain comprises a CDR3 having the sequence of SEQ ID NO: 57,

[0086] - The TCR β chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 60.

[0087] A more specific embodiment relates to an isolated TCR, wherein the TCR comprises:

[0088] a) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 5, a CDR 2 having the amino acid sequence of SEQ ID NO: 6 and a CDR 3 having the sequence of SEQ ID NO: 7,

[0089] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 8, a CDR 2 having the amino acid sequence of SEQ ID NO: 9, and a CDR 3 having the sequence of SEQ ID NO: 10; or

[0090] b) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 15, a CDR 2 having the amino acid sequence of SEQ ID NO: 16 and a CDR 3 having the sequence of SEQ ID NO: 17,

[0091] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 18, a CDR 2 having the amino acid sequence of SEQ ID NO: 19, and a CDR 3 having the sequence of SEQ ID NO: 20; or

[0092] c) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 25, a CDR 2 having the amino acid sequence of SEQ ID NO: 26, and a CDR 3 having the sequence of SEQ ID NO: 27,

[0093] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 28, a CDR 2 having the amino acid sequence of SEQ ID NO: 29, and a CDR 3 having the sequence of SEQ ID NO: 30;

[0094] d) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR 2 having the amino acid sequence of SEQ ID NO: 36, and a CDR 3 having the sequence of SEQ ID NO: 37,

[0095] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR 2 having the amino acid sequence of SEQ ID NO: 39, and a CDR 3 having the sequence of SEQ ID NO: 40; or

[0096] e) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 45, a CDR 2 having the amino acid sequence of SEQ ID NO: 46, and a CDR 3 having the sequence of SEQ ID NO: 47,

[0097] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 48, a CDR 2 having the amino acid sequence of SEQ ID NO: 49, and a CDR 3 having the sequence of SEQ ID NO: 50; or

[0098] f) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 55, a CDR 2 having the amino acid sequence of SEQ ID NO: 56, and a CDR 3 having the sequence of SEQ ID NO: 57,

[0099] - A TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:58, a CDR 2 having the amino acid sequence of SEQ ID NO:59 and a CDR 3 having the sequence of SEQ ID NO:60.

[0100] Preferred embodiments relate to an isolated TCR defined by the CDRs, in particular CDR 3 of the TCR alpha chain and TCR beta chain as described above, wherein the recombinant TCR sequence is modified to comprise murinized Calpha and Cbeta regions.

[0101] Some embodiments relate to an isolated TCR, wherein the TCR comprises

[0102] a) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO: 11 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO: 12; or

[0103] b) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO: 21 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO: 22; or

[0104] c) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO: 31 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO: 32; or

[0105] d) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:41 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:42; or

[0106] e) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:51 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:52; or

[0107] f) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:61 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:62.

[0108] As used herein, "at least 80% identical", and 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 shown amino acid sequence.

[0109] In some embodiments, the TCR comprises a TCR α chain and a TCR β chain, wherein

[0110] a) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 11, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 7,

[0111] - the variable TCR beta region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 12, and comprises a CDR3 region having the amino acid sequence shown in SEQ ID NO: 10; or

[0112] b) the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 21 and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 17,

[0113] - the variable TCR beta region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 22, and comprises a CDR3 region having the amino acid sequence shown in SEQ ID NO: 20; or

[0114] c) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:31, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:27;

[0115] - the variable TCR beta region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 32, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 30; or

[0116] d) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:41, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:37;

[0117] - the variable TCR beta region has an amino acid sequence that is 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

[0118] e) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:51, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:47;

[0119] - the variable TCR beta region has an amino acid sequence that is at least 80% identical to SEQ ID NO:52, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:50; or

[0120] f) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:61, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:57;

[0121] The variable TCRβ region has an amino acid sequence that is 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.

[0122] Exemplary embodiments relate to an isolated TCR, wherein the TCR comprises

[0123] a) a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 11 and a variable TCR beta region having the amino acid sequence of SEQ ID NO: 12; or

[0124] b) a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 21 and a variable TCR beta region having the amino acid sequence of SEQ ID NO: 22; or

[0125] c) a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 31 and a variable TCR beta region having the amino acid sequence of SEQ ID NO: 32; or

[0126] 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

[0127] e) a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 51 and a variable TCR beta region having the amino acid sequence of SEQ ID NO: 52; or

[0128] 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.

[0129] The following table shows an overview of exemplary TCRs of the present invention.

[0130] Table 1: Summary of exemplary TCRs of the invention

[0131] TCR CDR1_α CDR2_α CDR3_α TRAV TRAJ CDR1_β CDR2_β CDR3_β TRB TRBJ 1 DSASNY IRSNVGE CAASDLNF 13-1 41 SEHNR QUR CASSLVSRVDGYTF 7-9 1-2 2 DSASNY IRSNVGE CAAHTPGYSTLTF 13-1 11 SEHNR QUR CASSPRAGGETQYF 7-9 2-5 3 TSINN IRSNERE CATGDQTGANNLFF 17 36 SEHNR QUR CASSLTRTEKLFF 7-9 1-4 4 DSASNY IRSNVGE CAGRGKLTF 13-1 48 SEHNR QUR CASSLVRDEKLFF 7-9 1-4 5 SVFSS VVTGGEV CAGAGNNDMRF 27 43 SEHNR QUR CASSLVRGIEAFF 7-9 1-1 6 DSSSTY IFSNMDM WIF 5 30 SEHNR QUR CASSLTTPDGYTF 7-9 1-2

[0132] As can be seen from the examples, the TCR according to the present invention is effective for HA-1 H It is specific and shows only very low cross-reactivity to other epitopes or antigens.

[0133] Vector NTI Advance is preferred TM The determination of percent identity between multiple sequences was accomplished using the AlignX application of the 10 program (Invitrogen Corporation, Carlsbad CA, USA). This program uses a modified ClustalW algorithm (Thompson et al., 1994. Nucl Acids Res. 22: pp. 4673-4680; Invitrogen Corporation; Vector NTI Advance TM 10 DNA and protein sequence analysis software. User's Manual, 2004, pp. 389-662). The determination of percent identity was performed using the standard parameters of the AlignX application.

[0134] The TCR according to the present 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 that, for example, the TCR does not contain or is substantially free of other proteins and non-protein parts of the cell from which it originally originated.

[0135] In some embodiments, the amino acid sequence of a TCR may contain one or more phenotypically silent substitutions.

[0136] "Phenotypic 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 refers to codons encoding positively charged residues (H, K and R) being replaced with codons encoding positively charged residues, codons encoding negatively charged residues (D and E) being replaced with codons encoding negatively charged residues, codons encoding neutral polar residues (C, G, N, Q, S, T and Y) being replaced with codons encoding neutral polar residues, and codons encoding neutral non-polar residues (A, F, I, L, M, P, V and W) being replaced with codons encoding neutral non-polar residues. These variations can occur spontaneously, be introduced by random mutagenesis, or can be introduced by directed mutagenesis. These changes can be made without destroying the essential characteristics of these polypeptides. Ordinary technicians can easily 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 destroy ligand binding ability.

[0137] The technician understands that the nucleic acid encoding the TCR can also be modified. Useful modifications of the entire nucleic acid sequence include codon optimization of the sequence. Conservative substitution changes that lead to the expressed amino acid sequence can be made. These changes that do not affect function can be made in the complementary determining regions and non-complementary determining regions of the TCR chain amino acid sequence. Generally, additions and deletions should not be made in the CDR3 region.

[0138] According to some embodiments of the invention, the amino acid sequence of the TCR is modified to include a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety.

[0139] Non-limiting examples of detectable labels are radioactive labels, fluorescent labels, nucleic acid probes, enzymes and contrast agents. Therapeutic agents that can be associated with TCR include radioactive compounds, immunomodulators, enzymes or chemotherapeutic agents. The therapeutic agent can be encapsulated with a liposome connected to the TCR so that the compound can be slowly released at the target site. This will avoid damage during transport in the body and ensure that the therapeutic agent (e.g., toxin) has maximum effect after the TCR is bound to the relevant antigen presenting cell. Other examples of therapeutic agents are:

[0140] Peptide cytotoxins, i.e. proteins or peptides with the ability to kill mammalian cells, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase. The molecular weight of small molecule cytotoxic agents (i.e. compounds with the ability to kill mammalian cells) is less than 700 Daltons. Such compounds may contain toxic metals that can have cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e. compounds that degrade or transform under physiological conditions to release cytotoxic agents. Such agents may include, for example, docetaxel, gemcitabine, cisplatin, maytansine derivatives, rachelmycin, calicheamicin, etoposide, ifosfamide, irinotecan, porfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucoronate, mitoxantrone, 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 radionuclides with TCR or its derivatives may be carried out, for example, by a chelating agent; immunostimulators, also known as immunostimulants, are immune effector molecules that stimulate immune responses. 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-CD3, anti-CD28 or anti-CD16); alternative protein scaffolds with antibody-like binding properties; superantigens, i.e., antigens that cause nonspecific activation of T cells, resulting in polyclonal T cell activation and massive cytokine release, and mutants thereof; chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc.; complement activators; heterologous protein domains, allogeneic protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0141] Antigen receptor molecules (T cell receptor molecules) on human T lymphocytes are non-covalently associated with CD3 (T3) molecule complexes on the cell surface. Disturbance of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Therefore, some embodiments relate to TCRs as described herein that are associated with anti-CD3 antibodies or functional fragments or variants of the anti-CD3 antibodies (usually by fusion with the N or C terminus of the α or β chain). Antibody fragments and variants / analogs suitable for the compositions and methods described herein include minibodies, Fab fragments, F(ab<'>)2 fragments, dsFv and scFv fragments, nanobodies TM(Ablynx (Belgium), molecules comprising a synthetic single immunoglobulin variable heavy chain domain derived from camelid (e.g. camel or llama) antibodies) and Domain Antibodies (comprising affinity-matured single immunoglobulin variable heavy chain domains or immunoglobulin variable light chain domains (Domantis (Belgium)) or alternative protein scaffolds exhibiting antibody-like binding properties, such as Affibodies (comprising the engineered Protein A scaffold Affibody (Sweden)) or Anticalins (comprising the engineered anticalins Pieris (Germany)).

[0142] The therapeutic agent may preferably be selected from the group consisting of an immune effector molecule, a cytotoxic agent and a radionuclide. Preferably, the immune effector molecule is a cytokine.

[0143] The pharmacokinetic modifying portion can be, for example, at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group or a combination thereof. The association of at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group can be caused by various 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 can be modified by one or more pharmacokinetic modifying portions. In particular, the soluble form of the TCR is modified by one or more pharmacokinetic modifying portions. The pharmacokinetic modifying portion can achieve beneficial changes in the pharmacokinetic properties of the therapeutic agent, such as improved plasma half-life, reduced or enhanced immunogenicity and improved solubility.

[0144] TCR according to the present invention can be soluble or membrane-bound.Term " soluble " refers to TCR in soluble form (i.e., without transmembrane domain or cytoplasmic domain), for example, used as a targeting agent for delivering therapeutic agent to antigen presenting cells.For stability, soluble α β heterodimer TCR preferably has an introduced disulfide bond between the residues of the corresponding constant domain, such as described in WO 03 / 020763.One or both of the constant domains present in the α β heterodimer of the present invention can be truncated at the C-terminus or C-terminus, such as up to 15, up to 10 or up to 8 or less amino acids.In order to be used in adoptive therapy, α β heterodimer TCR can be, for example, transfected into a full-length chain with both cytoplasmic domain and transmembrane domain.TCR may include a disulfide bond corresponding to the corresponding α and β constant domains found in nature, and in addition or alternatively, non-natural disulfide bonds may be present.

[0145] Thus, a TCR, in particular a soluble form of a TCR according to the invention, can be modified by attaching additional functional moieties, e.g. to reduce immunogenicity, increase the hydrodynamic size (size in solution), solubility and / or stability (e.g. by enhanced protection from proteolytic degradation) and / or to extend serum half-life.

[0146] Other useful functional parts and modifications include "suicide" or "safety switches" that can be used to shut down effector host cells carrying the TCR of the invention in a patient. An example is the inducible Caspase 9 (iCasp9) "safety switch" described by Gargett and Brown Front Pharmacol. 2014; 5: 235. Briefly, effector host cells are modified by known methods to express Caspase 9 domains, whose dimerization depends on small molecule dimerizing drugs, such as AP1903 / CIP, and leads to rapid induction of apoptosis in modified effector cells. This system is described, for example, in EP2173869 (A2). Other examples of "suicide" "safety switches" are known in the art, such as herpes simplex virus thymidine kinase (HSV-TK), expression of CD20 followed by depletion using anti-CD20 antibodies, expression of truncated EGFR followed by depletion using anti-EGFR antibodies (Wang et al., Blood, 2011 Aug 4;118(5):1255-63), or the myc tag (Kieback et al., Proc Natl Acad Sci USA. 2008 Jan 15;105(2):623-8).

[0147] TCRs with altered glycosylation patterns are also contemplated herein. As known in the art, glycosylation patterns may depend on the amino acid sequence (e.g., the presence or absence of specific glycosylated amino acid residues discussed below) and / or the host cell or organism producing the protein. The glycosylation of a polypeptide is usually N-connected or O-connected. N-connection refers to the side chain of an asparagine residue to which a carbohydrate moiety is connected. By changing the amino acid sequence so that it includes one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine (wherein X is any amino group except proline), it is conveniently achieved that the glycosylation sites connected by N- are added to the binding molecule. O-connected glycosylation sites may be introduced by adding or replacing one or more serine or threonine residues in the starting sequence.

[0148] Another method of glycosylation of TCR is by chemical or enzymatic coupling of glycosides to proteins. Depending on the coupling method used, one or more sugars can 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) amide groups of glutamine. Similarly, deglycosylation (i.e., removal of carbohydrate moieties present on the binding molecule) can be achieved chemically, for example by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically by using endoglycosidases and exoglycosidases.

[0149] It is also contemplated to add drugs, such as small molecule compounds, to TCRs, particularly soluble forms of the present invention. Connection can be achieved by covalent bonds or non-covalent interactions (e.g., by electrostatic forces). To form drug conjugates, various linkers known in the art can be used.

[0150] TCR, in particular soluble forms of the invention TCR, can be additionally modified to introduce additional domains that facilitate identification, tracking, purification and / or isolation of individual molecules (tags). Thus, in some embodiments, the TCR alpha chain or TCR beta chain can be modified to include an epitope tag.

[0151] Epitope tags are useful examples of tags that can be incorporated into the TCR of the present invention. Epitope tags are short amino acid fragments that allow binding to specific antibodies and are therefore able to identify and track the binding and movement of soluble TCR or host cells in patients or cultured (host) cells. A variety of different techniques can be used to achieve epitope tags and therefore the detection of labeled TCRs.

[0152] The tag can be further used to stimulate and expand host cells carrying the TCR of the invention by culturing the cells in the presence of a binding molecule (antibody) specific for the tag.

[0153] Typically, in some cases, a TCR can be modified to have multiple mutations that change the affinity and dissociation rate of the TCR to the target antigen. In particular, the mutation can increase the affinity and / or reduce the dissociation rate. Thus, a TCR can be mutated in at least one CDR and its variable domain framework region.

[0154] However, in a preferred embodiment, no modification or in vitro affinity maturation is performed on the CDR regions of the TCR, such as for the TCR receptors in the examples. This means that the CDR regions have naturally occurring sequences. This may be advantageous because in vitro affinity maturation may lead to immunogenicity to the TCR molecule. This may lead to the production of anti-drug antibodies that reduce or inactivate the efficacy and treatment and / or cause adverse reactions.

[0155] The mutation may be one or more substitutions, deletions or insertions. These mutations may be introduced by any suitable method known in the art, such as polymerase chain reaction, restriction enzyme-based cloning, ligation-independent cloning procedures, which are described in, for example, Sambrook, Molecular Cloning—4th Edition (2012) Cold Spring Harbor Laboratory Press.

[0156] Theoretically, due to the mismatch between endogenous TCR chains and exogenous TCR chains, unpredictable TCR specificity with a risk of cross-reaction may occur. To avoid TCR sequence mismatch, the recombinant TCR sequence can be modified to include murinized Cα and Cβ regions, which has been shown to effectively enhance the correct pairing of TCR chains of various different transductions. Murinization of TCR (i.e., exchanging the human constant regions in α and β chains by their murine counterparts) is a commonly used technique for improving the cell surface expression of TCR in host cells. It is not desirable to be bound by a particular theory, and it is believed that murinized TCRs are more effectively associated with CD3 co-receptors; and / or preferentially paired with each other and less likely to form mixed TCRs on ex vivo genetically modified human T cells to express TCRs with desired antigen specificity, but still retain and express their "original" (i.e., endogenous) TCRs. Nine amino acids responsible for improving murinized TCR expression have been identified (Sommermeyer and Uckert, J Immunol. June 1, 2010; 184(11): 6223-31), and it is envisioned that at least one or all of the amino acid residues in the constant region of the TCR α and / or β chains are replaced with their mouse counterpart residues. This technology is also referred to as "minimum mouse humanization", and provides the advantages of enhancing cell surface expression while reducing the number of "foreign" amino acid residues in the amino acid sequence and thus reducing the risk of immunogenicity. Therefore, in some embodiments, the TCR sequence can be modified to include a minimum mouse humanized Cα region and Cβ region.

[0157] Some embodiments relate to an isolated TCR as described herein, wherein the TCR is a single chain type, wherein the TCR alpha chain and the TCR beta chain are connected by a linker sequence.

[0158] A suitable single-chain TCR format comprises a first segment consisting of an amino acid sequence corresponding to a variable TCR α region, a second segment consisting of an amino acid sequence corresponding to a variable TCR β region, an N-terminal fusion of the variable TCR β region and an amino acid sequence corresponding to an extracellular sequence of a constant region of the TCR β chain, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment. Alternatively, the first segment may be composed of an amino acid sequence corresponding to a variable region of the TCR β chain, the second segment may be composed of an amino acid sequence corresponding to a variable region sequence of the TCR α chain, and the N-terminal fusion of the variable region sequence of the TCR α chain and an amino acid sequence corresponding to an extracellular sequence of a constant region of the TCR α chain. The above-mentioned single-chain TCR may further comprise a disulfide bond between the first chain and the second chain, and wherein the length of the linker sequence and the position of the disulfide bond are such that the variable domain sequences of the first segment and the second segment are substantially oriented to each other as in a natural TCR. More specifically, the first segment may be composed of an amino acid sequence corresponding to a TCR α chain variable region sequence, which is fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a TCR α chain constant region, and the second segment may be composed of an amino acid sequence corresponding to a TCR β variable region, which is 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 chain and the second chain. The linker sequence may be any sequence that does not impair the function of the TCR.

[0159] In the context of the present invention, a "functional" TCR alpha and / or beta chain fusion protein should refer to a TCR or TCR variant that at least retains basic biological activity, for example, modified by the addition, deletion or substitution of amino acids. For the alpha and / or beta chains of the TCR, this means that both chains are still able to form a T cell receptor (with an unmodified alpha chain and / or beta chain or with another fusion protein alpha chain and / or beta chain of the present invention), which exerts its biological function, in particular binding to a specific peptide-MHC complex of the TCR, and / or functional signal transduction after a specific peptide:MHC interaction.

[0160] In a specific embodiment, the TCR can be modified to a functional TCR alpha and / or beta chain fusion protein, wherein the length of the epitope tag is 6 to 15 amino acids, preferably 9 to 11 amino acids. In another embodiment, the TCR can be modified to a functional TCR alpha and / or beta chain fusion protein, wherein the TCR alpha and / or beta chain fusion protein comprises two or more epitope tags spaced or directly connected in series. The embodiment of the fusion protein may comprise 2, 3, 4, 5 or even more epitope tags, as long as the fusion protein maintains its biological activity / multiple activities ("functionality").

[0161] Preferred is a functional TCR alpha and / or beta chain fusion protein according to the present invention, wherein the epitope tag is selected from but not limited to CD20 or Her2 / neu tags or other conventional tags such as myc tags, FLAG tags, T7 tags, HA (hemagglutinin) tags, His tags, S tags, GST tags or GFP tags. Myc, T7, GST, GFP tags are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, for example, U.S. Patent Nos. 4,703,004 and 4,851,341). The myc tag can be preferably used because high-quality reagents can be used for its detection. In addition to being recognized by antibodies, epitope tags can certainly 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.

[0162] TCR fragments and variants

[0163] 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 the following 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.

[0164] In specific embodiments, the polypeptide may be a functional portion of a TCR alone, for example in a soluble form. Alternatively, the polypeptide may be combined with other domains.

[0165] The functional portion can mediate the binding of the TCR to an antigen, particularly to an antigen-MHC complex.

[0166] In one embodiment, the functional portion comprises a TCR alpha variable chain and / or a TCR beta variable chain as described herein.

[0167] TCR variant molecules (i.e., molecules that combine polypeptides comprising functional portions of TCR and other domains) can have the binding properties of TCR receptors, but can be combined with signaling domains of effector cells (other than T cells), in particular, with signaling domains of NK cells. Therefore, some embodiments relate to proteins comprising a combination of a functional portion of a TCR as described herein with a signaling domain of an effector cell (e.g., NK cell).

[0168] "Binding" refers to the ability to specifically and non-covalently associate, bind or bind to a target.

[0169] Another aspect of the present 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 connected by a linker portion to form a multivalent complex. Preferably, the complex is water-soluble, so the linker portion should be selected accordingly. Preferably, the linker portion can be connected to a defined position on the TCR molecule, thereby minimizing the structural diversity of the complex formed. An embodiment of this aspect is provided by the TCR complex of the present invention, wherein the polymer chain or peptide linker sequence extends between the amino acid residues in the variable region sequence of each TCR that are not located in the TCR. Since the complex of the present invention can be used in medicine, the selection of the linker portion should be appropriately based on its pharmaceutical suitability, such as its immunogenicity. Examples of linker portions that meet the above-mentioned desired standards are known in the art, such as in the field of connecting antibody fragments.

[0170] Examples of linkers are hydrophilic polymers and peptide linkers. Examples of hydrophilic polymers are polyalkylene glycols. The most commonly used 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. Peptide linkers comprise amino acid chains and act on to produce simple linkers or multimeric domains on which TCR molecules can be connected.

[0171] One embodiment relates to a multivalent TCR complex wherein at least one of the TCRs is associated with a therapeutic agent.

[0172] Cytokine and chemokine release

[0173] 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 the TCR of the invention expressed on effector cells to the amino acid sequence of SEQ ID NO: 2 presented by the HLA-A*02:01 encoded molecule.

[0174] In 10 -7 [M] HA-1 peptide concentration, when the binding to the amino acid sequence of SEQ ID NO:2 presented by the HLA-A*02:01 encoded molecule is compared to the binding to SEQ ID NO:4 presented by the HLA-A*02:01 encoded molecule, the IFN-γ secretion induced by the binding of the TCR of the present invention expressed on effector cells to the amino acid sequence of SEQ ID NO:2 presented by the HLA-A*02:01 encoded molecule can be at least 3 times higher, preferably at least 10 times higher, more preferably at least 20 times higher, even more preferably at least 50 times higher, and most preferably at least 100 times higher.

[0175] In 10 -6 [M] HA-1 peptide concentration, when the binding to the amino acid sequence of SEQ ID NO:2 presented by the HLA-A*02:01 encoded molecule is compared to the binding to SEQ ID NO:4 presented by the HLA-A*02:01 encoded molecule, the IFN-γ secretion induced by the binding of the TCR of the present invention expressed on effector cells to the amino acid sequence of SEQ ID NO:2 presented by the HLA-A*02:01 encoded molecule can be at least 3 times higher, preferably at least 10 times higher, more preferably at least 20 times higher, even more preferably at least 50 times higher, and most preferably at least 100 times higher.

[0176] In 10 -5 [M] HA-1 peptide concentration, when the binding to the amino acid sequence of SEQ ID NO:2 presented by the HLA-A*02:01 encoded molecule is compared to the binding to SEQ ID NO:4 presented by the HLA-A*02:01 encoded molecule, the IFN-γ secretion induced by the binding of the TCR of the present invention expressed on effector cells to the amino acid sequence of SEQ ID NO:2 presented by the HLA-A*02:01 encoded molecule can be at least 3 times higher, such as at least 10 times higher, preferably at least 10 times higher, for example at least 20 times higher, at least 50 times higher, at least 100 times higher.

[0177] Therefore, for all HA-1 H -TCR transgenic T cells, to recognize HA-1 R Variant, requires HA-1 H The concentration of the peptide is at least 1,000 times, preferably at least 5,000 times, more preferably at least 8,000 times, most preferably at least 10,0000 times higher than that of the peptide.

[0178] In a specific embodiment, for example, when the ratio of TCR transgenic T cells to T2 cells is 2:1, at 10 -7 At an HA-1 peptide concentration of [M], the IFN-γ secretion induced by the binding of the TCR of the present invention expressed on the effector cells to the amino acid sequence of SEQ ID NO: 2 presented by the HLA-A*02:01 encoded molecule can be greater than 500 pg / ml, for example, greater than 1000 pg / ml.

[0179] pg / ml, more preferably greater than 2000pg / ml, most preferably greater than 3000pg / ml.

[0180] "Effector cells" may be peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). Typically, effector cells are immune effector cells, particularly T cells. Other suitable cell types include γ-δ T cells and NK-like T cells.

[0181] The present invention also relates to a method for identifying a TCR or a fragment thereof that binds to a target amino acid sequence of SEQ ID NO:2 or an HLA-A2 bound form thereof, preferably presented by an HLA-A*02:01 encoded molecule or an HLA-A*02:06 encoded molecule, preferably SEQ ID NO:2 presented by an HLA-A*02:01 encoded molecule or an HLA-A*02:06 encoded molecule, wherein the method comprises contacting a candidate TCR or a fragment thereof with the amino acid sequence of SEQ ID NO:2 or an HLA-A02 bound form thereof, preferably presented by an HLA-A*02:01 encoded molecule or an HLA-A*02:06 encoded molecule, preferably SEQ ID NO:2 presented by an HLA-A*02:01 encoded molecule, and determining whether the candidate TCR or a fragment thereof binds to the target and / or mediates an immune response.

[0182] Whether a candidate TCR or its fragment 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, wherein K562 cells (or other APCs) transfected with ivtRNA encoding the amino acid sequence SEQ ID NO: 2 are incubated with CD8+ enriched PBMCs expressing the TCR or a molecule containing the TCR fragment to be studied.

[0183] Nucleic acid, vector

[0184] 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.

[0185] Table 2: Nucleotide sequences encoding various peptide sequences

[0186]

[0187]

[0188]

[0189] "Nucleic acid molecule" generally refers to a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained from a natural source (e.g., isolated and / or purified), which can include natural, non-natural or altered nucleotides, and which can include natural, non-natural or altered internucleotide connections, such as phosphoramidate connections or phosphorothioate connections, rather than phosphodiester between nucleotides of unmodified oligonucleotides. Preferably, nucleic acid as described herein is recombinant. As used herein, the term "recombinant" refers to (i) molecules constructed outside living cells by connecting natural or synthetic nucleic acid fragments to nucleic acid molecules that can be replicated in living cells, or (ii) molecules obtained by the replication of those described in (i) above. For the purposes of this article, replication can be in vitro replication or in vivo replication. 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 similar companies such as Genscript, Thermo Fisher). See, e.g., Sambrook et al. 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 after hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Nucleic acids can comprise any nucleotide sequence encoding any recombinant TCR, polypeptide, or protein, or a functional portion or functional variant thereof.

[0190] The present disclosure also provides variants of isolated or purified nucleic acids, wherein the nucleic acid variants comprise 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 a nucleotide sequence encoding a TCR described herein. Such variant nucleotide sequences encode a nucleotide sequence that specifically recognizes HA-1 H Functional TCR for antigen.

[0191] The present disclosure also provides isolated or purified nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0192] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. "High stringency conditions" refer to nucleotide sequences and target sequences (nucleotide sequences of any nucleic acid described herein) with a more detectable amount specific hybridization than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides with precise complementary sequences or polynucleotides containing only a few scattered mismatches from random sequences that happen to have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such complementary small regions are more easily melted than the full-length complementation of 14-17 or more bases, and high stringency hybridization makes it easy to distinguish. Relatively high stringency conditions include, for example, low salt and / or high temperature conditions, such as conditions provided by about 0.02-0.1M NaCl or equivalents at a temperature of about 50-70°C. Such high stringency conditions allow very few (if any) mismatches between nucleotide sequences and template strands or target strands, and are particularly suitable for detecting the expression of any TCR described herein. It is generally believed that conditions can be made more stringent by adding an increased amount of formamide.

[0193] As described elsewhere herein, the nucleic acid encoding the TCR can be modified. Useful modifications in the entire nucleic acid sequence can be codon optimization. Conservative substitution changes that result in the expressed amino acid sequence can be made. These changes that do not affect function can be made in the complementary determining regions and non-complementary determining regions of the TCR chain amino acid sequence. Generally, additions and deletions should not be made in the CDR3 region.

[0194] Another embodiment relates to a vector comprising a nucleic acid encoding a TCR as described herein.

[0195] The vector is preferably a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, an adenoviral vector or a particle and / or a vector for gene therapy.

[0196] "Vector" is any molecule or composition with the ability to carry a nucleic acid sequence into a suitable host cell, in which the synthesis of the encoded polypeptide can occur. Generally and preferably, a vector is a nucleic acid that has been engineered to introduce a desired nucleic acid sequence (e.g., nucleic acid of the present 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, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, a lentiviral vector, an adenoviral vector, or a particle and / or a vector for gene therapy. The vector may comprise a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. The vector may also comprise one or more selective 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 present invention, and the nucleic acid is operably linked to a sequence that allows the expression of the nucleic acid.

[0197] Preferably, the vector is an expression vector. More preferably, the vector is a retrovirus, more particularly a γ-retrovirus or a lentivirus vector.

[0198] Cells and cell lines

[0199] Another aspect of the invention relates to a cell expressing a TCR as described herein.

[0200] In some embodiments, the cell is isolated or non-naturally occurring.

[0201] In specific embodiments, the cell may comprise a nucleic acid encoding a TCR as described herein or a vector comprising the nucleic acid.

[0202] In cells, the above-mentioned vector containing a nucleic acid sequence encoding the above-mentioned TCR can be introduced, or ivtRNA encoding the TCR can be introduced. The cell can be a peripheral blood lymphocyte, such as a T cell. The cloning and exogenous expression methods of TCR are described, for example, in Engels et al. (Relapse or eradication of cancer is predicted by peptide-majorhistocompatibility complex affinity. Cancer Cell, 23 (4), 516–26. 2013). The transduction of primary human T cells with lentiviral vectors is described, for example, in Cribbs "simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells" BMC Biotechnol. 2013; 13: 98.

[0203] The terms "transfection" and "transduction" are interchangeable and refer to the process of introducing exogenous nucleic acid sequences into a host cell (e.g., a eukaryotic host cell). Note that the introduction or transfer of nucleic acid sequences is not limited to the methods mentioned, but can be achieved by any number of means, including electroporation, microinjection, gene gun delivery, lipofection, superfection, and infection by retrovirus or other viruses suitable for transduction or transfection.

[0204] Some embodiments relate to a cell comprising:

[0205] a) an expression vector comprising at least one nucleic acid as described herein, or

[0206] 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.

[0207] In some embodiments, the cell is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The cell can be a natural killer cell or a T cell. Preferably, the cell is a T cell. The T cell can be a CD4+ or CD8+ T cell.

[0208] In some embodiments, the cell is a stem cell-like memory T cell.

[0209] Stem cell-like memory T cells (TSCM) are subpopulations with a low degree of differentiation of CD8+T cells, with the ability of self-renewal and long-term persistence. 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 TSCM remain static (Flynn et al., Clinical & Translational Immunology (2014)). These remaining TSCM cells show the ability to establish persistent immune memory in vivo, and are therefore considered to be important T cell subsets for adoptive T cell therapy (Lugli et al., Nature Protocols 8, 33–42 (2013) Gattinoni et al., Nat. Med. October 2011; 17 (10): 1290–1297). Immuno-magnetic selection can be used to limit the T cell pool to stem cell memory T cell subtypes, see (Riddell et al. 2014, Cancer Journal 20 (2): 141–44).

[0210] Antibodies targeting TCR

[0211] Another aspect of the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to a portion of a TCR described herein that mediates the binding to HA-1 H In one embodiment, TCR-mediated HA-1 H The specific portion 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 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.

[0212] Antibodies or antigen-binding fragments thereof can modulate the activity of TCR. They may or may not block the binding of TCR to HA-1 H It can be used to modulate the therapeutic activity of TCR or for diagnostic purposes.

[0213] Pharmaceutical compositions, drug treatments and kits

[0214] Another aspect of the present 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 the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or an antibody that specifically binds to a portion of the TCR described herein.

[0215] Those active ingredients of the present invention are preferably used in such pharmaceutical compositions in a dosage mixed with an acceptable carrier or carrier material, so that the disease can be treated or at least alleviated. Such compositions may (in addition to the active ingredient and carrier) contain filler materials, salts, buffers, stabilizers, solubilizers and other materials, which are known in the prior art.

[0216] The term "pharmaceutically acceptable" defines a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The choice of carrier depends on the application.

[0217] The pharmaceutical composition may contain additional ingredients that enhance the activity of the active ingredient or supplement the treatment. 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.

[0218] Techniques for formulation or preparation and administration / dosing of the active ingredients of the invention are disclosed in the latest edition of "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA. Suitable administration is parenteral administration, such as intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injection. Intravenous injection is the preferred treatment for patients.

[0219] According to a preferred embodiment, the pharmaceutical composition is an infusion or an injection.

[0220] Injectable compositions are pharmaceutically acceptable fluid compositions comprising at least one active ingredient, such as a population of amplified T cells expressing TCR (e.g., autologous or allogeneic for the patient to be treated). The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition may additionally comprise a small amount of one or more non-toxic auxiliary substances, such as emulsifiers, preservatives, and pH buffers, etc. Such injectable compositions that can be used 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.

[0221] Typically, pharmaceutical compositions comprise at least one pharmaceutically acceptable carrier.

[0222] Therefore, another aspect of the present invention relates to 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 the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or an antibody that specifically binds to a portion of the TCR as described herein, for use as a drug.

[0223] Some embodiments relate to 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 the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, for use in treating cancer.

[0224] In one embodiment, the cancer is a hematological cancer.

[0225] Hematological cancers, also known as blood cancers, do not form solid tumors and are therefore primarily dispersed throughout the body.

[0226] The hematological cancer may be selected from the group consisting of: non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), multiple myeloma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell polymorphic 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, 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. Other hematological cancers or diseases include myelodysplasia.

[0227] 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 modified to express a recombinant TCR as described herein, such as a T cell or NK cell; (iv) a nucleic acid encoding a recombinant TCR as described herein. In some embodiments, the present disclosure provides a composition comprising a lentiviral vector particle containing a nucleotide sequence encoding a recombinant TCR as described herein (or a T cell that has been modified using a vector particle as described herein to express a recombinant TCR). Such a composition can be administered to a subject using the methods of the present disclosure as further described herein.

[0228] Compositions comprising the modified T cells described herein can be used in methods and compositions for adoptive immunotherapy according to known techniques or variations thereof that will be apparent to those skilled in the art based on this disclosure.

[0229] In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells in a therapeutically effective amount in a medium or container system suitable for administration (a "pharmaceutically acceptable" carrier). A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% dextrose in water or Ringer's lactate can also be used. The infusion medium can be supplemented with human serum albumin.

[0230] The number of cells in the composition for effective treatment is generally greater than 10 cells, and up to 10 6 up to and including 10 8 or 10 9 cells, and can be greater than 10 10 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, which may be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually greater than 10 8 Clinically relevant numbers of immune cells can be distributed over multiple infusions, accumulating to be equal to or greater than 10 9 , 10 10 or 10 11 Cells. The pharmaceutical compositions provided herein can be in various 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 can be formulated by conventional methods and can be administered to a subject at a suitable dose. Stabilizers such as lipids, nuclease inhibitors, polymers and chelating agents can protect the composition from degradation in vivo. In compositions intended to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents may be included.

[0231] The recombinant TCR described herein or the viral vector particles comprising the nucleotide sequence encoding the recombinant TCR provided herein can be packaged into a test kit. The test kit may optionally include one or more components, such as instructions for use, devices and other reagents, and components, such as tubes, containers and syringes for implementing the method. An exemplary test kit may include nucleic acids, recombinant TCR polypeptides or viruses encoding the recombinant TCR provided herein, and may optionally include instructions for use, devices for detecting viruses in a subject, devices for applying the composition to a subject, and devices for applying the composition to a subject.

[0232] Also contemplated herein are kits comprising polynucleotides encoding a target gene (e.g., a recombinant TCR). Also contemplated herein are kits comprising a viral vector encoding a target sequence (e.g., a recombinant TCR) and optionally a polynucleotide sequence encoding an immune checkpoint inhibitor.

[0233] The kit contemplated herein also includes a kit for implementing a method for detecting the presence of a polynucleotide encoding any one or more TCR disclosed herein. Specifically, such a diagnostic kit may include a suitable amplification and detection primer set and other related reagents for performing deep sequencing to detect the polynucleotide encoding the TCR disclosed herein. In other embodiments, the kit herein may include a reagent for detecting TCR disclosed herein, such as an antibody or other binding molecules. The diagnostic kit may also include instructions for determining the presence of a polynucleotide encoding the TCR disclosed herein or for determining the presence of the TCR disclosed herein. The kit may also include instructions. Instructions generally include descriptions of the following tangible expressions: the components included in the kit, and methods of administration, including methods for determining the appropriate state of a subject, suitable dosage, and suitable methods of administration. Instructions may also include guidance for monitoring a subject during treatment.

[0234] The kits provided herein may also include a device for administering the compositions described herein to a subject. Any of a variety of devices known in the art for administering a drug or vaccine may 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 eyedroppers. Typically, the device for administering the virus of the kit will be compatible with the virus of the kit; for example, a needleless injection device such as a high-pressure injection device may be included in a kit containing a virus that is not destroyed by high-pressure injection, but is generally not included in a kit containing a virus that is destroyed by high-pressure injection.

[0235] 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 the various devices known in the art for administering a drug to a subject may be included in the kit provided herein. Exemplary devices include a hypodermic needle, an intravenous needle, a catheter, a needle-free injection, but are not limited to a hypodermic needle, an intravenous needle, a catheter, a needle-free injection device, an inhaler, and a liquid dispenser, such as a dropper. Typically, the device for administering the compound of the kit will be compatible with the desired method of administering the compound.

[0236] experiment

[0237] Example:

[0238] Example 1: HA-1 H -TCR transgenic T cells bind HA-1 H Polymer

[0239] An in vitro priming method was used to isolate HA-1 HThe priming system uses mature dendritic cells (mDC) from HLA-A*02:01 positive donors as antigen presenting cells and autologous CD8 + Enriched T cells serve as responder cells. In vitro transcribed RNA (ivtRNA) encoding 31 amino acids of the human HMHA1 gene (ARFAEGLEKLKECVLHDDLLEARRPRAHECL; SEQ ID NO: 126) was used as a source of specific antigen. After electroporation into mDC, the HMHA1 encoding ivtRNA was translated into protein, which was subsequently processed and presented as a peptide by the HLA-A*02:01 molecule on the mDC. In vitro co-culture of T cells with mDC transfected with ivtRNA from the same donor resulted in the de novo induction of antigen-specific T cells, which served as a source of corresponding TCRs. Antigen-specific T cells can be enriched by a variety of methods and cloned by limiting dilution or FACS-based single-cell sorting. HA-1 was sequenced by next-generation sequencing. H The sequences of the TCR alpha and TCR beta chains of reactive T cell clones were identified and cloned into the retroviral vector pES.12-6 after the constant TCR region was exchanged with its mouse counterpart. PBMCs from healthy donors were isolated by ficoll gradient centrifugation. CD8 T cells were enriched by negative magnetic selection (Miltenyi) and stimulated with anti-CD3 and anti-CD28 mAb (BD Pharmingen, Heidelberg, Germany) in non-tissue culture 24-well plates. Double-tropic retroviral particles were produced by transfecting HEK293T cells with the corresponding TCR encoding retroviral plasmids and two expression plasmids. The next day after stimulation, CD8 T cells were transduced, and the transduced CD8 were enriched by FACS using the mouse constant beta region as a transduction marker on the twelfth day. + The cells were then expanded using a rapid expansion protocol (Riddell SR, Science, 1992 Jul 10; 257(5067):238-41).

[0240] result:

[0241] Use from HA-1 H Six different TCRs and one that failed to recognize HA-1 were isolated from reactive T cell clones H CD8 T cells were transduced with a control TCR. GeneOptimizer TM The nucleotide sequence of the isolated TCR was codon optimized using the PCR algorithm (ThermoFisher), and the constant region was murinized. The codon-optimized and murinized TCR was used for all other experiments described herein. H- MHC multimers (immunAware) and staining with antibodies against CD8 and mouse constant β region. All HA-1 except one H - TCR transgenic T cell populations all bind HA-1 very efficiently H -MHC multimers (>90%); only TCR_6 showed a lower percentage of HA-1 H -MHC multimer positive cells (37%). No HA-1 was observed in the control TCR H -MHC multimer staining. These results indicate that from HA-1 H TCRs isolated from reactive T cell clones can be expressed transgenically in T cells from healthy donors. Figure 1 )

[0242] Example 2: HA-1 H -TCR transgenic T cells recognize HA-1 H Positive target cells

[0243] Confirm HA-1 of TCR transgenic T cells according to the following protocol H Specificity:

[0244] Towards T2 cells (HLA-A*02:01 阳 ) Load saturation (10 -5 M) HA-1 H peptide (SEQ ID NO: 2) or control peptide Astn1 P1268L (KLYGLDWAEL). H K562 cells were transduced with a portion of the HMHA1 gene that expressed an epitope. K562 cells transduced with only HLA-A*02:01 were used as controls. Each target cell line and TCR transgenic T cells were co-cultured with 20,000 T cells and 10,000 target cells at a ratio of 2:1. After 20-24 hours, the IFN-γ concentration in the co-culture supernatant was analyzed by standard sandwich ELISA (BD human IFN-γ ELISA device).

[0245] result:

[0246] HA-1 H -TCR transgenic T cells recognize HA-1 H T cells expressing the control TCR only recognized T2 cells loaded with the control peptide. These results suggest that HA-1 H TCRs isolated from reactive T cell clones were functional after transfer into T cells from healthy donors. Figure 2 )

[0247] Example 3: HA-1 H -TCR transgenic T cells cannot recognize HA-1 R -Peptide target cells

[0248] In order to analyze the two variants of HA-1 H and HA-1 R The differences in HA-1 H - Functional avidity of TCR transgenic T cells for the two variants loaded on HLA-A*02:01:

[0249] T2 cells were externally loaded with different concentrations (10 -11 M-10 -5 M) HA-1 H -peptide and (10 -8 M-10 -5 M) HA-1 R -peptide and co-cultured with TCR transgenic T cells using 20,000 T cells and 10,000 T2 cells at a ratio of 2: 1. After 20-24 hours, the IFN-γ concentration in the co-culture supernatant was analyzed by standard sandwich ELISA (BD human IFN-γ ELISA instrument).

[0250] result:

[0251] TCR_3 and TCR_4 showed binding to HA-1 loaded on HLA-A*02:01 encoding molecules. H -peptide showed the highest functional affinity, and TCR_2 showed the lowest functional affinity. At non-physiological high peptide concentrations (10 -5 M) were observed to have some HA-1 H -TCR transgenic T cells against HA-1 R There are only very few recognitions, and for all HA-1 H -TCR transgenic T cells, to recognize HA-1 R - variant, needs to be relative to HA-1 H These results suggest that HA-1 H -TCR for HA-1 H - variants are highly specific. In addition, the HA-1 loaded on the HLA-A*02:01 encoding molecule R The functional affinity of the peptide was compared with the TCR described in WO2018058002A1 (TCR2). In contrast to the TCR described herein, this TCR also -6 M concentration to recognize HA-1 R-peptide, and in 10 -5 M concentration, significantly more IFN-γ was released, indicating that all TCRs described herein have significantly higher specificity than the TCRs described in WO2018058002A1. Figure 3 A and 3B)

[0252] Example 4: HA-1 H -TCR transgenic T cells recognize HA-1 H Positive LCL

[0253] To analyze HA-1 H -TCR transgenic T cells recognize physiological levels of HA-1 on unmodified target cells H To test the ability of T cells to express T cells, we co-cultured T cells with different lymphoblastoid cell lines (LCLs) and analyzed the cytokine release and cytotoxicity of TCR transgenic T cells:

[0254] DNA of seven LCLs was isolated (Quick Extract DNA Extract Solution, Illumina), and a portion of the HMHA1 gene encoding HA-1 was amplified by PCR. PCR products were separated from agarose gels and sent for sequencing (Eurofins Genomics). Based on the sequencing results, LCLs were assigned to HA-1. H Positive (HA-1 H / H and HA-1 H / R ) or HA-1 H Negative (HA-1 R / R ) group. To analyze cytokine release, 20,000 T cells and 10,000 T2 cells were used to co-culture TCR transgenic T cells and LCLs at a ratio of 2:1. After 20-24 hours, IFN-γ concentrations in the co-culture supernatants were analyzed by standard sandwich ELISA (BD human IFN-γ ELISA device). For cytotoxicity assays, co-cultures were established using 100,000 TCR transgenic T cells that had been transduced with a fluorescent marker gene and 20,000 LCLs at an effector to target ratio of 5:1. Live cell monitoring ( The reduction of fluorescent target cells (total integrated intensity in RCU × μm) was measured every two hours over a total period of 20 hours. 2 / image, RCU=Red Calibration Unit).

[0255] result:

[0256] All HA-1 H -TCR transgenic T cells in the presence of HA-1 HPositive LCL (LCL 1-5) released IFN-γ when co-cultured, but did not release IFN-γ when co-cultured with HA-1 H Negative LCL (LCL 6-7) after co-culture. T cells expressing control TCR did not recognize any LCL. In addition, HA-1 H Positive LCL was stained with HA-1 H -TCR transgenic T cells lysed. Untransduced CD8 T cells were used as a negative control and did not lyse LCLs. These results suggest that HA-1 H -TCR can recognize HA-1 loaded on HLA-A*02:01 encoded molecules at physiological levels H . ( Figure 4A and 4B )

[0257] Example 5: HA-1H-TCR transgenic T cells recognize HA-1H positive tumor cell lines

[0258] To analyze HA-1 H -TCR transgenic T cells recognize HA-1 presented on tumor cell lines H The ability of T cells to bind to different HA-1 H Co-culture of positive tumor cell lines.

[0259] DNA from the tumor cell line was isolated (Quick Extract DNA Extract Solution, Illumina), and a portion of the HMHA1 gene encoding HA-1 was amplified by PCR. The PCR product was isolated from an agarose gel and extracted from the gel for subsequent sequencing (Eurofins Genomics). H Positive (HA-1 H / H and HA-1 H / R ) tumor cell lines were used for the experiments. To analyze cytokine release, 20,000 T cells and 10,000 T2 cells were used to co-culture TCR transgenic T cells and tumor cell lines at a ratio of 2:1. After 20-24 hours, the IFN-γ concentration in the co-culture supernatant was analyzed by standard sandwich ELISA (BD human IFN-γ ELISA device). For cytotoxicity assays, co-cultures were established using 100,000 TCR transgenic T cells that had been transduced with a fluorescent marker gene and 20,000 tumor cells at an effector to target ratio of approximately 5:1. Live cell monitoring ( The reduction in fluorescent target cells (total integrated intensity in RCU x μm) was measured every two hours over a total period of 20 hours (ZOOM). 2 / image, RCU=Red Calibration Unit).

[0260] result:

[0261] When co-cultured with tumor cell lines, all HA-1 except TCR_2 H -TCR transgenic T cells all release IFN-γ. T cells expressing control TCR do not recognize tumor cell lines. HA-1 H -TCR transgenic T cells also showed cytotoxic activity against tumor cell lines. Consistent with the cytokine release data, TCR_2 lysed target cells with lower efficiency. Untransduced CD8 T cells were used as negative control. ( Figure 5A and 5B )

[0262] Example 6: T cells expressing TCR_3 and TCR_4 recognize HA-1 at levels comparable to previously described TCRs H Positive tumor cell lines

[0263] In order to H -TCR transgenic T cells against HA-1 H Identification of positive tumor cell lines was compared with T cells expressing TCR2 described in WO2018058002A1, and 20,000 T cells and 10,000 tumor cells were co-cultured. After 20 hours, the IFN-γ concentration in the co-culture supernatant was analyzed by standard sandwich ELISA (BD human IFN-γ ELISA device). For cytotoxicity assays, 10,000 HA-1 H 10,000 cells of negative / GFP-positive K562 cells and tumor cell lines expressing mCherry were co-cultured with 20,000 TCR transgenic T cells. After 45 hours, samples were analyzed by flow cytometry and mCherry-positive (HA-1 H positive) and GFP positive (HA-1 H The ratio of TCR-negative cells was normalized to that measured for control TCR.

[0264] result:

[0265] In the case of HA-1 H After co-culture with positive tumor cell lines, T cells expressing TCR_3 and TCR_4 released similar amounts of IFN-γ as T cells expressing TCR2 described in WO2018058002A1 ( Figure 6 A). T cells expressing TCR_3 and TCR_4 also showed cytotoxic activity against different tumor cell lines comparable to that of TCR2 ( Figure 6 B).

[0266] Example 7: TCR recognition motif

[0267] In order to analyze the specific recognition motif of TCR, serine replacement scanning was performed. By replacing the original amino acid of the epitope with serine, the position in the peptide that is essential for TCR-mediated recognition can be identified. In order to define the recognition motif, effector T cells expressing different HA-1TCRs (TCR_3, TCR_4, and TCR2 described in WO2018058002A1) were co-cultured with T2 cells, and the T2 cells were loaded with HA-1H peptides (SEQID NO: 2) in which each individual amino acid residue was successively replaced by serine, or loaded with control peptides. T2 cells were loaded with saturated concentrations (10 -5 M) peptide, washed, and co-cultured with effector cells at an E:T ratio of 1: 1. The supernatant was harvested after about 20 hours of co-culture and secreted IFN-γ was analyzed by ELISA. T cells expressing a control TCR were used as a negative control.

[0268] result:

[0269] Compared with TCR2 (WO2018058002A1), TCR_3 and TCR_4 showed a more specific recognition pattern at serine, such as recognizing fewer peptides.

[0270] Comparison of different constant TCR regions

[0271] To compare the effects of different constant TCR regions, TCR-3 was cloned with murine, mouse humanized (Sommermeyer and Uckert, 2010, J. Immunol.) and human constant regions. HA-1 with different constant TCR regions was tested by co-culturing 20,000 T cells and 10,000 tumor cells. H -TCR transgenic T cells against HA-1 H - Recognition of TCR-positive tumor cell lines or LCLs. After 20-24 hours, the IFN-γ concentration in the co-culture supernatant was analyzed by standard sandwich ELISA (BD human IFN-γ ELISA kit). For cytotoxicity assays, 10,000 HA-1 H 10,000 cells of negative / GFP-positive K562 cells and tumor cell lines expressing mCherry were co-cultured with 20,000 TCR transgenic T cells. After 45 hours, samples were analyzed by flow cytometry and mCherry-positive (HA-1 H positive) and GFP positive (HA-1 H The ratio of TCR-negative cells was normalized to that measured for control TCR.

[0272] result:

[0273] As expected, HA-1 H - TCR transgenic T cells have slightly reduced responsiveness ( Figure 8 A and B). However, even with human constant TCR regions, in the presence of HA-1 H After co-culture with positive target cells, T cells still specifically released IFN-γ and showed cytotoxic effects.

[0274] The description also includes the following embodiments:

[0275] Embodiment 1. An isolated T cell receptor (TCR) specific for an allelic variant of minor histocompatibility antigen 1 (HA-1).

[0276] Embodiment 2. The isolated TCR according to embodiment 1, wherein the allelic variant of HA-1 is HA-1 H .

[0277] Embodiment 3. An isolated TCR according to any of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence SEQ ID NO: 2 or a fragment thereof.

[0278] Embodiment 4. An isolated TCR according to any of the preceding embodiments, wherein the TCR does not recognize the amino acid sequence SEQ ID NO: 4 or a fragment thereof.

[0279] Embodiment 5. An isolated TCR according to any of the preceding embodiments, wherein the recognition motif of the TCR comprises at least the sequence shown in SEQ ID NO:127.

[0280] Embodiment 6. An isolated TCR according to any of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 2 in an HLA-A2-bound form.

[0281] Embodiment 7. An isolated TCR according to any of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 2 presented by the HLA-A*02:01 encoded molecule.

[0282] Embodiment 8. An isolated TCR according to any of the preceding embodiments, wherein the TCR comprises a TCR α chain, the TCR α chain comprises a complementarity determining region 3 (CDR3), and the CDR3 has 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.

[0283] Embodiment 9. An isolated TCR according to any of the preceding embodiments, wherein the TCR comprises a TCRβ chain, the TCRβ chain comprises a CDR3, and the CDR3 has an amino acid sequence selected from the group consisting 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.

[0284] Embodiment 10. An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

[0285] 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,

[0286] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 8, a CDR2 having the amino acid sequence of SEQ ID NO: 9, and a CDR3 having the sequence of SEQ ID NO: 10; or

[0287] 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,

[0288] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 18, a CDR2 having the amino acid sequence of SEQ ID NO: 19, and a CDR3 having the sequence of SEQ ID NO: 20; or

[0289] 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,

[0290] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 28, a CDR2 having the amino acid sequence of SEQ ID NO: 29, and a CDR3 having the sequence of SEQ ID NO: 30;

[0291] 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,

[0292] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the sequence of SEQ ID NO: 40; or

[0293] 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,

[0294] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 48, a CDR2 having the amino acid sequence of SEQ ID NO: 49, and a CDR3 having the sequence of SEQ ID NO: 50; or

[0295] 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,

[0296] - A TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:58, a CDR2 having the amino acid sequence of SEQ ID NO:59 and a CDR3 having the sequence of SEQ ID NO:60.

[0297] Embodiment 11. An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises a TCR α chain and a TCR β chain, wherein

[0298] a) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 11, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 7,

[0299] - the variable TCR beta region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 12, and comprises a CDR3 region having the amino acid sequence shown in SEQ ID NO: 10; or

[0300] b) the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 21 and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 17,

[0301] - the variable TCR beta region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 22, and comprises a CDR3 region having the amino acid sequence shown in SEQ ID NO: 20; or

[0302] c) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:31, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:27;

[0303] - the variable TCR beta region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 32, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 30; or

[0304] d) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:41, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:37;

[0305] - the variable TCR beta region has an amino acid sequence that is 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

[0306] e) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:51, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:47;

[0307] - the variable TCR beta region has an amino acid sequence that is at least 80% identical to SEQ ID NO:52, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:50; or

[0308] f) - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:61, and comprises a CDR3 region having an amino acid sequence as shown in SEQ ID NO:57;

[0309] The variable TCRβ region has an amino acid sequence that is 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.

[0310] Embodiment 12. An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

[0311] a) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO: 11 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO: 12; or

[0312] b) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO: 21 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO: 22; or

[0313] c) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO: 31 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO: 32; or

[0314] d) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:41 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:42; or

[0315] e) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:51 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:52; or

[0316] f) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:61 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:62.

[0317] Embodiment 13: An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

[0318] a) a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 11 and a variable TCR beta region having the amino acid sequence of SEQ ID NO: 12; or

[0319] b) a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 21 and a variable TCR beta region having the amino acid sequence of SEQ ID NO: 22; or

[0320] c) a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 31 and a variable TCR beta region having the amino acid sequence of SEQ ID NO: 32; or

[0321] 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

[0322] e) a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 51 and a variable TCR beta region having the amino acid sequence of SEQ ID NO: 52; or

[0323] 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.

[0324] Embodiment 14. An isolated TCR according to any of the preceding embodiments, wherein the TCR is purified.

[0325] Embodiment 15. An isolated TCR according to any of the preceding embodiments, wherein the amino acid sequence of the TCR comprises one or more phenotypically silent substitutions.

[0326] Embodiment 16. An isolated TCR according to any of the preceding embodiments, wherein the amino acid sequence of the TCR is modified to include a detectable label, a therapeutic agent, or a pharmacokinetic modifying portion.

[0327] Embodiment 17. An isolated TCR according to Embodiment 16, wherein the therapeutic agent is selected from the group consisting of an immune effector molecule, a cytotoxic agent, and a radionuclide.

[0328] Embodiment 18. An isolated TCR according to embodiment 17, wherein the immune effector molecule is a cytokine.

[0329] Embodiment 19. An isolated TCR according to any of the preceding embodiments, wherein the TCR is soluble or membrane-bound.

[0330] Embodiment 20: An isolated TCR according to Embodiment 16, wherein the pharmacokinetic modifying moiety is at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group, or a combination thereof.

[0331] Embodiment 21. An isolated TCR according to any one of the preceding embodiments, wherein the TCR is a single-chain type, wherein the TCR α chain is connected to the TCR β chain via a linker sequence.

[0332] Embodiment 22. An isolated TCR according to any of the preceding embodiments, wherein the TCR α chain or the TCR β chain is modified to comprise an epitope tag.

[0333] Embodiment 23. An isolated TCR according to any of the preceding embodiments, wherein the recombinant TCR sequence can be modified to contain a most humanized Cα region and Cβ region.

[0334] Embodiment 24. An isolated polypeptide comprising a functional portion of a TCR according to 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.

[0335] Embodiment 25. An isolated polypeptide according to embodiment 24, wherein the functional portion comprises a TCR alpha variable chain and / or a TCR beta variable chain.

[0336] Embodiment 26. A multivalent TCR complex comprising at least two TCRs as embodied in any one of embodiments 1 to 23.

[0337] Embodiment 27. A multivalent TCR complex wherein at least one of the TCRs is associated with a therapeutic agent.

[0338] Embodiment 28. An isolated TCR according to embodiments 1 to 23, a polypeptide according to embodiments 24 and 25, a multivalent TCR complex 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 the HLA-A*02:01 encoded molecule.

[0339] 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.

[0340] Embodiment 30. The nucleic acid of embodiment 29, wherein the nucleic acid is codon optimized.

[0341] Embodiment 31. A vector comprising the nucleic acid of embodiment 29 or 30.

[0342] Embodiment 32. A vector according to embodiment 31, wherein the vector is an expression vector.

[0343] Embodiment 33. The vector of Embodiment 31 or 32, wherein the vector is a retroviral vector.

[0344] Embodiment 34. The vector of Embodiment 31 or 32, wherein the vector is a lentiviral vector.

[0345] Embodiment 35. A cell expressing a TCR according to embodiments 1 to 23.

[0346] Embodiment 36. A cell according to embodiment 34, wherein the cell is isolated or non-naturally occurring.

[0347] Embodiment 37. A cell comprising the nucleic acid according to embodiment 29 or 30 or the vector according to embodiments 31 to 35.

[0348] Embodiment 38. The cell of any one of embodiments 35 to 37, wherein the cell comprises:

[0349] a) an expression vector comprising at least one nucleic acid as embodied in embodiment 29 or 30,

[0350] b) a first expression vector comprising a nucleic acid encoding the α chain of the TCR as embodied in any one of embodiments 1 to 23, and a second expression vector comprising a nucleic acid encoding the β chain of the TCR as embodied in any one of embodiments 1 to 23.

[0351] 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).

[0352] Embodiment 40. A cell according to any one of embodiments 35 to 38, wherein the cell is a T cell.

[0353] Embodiment 41. An antibody or antigen-binding fragment thereof that specifically binds to a portion of the TCR according to embodiments 1 to 23, said portion mediating specificity for an allelic variant of HA-1.

[0354] Embodiment 42. An antibody or antigen-binding fragment thereof according to Embodiment 40, wherein the portion of the TCR that mediates specificity for an 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.

[0355] Embodiment 43. The antibody or antigen-binding fragment thereof according to Embodiment 41 or 42, wherein the allelic variant of HA-1 is H .

[0356] 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.

[0357] Embodiment 45: A pharmaceutical composition according to Embodiment 44, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

[0358] Embodiment 46: The TCR according to embodiments 1 to 23, the polypeptide according to embodiments 24 to 25, the multivalent TCR complex according to any one of embodiments 27 to 28, the nucleic acid according to embodiment 29 or 30, the vector according to embodiments 31 to 34, the cell according to any one of embodiments 34 to 39, or the antibody according to embodiments 41 to 43, for use as a medicament.

[0359] Embodiment 47: A TCR according to embodiments 1 to 23, a polypeptide according to embodiments 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.

[0360] 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.

[0361] Embodiment 49: The TCR, the polypeptide, the multivalent TCR complex, the nucleic acid or the cell for use according to Embodiment 45, wherein the hematological cancer is selected from the group consisting of non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), multiple myeloma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell polymorphic lymphoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), central nervous system lymphoma, C D37+ dendritic cell lymphoma, lymphoblastic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, extraskeletal 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, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma / leukemia, B-cell proliferation of unknown malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, and myelodysplasia. Sequence Listing <110> Gene Medical Immunotherapy, LLC <120> HA-1 specific T cell receptor and its use <130> M11350WO <150> EP18187539.4 <151> 2018-08-06 <160> 127 <170> PatentIn Version 3.5 <210> 1 <211> 1136 <212> PRT <213> Homo sapiens <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> Homo sapiens <400> 2 Val Leu His Asp Asp Leu Leu Glu Ala 1 5 <210> 3 <211> 1136 <212> PRT <213> Homo sapiens <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> Homo sapiens <400> 4 Val Leu Arg Asp Asp Leu Leu Glu Ala 1 5 <210> 5 <211> 6 <212> PRT <213> Homo sapiens <400> 5 Asp Ser Ala Ser Asn Tyr 1 5 <210> 6 <211> 7 <212> PRT <213> Homo sapiens <400> 6 Ile Arg Ser Asn Val Gly Glu 1 5 <210> 7 <211> 8 <212> PRT <213> Homo sapiens <400> 7 Cys Ala Ala Ser Asp Leu Asn Phe 1 5 <210> 8 <211> 5 <212> PRT <213> Homo sapiens <400> 8 Ser Glu His Asn Arg 1 5 <210> 9 <211> 6 <212> PRT <213> Homo sapiens <400> 9 Phe Gln Asn Glu Ala Gln 1 5 <210> 10 <211> 14 <212> PRT <213> Homo sapiens <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> Homo sapiens <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> Homo sapiens <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 murinized 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 murinized 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> Homo sapiens <400> 15 Asp Ser Ala Ser Asn Tyr 1 5 <210> 16 <211> 7 <212> PRT <213> Homo sapiens <400> 16 Ile Arg Ser Asn Val Gly Glu 1 5 <210> 17 <211> 13 <212> PRT <213> Homo sapiens <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> Homo sapiens <400> 18 Ser Glu His Asn Arg 1 5 <210> 19 <211> 6 <212> PRT <213> Homo sapiens <400> 19 Phe Gln Asn Glu Ala Gln 1 5 <210> 20 <211> 14 <212> PRT <213> Homo sapiens <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> Homo sapiens <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> Homo sapiens <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> twenty three <211> 268 <212> PRT <213> Artificial sequence <220> <223> Human with murinized constant region <400> twenty three 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 a 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> Homo sapiens <400> 25 Thr Ser Ile Asn Asn 1 5 <210> 26 <211> 7 <212> PRT <213> Homo sapiens <400> 26 Ile Arg Ser Asn Glu Arg Glu 1 5 <210> 27 <211> 14 <212> PRT <213> Homo sapiens <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> Homo sapiens <400> 28 Ser Glu His Asn Arg 1 5 <210> 29 <211> 6 <212> PRT <213> Homo sapiens <400> 29 Phe Gln Asn Glu Ala Gln 1 5 <210> 30 <211> 13 <212> PRT <213> Homo sapiens <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> Homo sapiens <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> Homo sapiens <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 a 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 a 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> Homo sapiens <400> 35 Asp Ser Ala Ser Asn Tyr 1 5 <210> 36 <211> 7 <212> PRT <213> Homo sapiens <400> 36 Ile Arg Ser Asn Val Gly Glu 1 5 <210> 37 <211> 9 <212> PRT <213> Homo sapiens <400> 37 Cys Ala Gly Arg Gly Lys Leu Thr Phe 1 5 <210> 38 <211> 5 <212> PRT <213> Homo sapiens <400> 38 Ser Glu His Asn Arg 1 5 <210> 39 <211> 6 <212> PRT <213> Homo sapiens <400> 39 Phe Gln Asn Glu Ala Gln 1 5 <210> 40 <211> 13 <212> PRT <213> Homo sapiens <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> Homo sapiens <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> Homo sapiens <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 murinized 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 a 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> Homo sapiens <400> 45 Ser Val Phe Ser Ser 1 5 <210> 46 <211> 7 <212> PRT <213> Homo sapiens <400> 46 Val Val Thr Gly Gly Glu Val 1 5 <210> 47 <211> 11 <212> PRT <213> Homo sapiens <400> 47 Cys Ala Gly Ala Gly Asn Asn Asp Met Arg Phe 1 5 10 <210> 48 <211> 5 <212> PRT <213> Homo sapiens <400> 48 Ser Glu His Asn Arg 1 5 <210> 49 <211> 6 <212> PRT <213> Homo sapiens <400> 49 Phe Gln Asn Glu Ala Gln 1 5 <210> 50 <211> 13 <212> PRT <213> Homo sapiens <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> Homo sapiens <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> Homo sapiens <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 murinized 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 murinized 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> Homo sapiens <400> 55 Asp Ser Ser Ser Thr Tyr 1 5 <210> 56 <211> 7 <212> PRT <213> Homo sapiens <400> 56 I'm so tired I'm so tired 1 5 <210> 57 <211> 8 <212> PRT <213> Homo sapiens <400> 57 Cys Ala Glu Lys Trp Ile Ile Phe 1 5 <210> 58 <211> 5 <212> PRT <213> Homo sapiens <400> 58 Ser Glu His Asn Arg 1 5 <210> 59 <211> 6 <212> PRT <213> Homo sapiens <400> 59 Phe Gln Asn Glu Ala Gln 1 5 <210> 60 <211> 13 <212> PRT <213> Homo sapiens <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> Homo sapiens <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> Homo sapiens <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 Murinized 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 murinized 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 Astn1 P1268L <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> twenty one <212> DNA <213> Artificial sequence <220> <223> Codon-optimized Homo sapiens sequence <400> 67 atcagatcca acgtgggcga g 21 <210> 68 <211> twenty four <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 tggggagccga 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 Murinized Constant Region (Codon Optimized) <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 (codon-optimized) with murine constant region <400> 75 atgggcacaa gcctgctgtg ttggatggcc ctgtgtctgc tgggagccga tcatgccgat 60 acgggagtgt ctcagaaccc cagacacaag atcaccaagc ggggccagaa cgtgaccttc 120 agatgcgacc ctatcagcga gcaaaccgg ctgtactggt agacagac actcggccag 180 ggacctgagt tcctgaccta cttccagaac gaggcccagc tggaaagag cagactgctg 240 agcgacagat tcagcgccga aagaccaag 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 aagagcaa 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> twenty one <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 tggggagccga tcatgccgat 60 acggggagtgt 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 murinized constant regions (codon optimized) <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 gcgaagaa 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 480 540 ctggacatga aggccatgga cagcaagtcc aacggcgcaa tcgcctggtc caaccagacc 600 agcttcacat gccaaggacat cttcaaagag acaaacgcca cataccccag cagcgacgtg 660 ccctgtgatg ccaccctgac agaaagtcc 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 (codon-optimized) with murine constant region <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> twenty one <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> Homo sapiens sequence, codon-optimized <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 Murinized Constant Region (Codon Optimized) <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 - derived constant region (codon - optimized) <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 attackgcg 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> twenty one <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 tggggagccga tcatgccgat 60 acggggagtgt 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 murinized constant regions (codon optimized) <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 Murinized Constant Region (Codon Optimized) <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> twenty one <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> Homo sapiens codon-optimized 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-optimized) <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 Murinized Constant Region (Codon Optimized) <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 attackgcg 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> twenty one <212> DNA <213> Artificial sequence <220> <223> Codon-optimized Homo sapiens sequence <400> 117 attttttcaa atatggacat g 21 <210> 118 <211> twenty four <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 tggggagccga tcatgccgat 60 acggggagtgt 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 murinized constant regions (codon optimized) <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 (codon-optimized) with murine constant region <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> Homo sapiens <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> Homo sapiens <400> 127 Asp Asp Leu Leu Glu 1 5

Claims

1. HA-1 H ) has a specific isolated T cell receptor (TCR), wherein the TCR comprises - a TCR alpha chain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 25, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 26, and a CDR3 having a sequence as shown in SEQ ID NO: 27, - A TCR β chain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:28, a CDR2 having an amino acid sequence as shown in SEQ ID NO:29, and a CDR3 having a sequence as shown in SEQ ID NO:

30.

2. The isolated TCR according to claim 1, wherein the TCR specifically recognizes the amino acid sequence SEQ ID NO:

2.

3. The isolated TCR according to claim 1, wherein the TCR does not recognize the amino acid sequence SEQ ID NO:

4.

4. The isolated TCR according to claim 1, wherein the recognition motif of the TCR comprises at least the sequence shown in SEQ ID NO:

127.

5. The isolated TCR of claim 1, wherein the TCR comprises: a) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:31 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:32, wherein the CDRs of the TCR are not modified.

6. The isolated TCR of claim 1, wherein the TCR comprises a) a variable TCR alpha region having the amino acid sequence shown in SEQ ID NO:31 and a variable TCR beta region having the amino acid sequence shown in SEQ ID NO:

32.

7. The isolated TCR of claim 1, wherein the TCR comprises a) a TCR alpha chain having an amino acid sequence as shown in SEQ ID NO:33 and a TCR beta chain having an amino acid sequence as shown in SEQ ID NO:

34.

8. An isolated polypeptide comprising a functional portion of the TCR according to any one of claims 1 to 7, wherein the functional portion comprises a variable TCR α region having an amino acid sequence as shown in SEQ ID NO: 31 and a variable TCR β region having an amino acid sequence as shown in SEQ ID NO:

32.

9. A multivalent TCR complex comprising the TCR according to any one of claims 1 to 7.

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

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

12. A host cell expressing the TCR according to any one of claims 1 to 7.

13. Use of the TCR according to any one of claims 1 to 7, the polypeptide according to claim 8, the multivalent TCR complex according to claim 9, the nucleic acid according to claim 10, the vector according to claim 11 or the cell according to claim 12 in the preparation of a medicament for treating cancer.

14. Use according to claim 13, wherein the cancer is a hematological cancer.

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