Chimeric antigen receptors targeting cd19 and uses thereof

Abstract

The presently disclosed subject matter provides chimeric antigen receptors (CARs) that specifically target CD19 and cells comprising such CARs that target CD19. The presently disclosed subject matter also provides the use of CARs that target CD19 for therapy, e.g., for the treatment of blood cancers.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 015,362, filed April 24, 2020, and U.S. Provisional Application No. 63 / 073,133, filed September 1, 2020, the contents of which are incorporated herein by reference in their entirety, and claims priority to each thereof. Technical Field

[0003] The subject matter of this disclosure provides a method for treating vegetations (e.g., cancer) using cell therapy containing a chimeric antigen receptor (CAR) that specifically targets CD19. Background Technology

[0004] Cell-based immunotherapy is a potentially curative treatment for cancer. T cells and other immune cells are modified to target tumor antigens by introducing genetic material encoding artificial or synthetic receptors that target the antigens. These receptors are called chimeric antigen receptors (CARs) and are specific to the selected antigens. CAR-based targeted T-cell therapy has recently shown clinical success in treating hematologic malignancies.

[0005] T cells expressing CD19-specific CARs with binding domains derived from mouse monoclonal antibodies have been shown to treat B-cell malignancies. T cell-mediated immune responses specific to the mouse scFv antigen-binding domain of CARs can occur in human patients, leading to premature elimination of CAR T cells and increasing the risk of tumor recurrence. Therefore, there is a need for improved CD19-targeting CARs with reduced toxicity and immunogenicity and / or enhanced safety and efficacy. Summary of the Invention

[0006] The subject matter of this disclosure provides a chimeric antigen receptor (CAR) targeting CD19, cells containing the CD19-targeting CAR, and the use of said cells for treatment, such as for treating vegetations.

[0007] In some embodiments, the CAR includes an extracellular antigen-binding domain specifically binding to CD19, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain includes a single-chain variable fragment (scFv), Fab, or F(ab)2. In some embodiments, the extracellular antigen-binding domain includes scFv. In some embodiments, the scFv is human scFv. In some embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region comprising CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or a conserved modification thereof, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or a conserved modification thereof, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or a conserved modification thereof; and / or (b) a light chain variable region comprising:

[0008] i) CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modification thereof, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modification thereof, and CDR3 containing the amino acid sequence shown in SEQ ID NO:13 or its conservative modification thereof;

[0009] ii) CDR1 containing the amino acid sequence shown in SEQ ID NO:16 or its conservative modification thereof, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modification thereof, and CDR3 containing the amino acid sequence shown in SEQ ID NO:17 or its conservative modification thereof;

[0010] iii) CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modification thereof, CDR2 containing the amino acid sequence shown in SEQ ID NO:20 or its conservative modification thereof, and CDR3 containing the amino acid sequence shown in SEQ ID NO:21 or its conservative modification thereof;

[0011] iv) CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modification thereof, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modification thereof, and CDR3 containing the amino acid sequence shown in SEQ ID NO:57 or its conservative modification thereof;

[0012] v) CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modification thereof, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modification thereof, and CDR3 containing the amino acid sequence shown in SEQ ID NO:59 or its conservative modification thereof;

[0013] vi) CDR1 containing the amino acid sequence shown in SEQ ID NO:16 or its conservative modification thereof, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modification thereof, and CDR3 containing the amino acid sequence shown in SEQ ID NO:61 or its conservative modification thereof;

[0014] vii) CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservatively modified form, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservatively modified form, and CDR3 containing the amino acid sequence shown in SEQ ID NO:63 or its conservatively modified form; or

[0015] viii) CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modification thereof, CDR2 containing the amino acid sequence shown in SEQ ID NO:65 or its conservative modification thereof, and CDR3 containing the amino acid sequence shown in SEQ ID NO:66 or its conservative modification thereof.

[0016] In some embodiments, the heavy chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9.

[0017] In some embodiments, the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:13.

[0018] In some embodiments, the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:17.

[0019] In some embodiments, the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:20, and CDR3 containing the amino acid sequence shown in SEQ ID NO:21.

[0020] In some embodiments, the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:57.

[0021] In some embodiments, the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:59.

[0022] In some embodiments, the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:61.

[0023] In some embodiments, the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:63.

[0024] In some embodiments, the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:65, and CDR3 containing the amino acid sequence shown in SEQ ID NO:66.

[0025] In some embodiments, the heavy chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:13.

[0026] In some embodiments, the heavy chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:17.

[0027] In some embodiments, the heavy chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:20, and CDR3 containing the amino acid sequence shown in SEQ ID NO:21.

[0028] In some embodiments, the heavy chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:57.

[0029] In some embodiments, the heavy chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:59.

[0030] In some embodiments, the heavy chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:61.

[0031] In some embodiments, the heavy chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:63.

[0032] In some embodiments, the heavy chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:65, and CDR3 containing the amino acid sequence shown in SEQ ID NO:66.

[0033] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or homology with the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10.

[0034] In some embodiments, the light chain variable region comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 96%, about 97%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or homology with the amino acid sequence shown in SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:67. In some embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:14, SEQ ID NO:18, or SEQ ID NO:22.

[0035] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or homology with the amino acid sequence shown in SEQ ID NO:10; and the light chain variable region comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or homology with the amino acid sequence shown in SEQ ID NO:10; and the light chain variable region comprises an amino acid sequence having at least 80%, about 81%, about 82%, about 83%, about 8 ...94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or homology with the amino acid sequence shown in SEQ ID NO:10; and the light chain variable region comprises an amino acid sequence having at least 80%, about 81%, about 82%, about 83%, about 84%, about 9 The amino acid sequence shown in NO:67 has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or homology. In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:67. In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:14, SEQ ID NO:18, or SEQ ID NO:22.

[0036] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:14.

[0037] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:18.

[0038] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:22.

[0039] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:58.

[0040] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:60.

[0041] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:62.

[0042] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:64.

[0043] In some embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:67.

[0044] In some embodiments, the extracellular antigen-binding domain includes a linker between the heavy chain variable region and the light chain variable region. In some embodiments, the linker consists of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the positions of the heavy chain variable region and the light chain variable region from the N-terminus to the C-terminus are: V L -V H .

[0045] In some embodiments, the extracellular antigen-binding domain includes or is composed of the scFv, which contains or consists of the amino acid sequence shown in SEQ ID NO:15, SEQ ID NO:19 or SEQ ID NO:23.

[0046] In some embodiments, the transmembrane domain comprises a CD8 peptide, CD28 peptide, CD3ζ peptide, CD4 peptide, 4-1BB peptide, OX40 peptide, ICOS peptide, CTLA-4 peptide, PD-1 peptide, LAG-3 peptide, 2B4 peptide, or BTLA peptide. In some embodiments, the transmembrane domain comprises a CD28 peptide.

[0047] In some embodiments, the intracellular signal transduction domain includes a CD3ζ polypeptide. In some embodiments, the CD3ζ polypeptide is a modified CD3ζ polypeptide. In some embodiments, the modified CD3ζ polypeptide comprises native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 consisting of two loss-of-function mutations. In some embodiments, native ITAM1 consists of the amino acid sequence shown in SEQ ID NO:31. In some embodiments, the ITAM2 variant consists of the amino acid sequence shown in SEQ ID NO:37. In some embodiments, the ITAM3 variant consists of the amino acid sequence shown in SEQ ID NO:41. In some embodiments, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:43.

[0048] In some embodiments, the intracellular signal transduction domain further includes at least one co-stimulatory signal transduction region. In some embodiments, the at least one co-stimulatory signal transduction region includes a CD28 peptide, a 4-1BB peptide, an OX40 peptide, an ICOS peptide, a DAP-10 peptide, or a combination thereof. In some embodiments, the at least one co-stimulatory signal transduction region includes an intracellular domain or a portion thereof of CD28, an intracellular domain or a portion thereof of 4-1BB, an intracellular domain or a portion thereof of OX40, an intracellular domain or a portion thereof of ICOS, or an intracellular domain or a portion thereof of DAP-10. In some embodiments, the at least one co-stimulatory signal transduction region includes a CD28 peptide.

[0049] In some implementations, CAR is expressed by a vector. In some implementations, the vector is a retroviral vector.

[0050] This disclosure also provides cells comprising the CAR disclosed herein. In some embodiments, the cells are transduced by the CAR. In some embodiments, the CAR is constitutively expressed on the cell surface. In some embodiments, the cells are immune-responding cells. In some embodiments, the cells are lymphoid or myeloid lineage cells. In some embodiments, the cells are selected from T cells, natural killer (NK) cells, stem cells from which lymphoid cells can differentiate, and stem cells from which myeloid cells can differentiate. In some embodiments, the cells are T cells. In some embodiments, the T cells are selected from helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. In some embodiments, the cells are NK cells. In some embodiments, the NK cells are derived from stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells.

[0051] Furthermore, the subject matter of this disclosure provides nucleic acid molecules encoding the CARs disclosed herein. In some embodiments, the nucleic acid molecule further comprises a promoter operatively linked to the CAR. The promoter may be endogenous or exogenous. In some embodiments, the promoter is an exogenous promoter. In some embodiments, the exogenous promoter is selected from the elongation factor (EF)-1 promoter, the cytomegalovirus immediate early promoter (CMV) promoter, the simian virus 40 early promoter (SV40) promoter, the phosphoglycerate kinase (PGK) promoter, the metallothionein promoter, and the ubiquitin C promoter. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the endogenous promoter is selected from the TCRα promoter, the TCRβ promoter, and the β2-microglobulin promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, IL-2 promoter, 4-1BB promoter, PD1 promoter, and LAG3 promoter.

[0052] This disclosure also provides vectors comprising the nucleic acid molecules disclosed herein. In some embodiments, the vector is a retroviral vector.

[0053] This disclosure also provides for cells expressing the nucleic acid molecules disclosed herein. In some embodiments, said cells are T cells or natural killer (NK) cells.

[0054] The subject matter of this disclosure provides compositions comprising the cells disclosed herein. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the composition comprises about 1 × 10⁻⁶ cells. 6 Approximately 5×10 8 Cells. In some embodiments, the composition contains about 1 × 10⁶ cells. 6 To approximately 1×10 8 Cells. In some embodiments, the composition contains about 1 × 10⁶ cells. 6 Approximately 5×10 7 10 cells. In some embodiments, the composition comprises about 2.5 × 10⁶ cells. 7 Each cell.

[0055] The subject matter of this disclosure also provides various methods for using the cells of this disclosure. The subject matter of this disclosure provides methods for reducing tumor burden in a subject. In some embodiments, the methods include administering the cells or compositions disclosed herein to the subject. In some embodiments, the methods reduce the number of tumor cells in the subject, reduce the size of the tumor in the subject, and / or eradicate the tumor in the subject.

[0056] The subject matter of this disclosure provides methods for increasing or prolonging the survival of subjects suffering from vegetations. In some embodiments, the method includes administering the cells or compositions disclosed herein to the subject.

[0057] This disclosure provides methods for treating and / or preventing vegetations in a subject. In some embodiments, the methods include administering the cells or compositions disclosed herein to the subject.

[0058] The cells and compositions disclosed herein can be used for treatment. In some embodiments, the cells and compositions disclosed herein are used to treat and / or prevent vegetations in a subject. In some embodiments, the cells and compositions disclosed herein are used to increase or prolong the survival of a subject suffering from vegetations. In some embodiments, the cells and compositions disclosed herein are used to treat and / or prevent vegetations in a subject.

[0059] In some embodiments, the tumor and / or vegetation is CD19-associated. In some embodiments, the tumor and / or vegetation is a blood cancer. In some embodiments, the blood cancer is selected from multiple myeloma, leukemia, and lymphoma. In some embodiments, the leukemia is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. In some embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In some embodiments, the tumor and / or vegetation is a B-cell malignancy. In some embodiments, the B-cell malignancy is selected from B-cell non-Hodgkin lymphoma (NHL), B-cell Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter transformation, and CNS lymphoma. In some embodiments, the tumor and / or vegetation is B-cell lymphoma. In some embodiments, the B-cell lymphoma is relapsed or refractory (R / R) B-cell lymphoma. In some embodiments, the subject is a human subject.

[0060] Furthermore, the subject matter of this disclosure provides methods for producing cells comprising the CAR disclosed herein. In some embodiments, the methods include introducing a nucleic acid molecule encoding the CAR disclosed herein into the cell.

[0061] Furthermore, the subject matter of this disclosure provides kits for reducing tumor burden in subjects, treating and / or preventing vegetations in subjects, and / or increasing or prolonging the survival of subjects with vegetations. In some embodiments, the kits include the cells disclosed herein. In some embodiments, the kits also include written instructions for using cells to reduce tumor burden in subjects, treat and / or prevent vegetations in subjects, and / or increase or prolong the survival of subjects with vegetations. Attached Figure Description

[0062] Figure 1A and 1B The cytotoxicity of T cells containing a CD19-targeting CAR against CD19-expressing cells was described. Figure 1A It demonstrated killing effect on the CD19-expressing Raji cancer cell line. Figure 1B The study demonstrated cytotoxicity against CD19-knockout Raji cells. The positive control was 1928z-1XX CAR.

[0063] Figure 2A and 2B The cytotoxicity of T cells containing a CD19-targeting CAR against CD19-expressing cells was described, measured by flow cytometry-based killing assessment. Figure 2A It demonstrated killing effect on the CD19-expressing Raji cancer cell line. Figure 2B The study demonstrated cytotoxicity against CD19-knockout Raji cells. The positive control was 1928z-1XX CAR.

[0064] Figure 3A and 3B The secretion of IL-2 cytokine by T cells containing CARs that target CD19 is described. Figure 3A This study demonstrates the secretion of IL-2 cytokines by T cells containing CD19-targeting CARs co-cultured with the Raji cancer cell line expressing CD19. Figure 3B This study demonstrates the secretion of IL-2 cytokines by T cells containing a CD19-targeting CAR, co-cultured with CD19-knockout Raji cells. The positive control was 1928z-1XX CAR.

[0065] Figure 4A and 4B The secretion of IFN-γ cytokines by T cells containing CARs that target CD19 is described. Figure 4A This study demonstrates the secretion of IFN-γ cytokines by T cells containing CD19-targeting CARs co-cultured with the Raji cancer cell line expressing CD19. Figure 4BThis study demonstrates the secretion of IFN-γ cytokines by T cells containing a CD19-targeting CAR, co-cultured with CD19-knockout Raji cells. The positive control was the 1928z-1XX CAR.

[0066] Figure 5 The in vivo antitumor activity of T cells containing a CD19-targeting CAR was demonstrated. #17 is a negative control, which is a CD19-targeting CAR with no detectable in vivo activity.

[0067] Figure 6 The amino acid sequence of CD19-HSA-His10 is shown.

[0068] Figure 7 Binding data for #2 scFv, SJ25c1 scFv, and FMC63 scFv are shown. Relative competition for common epitopes in soluble CD19 antigens was evaluated in a Biacore surface plasmon resonance (SPR) assay. A mixture of soluble CD19-specific antibodies (FMC63 or SJ25c1) and CD19 antigen was flown onto a chip containing #2 scFv to determine if #2 scFv cross-competes with the same epitopes as SJ25c1 scFv and FMC63-scFv. 19(T2) represents "#2 scFv".

[0069] Figure 8 A gamma retroviral vector containing #2 CAR is described. “19(T2)” represents “#2”. #2 CAR contains a CD19-specific T2 (19(T2)) scFv (or “#2 scFv”), a CD8 α leader peptide, a CD28 gene fragment, and a modified CD3ζ intracellular signaling domain. The CD28 gene fragment includes its extracellular hinge, transmembrane, and intracellular domains. The modified CD3ζ chain has one functional wild-type and two mutant immune receptor tyrosine activation motifs (ITAMs), termed “1XX”. The amino acid sequence of the modified CD3ζ intracellular signaling domain is shown in SEQ ID NO:43. The amino acid sequence alignment of CD3ζ-1XX with CD3ζ-wild-type is shown below. Figure 9 As shown. The amino acid differences between the two sequences are labeled.

[0070] Figure 9 The amino acid sequence of CD3ζ-1XX was compared with that of CD3ζ-wild type.

[0071] Figure 10 The in vitro cytotoxic activities of #2 CAR-T cells and 1928z-1XX CAR-T cells were described. “19(T2)28z1xx” represents “#2 CAR”.

[0072] Figure 11 The tumor elimination outcomes of #2 CAR T cells and 1928z-1XX CAR T cells are described. “19(T2)28z1xx” represents “#2 CAR”.

[0073] Figure 12 A gamma retroviral vector containing the 1928z CAR is described. The amino acid sequence of wild-type hCD3ζ is shown in... Figure 9 It is shown as SEQ ID NO:30.

[0074] Figure 13 Describes CD4 transduction by gamma retroviruses + and CD8 + The expression of CAR in T cells. “19(T2)28z1XX” represents “#2 CAR”.

[0075] Figure 14 Phenotypic 1928z CAR T cells and #2 CAR T cells are described. “19(T2)28z1XX” represents “#2CAR”.

[0076] Figure 15 The kinetics of tumor elimination by #2 CAR T cells and 1928z CAR T cells are described. “19(T2)28z1XX” represents “#2 CAR”. Each curve represents the tumor burden of one mouse.

[0077] Figure 16 The survival of mice carrying NALM6 leukemia after intravenous infusion of #2 CAR T cells or 1928z CAR T cells is described. “19(T2)28z1XX” represents “#2 CAR”.

[0078] Figure 17 The quantification and phenotype of CAR T cells in the bone marrow of mice treated with #2 CAR T cells or 1928z CAR T cells (day 17) are described. All data are presented as mean ± standard error, and p-values ​​were calculated using a two-tailed unpaired Student's t-test. “19(T2)28z1XX” represents “#2 CAR”.

[0079] Figure 18 The text describes the body weight of mice after treatment with #2 CAR T cells or 1928z CAR T cells. "19(T2)28z1XX" represents "#2 CAR". An asterisk indicates the time point at which unplanned death occurred. Shorter curves represent the loss of all mice at the specified time points.

[0080] Figure 19 Describes the use of 1×10 on days 10 and 27 / 28 after infusion. 6Quantitative analysis of serum liver enzymes in mice treated with CAR T-cell therapy. Statistical comparisons were performed between two cohorts on the same day. All data are presented as mean ± sem; p-values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR".

[0081] Figure 20 The text describes the use of 1×10 on days 10 and 27 / 28 after infusion. 6 Quantification of human cytokine levels in mice treated with #2 CAR T cells or 1928z CAR cells. All data are mean ± sem; p-values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR".

[0082] Figure 21 Administer 1×10 on days 10 and 27 / 28 after infusion. 6 Quantification of mouse cytokine levels in mice treated with #2 CAR T cells or 1928z CAR T cells. All data are mean ± standard error; p-values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR".

[0083] Figure 22 The quantification of basic hematologic cells in mice treated with #2 CAR T cells and 1928z CAR T cells 27 / 28 days post-infusion is described. All data are mean ± sem; p-values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR".

[0084] Figure 23 This study compares the body weight and critical organ weights of mice treated with #2 CAR T cells and 1928z CAR T cells 27 / 28 days post-infusion. The number of mice in each group is shown in the legend in the figure. All data are presented as mean ± standard error; p-values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR".

[0085] Figure 24 This study describes the quantification of lymphocyte and tumor infiltration in the liver, spleen, and bone marrow of mice treated with CAR T cells. All data are presented as mean ± sem; p-values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR".

[0086] Figure 25Exemplary flow cytometry chromatograms depict anti-CD19 antibodies binding to wild-type and CD19 knockout Raji and NALM-6 cells expressing CD19.

[0087] Figures 26A-26C An exemplary binding curve of an anti-CD19 antibody that binds to CD19 on NALM-6 cells was depicted. Detailed Implementation

[0088] The subject matter of this disclosure provides a chimeric antigen receptor (CAR) specifically targeting CD19 and cells comprising such a CD19-targeting CAR. The cells may be immune-responding cells, such as genetically modified immune-responding cells (e.g., T cells or NK cells). The subject matter of this disclosure also provides the use of the cells and compositions comprising them for treating, for example, vegetations. The CD19-targeting CAR of this disclosure has shown to be safer and / or more effective than other CD19-targeting CARs, for example, as demonstrated by its greater persistence (see Examples 6 and 7). This greater persistence has also been shown not to pose a safety risk. Therefore, the CD19-targeting CAR T cells of this disclosure can be used at lower doses than most CD19-targeting CAR T cells currently used in immunotherapy. The CD19-targeting CAR T cells of this disclosure are expected to be safer, for example, and therefore, with fewer side effects, including but not limited to cytokine release syndrome (CRS) and a lower incidence of neurotoxicity (NT).

[0089] Non-limiting embodiments of this disclosure are described in this specification and examples.

[0090] For the purpose of clarity of this disclosure, and not in a restrictive manner, the detailed description is divided into the following sections:

[0091] 5.1. Definition;

[0092] 5.2.CD19;

[0093] 5.3. Chimeric antigen receptor (CAR);

[0094] 5.4. Cells;

[0095] 5.5. Nucleic acid molecules and vectors;

[0096] 5.6. Dosage form and administration; and

[0097] 5.7. Treatment methods.

[0098] 5.1. Definition

[0099] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0100] As used herein, the term "about" or "approximately" refers to an acceptable margin of error for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to convention in the art, "about" may refer to within three or more standard deviations. Alternatively, "about" may indicate a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may indicate within an order of magnitude of the numerical value, preferably within five times, more preferably within two times.

[0101] As used herein, the term "immune response cell" refers to a cell, its progenitor cell, or its progeny that plays a role in an immune response. In some embodiments, the immune response cell is a cell of the lymphoid lineage. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can differentiate. In some embodiments, the immune response cell is a bone marrow lineage cell.

[0102] As used herein, the term "activated immune response cells" refers to the induction of signal transduction or changes in protein expression within cells that lead to the initiation of an immune response. For example, a signal transduction cascade occurs when the CD3 chain responds to ligand binding and aggregates an immunoreceptor tyrosine inhibitory motif (ITAM). In some embodiments, when a CAR binds to an antigen, an immune synapse forms, comprising the aggregation of numerous molecules near the binding receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This aggregation of membrane-bound signaling molecules phosphorylates the ITAM motif contained within the CD3 chain. This phosphorylation, in turn, initiates T cell activation pathways, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce overall gene expression on T cells, thereby increasing IL-2 production, promoting the proliferation and expression of master-regulating T cell proteins, and subsequently initiating a T cell-mediated immune response.

[0103] As used herein, the term "stimulated immune response cells" refers to signals that lead to a strong and sustained immune response. In some embodiments, this occurs after activation of immune response cells (e.g., T cells) or is simultaneously mediated by receptors including, but not limited to, CD28, 4-1BB, OX40, CD40, and ICOS. Receiving multiple stimulating signals can be important for establishing a strong and long-lasting T cell-mediated immune response. T cells can be rapidly suppressed and become unresponsive to antigens. Although the effects of these co-stimulating signals may vary, they generally lead to increased gene expression, resulting in long-lived, proliferative, and anti-apoptotic T cells that respond strongly to antigens and can eradicate them completely and sustainably.

[0104] As used herein, a “complementarity-determining region” or “CDR” is defined as the amino acid sequence of the complementarity-determining region of an antibody, which is a hypervariable region of the immunoglobulin heavy and light chains. In some embodiments, the “CDR” of a variable domain is an amino acid residue within the variable region identified according to the cumulative, AbM, contact, and / or conformational definitions of Kabat, Chothia, Kabat, and Chothia, or any CDR determination method well known in the art. An antibody CDR can be identified as a hypervariable region originally defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, NIH, Washington DC. The location of the CDR can also be identified as a structural loop structure originally described by Chothia et al. See, for example, Chothia et al., Nature 342:877-883, 1989. Other methods for CDR identification include the “AbM definition,” a compromise between Kabat and Chothia, derived using Oxford Molecular’s ​​AbM antibody modeling software (now Accelrys®) or based on the “contact definition” of CDRs based on observed antigen contact, cited by MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to here as the “conformation definition” of CDRs, the position of a CDR can be identified as a residue that contributes enthalpy to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Other CDR boundary definitions do not strictly follow one of the methods described above, but nonetheless overlap with at least a portion of the Kabat CDR; however, they can be shortened or lengthened based on predictions or experimental findings that specific residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding. In some embodiments, a CDR can refer to any CDR defined by methods known in the art, including combinations of methods. The methods used herein can be applied to CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, a CDR can be defined according to any of the following: Kabat, Chothia, extended, AbM, contact, and / or conformational definitions. Typically, an antibody contains three heavy chain and three light chain CDRs or CDR regions within its variable region. The CDR provides most of the contact residues to enable the antibody to bind to an antigen or epitope. In some embodiments, the Kabat numbering system is used to delineate the CDR regions.

[0105] The term “single-chain variable fragment” or “scFv” as used in this article refers to the heavy chain (V) of immunoglobulins (e.g., mouse or human). H ) and light chains (V L A fusion protein consisting of the variable region of a protein, covalently linked to form a V H ::V L Heterodimer. Heavy chain (V H ) and light chains (V L It is directly linked or linked via a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), which will link V H N-terminus and V L The C-terminal connection or V H C-terminus and V L The adapter is an N-terminal linker. It is typically enriched with glycine for flexibility and serine or threonine for solubility. The adapter can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of adapters are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the adapter is a G4S adapter.

[0106] In some embodiments, the adapter comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, which is provided below:

[0107]

[0108] In some embodiments, the adapter comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, which is provided below:

[0109]

[0110] In some embodiments, the adapter comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, which is provided below:

[0111]

[0112] In some embodiments, the adapter comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, which is provided below:

[0113]

[0114] As used herein, the terms "substantially identical" or "substantially homologous" refer to a polypeptide or nucleic acid molecule that exhibits at least about 50% identity or homology with a reference amino acid sequence (e.g., any amino acid sequence described herein) or a reference nucleic acid sequence (e.g., any nucleic acid sequence described herein). In some embodiments, such a sequence has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% identity or homology with the amino acid sequence or nucleic acid sequence used for comparison.

[0115] Sequence identity can be measured using sequence analysis software, such as the sequence analysis software package from the Genetics Computing Group at the University of Wisconsin-Madison Biotechnology Center, 1710 University Avenue, 53705, Wisconsin, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs. Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conserved substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine, lysine, arginine; and phenylalanine, tyrosine. In exemplary methods for determining the degree of identity, the BLAST program can be used, where probability scores between e-3 and e-100 indicate closely related sequences.

[0116] The percentage of homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4. Alternatively, the percentage of homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, with vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Alternatively, the amino acid sequence of the subject matter of this disclosure can be further used as a “query sequence” to search public databases for, for example, to identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. ((1990) J. Mol. Biol. 215:403-10). BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the specified sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFv703). For vacancy alignments for comparative purposes, Gapped BLAST can be used as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0117] As used herein, the term "conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the CD19-targeting CAR (e.g., the extracellular antigen-binding domain of the CAR) comprising an amino acid sequence disclosed herein. Conserved modifications may include amino acid substitutions, additions, and deletions. Modifications can be introduced into the extracellular antigen-binding domain of the CAR disclosed herein using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be grouped according to their physicochemical properties (e.g., charge and polarity). A conserved amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified according to polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Therefore, one or more amino acid residues within the CDR region can be substituted by other amino acid residues from the same group, and the retained function of the altered antibody (i.e., the functions listed in (c) to (l) above) can be tested using the functional assays described herein. In some embodiments, no more than one, two, three, four, or five residues within a specified sequence or CDR region are altered.

[0118] As used herein, the term "effective amount" is an amount sufficient to affect a beneficial or desired clinical outcome during treatment. An effective amount may be administered to a subject at one or more doses. In some implementations, an effective amount may be an amount sufficient to alleviate, improve, stabilize, reverse, or slow disease progression or otherwise reduce the pathological consequences of the disease. The effective amount can be determined by a physician on a case-by-case basis and within the skill of a person skilled in the art. Several factors are typically considered when determining an appropriate dose to achieve an effective amount. These factors include the subject's age, sex, and weight; the disease being treated; the severity of the disease; and the form and effective concentration of the cells administered.

[0119] As used herein, the term "vesicle" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Tumor formation is typically uncontrolled and progressive, and occurs without causing or terminating the proliferation of normal cells. Vesicles can affect a wide range of cell types, tissues, or organs, including but not limited to those selected from: bladder, bone, brain, breast, cartilage, glial tissue, esophagus, fallopian tubes, gallbladder, heart, intestine, kidneys, liver, lungs, lymph nodes, nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, trachea, genitourinary tract, ureter, urethra, uterus, and vagina, or their tissues or cell types. Vesicles include cancers such as sarcomas, tumors, or plasmacytomas (malignant tumors of plasma cells). Vesicles can be primary tumors or primary cancers.

[0120] As used herein, the term “signal sequence” or “leader sequence” refers to a peptide sequence (e.g., 5, 10, 15, 20, 25, or 30 amino acids) present at the N-terminus of newly synthesized proteins that guides them into the secretory pathway.

[0121] The terms “comprising” and “including” are intended to have the broad meaning given to them under U.S. patent law and can mean “containing”, “including”, etc.

[0122] As used herein, “treatment” refers to a clinical intervention that attempts to alter the course of a disease in the treated individual or cells, and may be used for prevention or in clinicopathological processes. The therapeutic effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. By preventing the progression of disease or condition, treatment can prevent deterioration of the condition not only in affected or diagnosed or suspected subjects, but also in subjects with the condition or suspected at risk of having the condition, and prevent the onset or symptoms of the condition.

[0123] In this article, “individual” or “subject” refers to a vertebrate, such as a human, or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, primates, farm animals, locomotor animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0124] 5.2.CD19

[0125] CD19 is a 95 kDa cell surface glycoprotein present in the early stages of normal B cell development until differentiation into plasma cells. CD19 is absent from other normal tissues, including pluripotent hematopoietic stem cells. It is expressed in B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and a group of acute myeloid leukemias, making it an attractive target for immunotherapy with minimal risk of autoimmune diseases (excluding B-cell aplasia) or irreversible myelotoxicity.

[0126] CD19 is a member of the immunoglobulin superfamily and a component of the cell surface signal transduction complex, which includes Leu13, CD81, and CD21, and actively regulates signal transduction through B cell receptors.

[0127] In some embodiments, the CAR of this disclosure binds to human CD19. In some embodiments, human CD19 comprises or consists of the amino acid sequence of NCBI reference number: NP_001171569.1 (SEQ ID NO:5).

[0128] SEQ ID NO: 5 is provided as follows:

[0129]

[0130] In some embodiments, human CD19 comprises or consists of the amino acid sequence of NCBI reference number: NP_001761.3 (SEQ ID NO:6).

[0131] SEQ ID NO:6 is provided below:

[0132]

[0133] In some embodiments, the CAR binds to the extracellular domain of CD19. In some embodiments, the CAR binds to the extracellular domain of human CD19. In some embodiments, the extracellular domain of human CD19 comprises or is composed of amino acids 20-291 of SEQ ID NO:5. In some embodiments, the extracellular domain of human CD19 comprises or is composed of amino acids 20-291 of SEQ ID NO:6.

[0134] In some embodiments, CD19 comprises or consists of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity with the amino acid sequence or fragment thereof shown in SEQ ID NO:5.

[0135] In some embodiments, CD19 comprises or consists of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity with the amino acid sequence or fragment thereof shown in SEQ ID NO:6.

[0136] 5.3. Chimeric antigen receptor (CAR)

[0137] CARs are engineered receptors that specifically transplant or confer a target onto immune effector cells. CARs can be used to specifically transplant monoclonal antibodies onto cells, such as immune response cells, like T cells or NK cells; and to facilitate the transfer of their coding sequences via retroviral vectors.

[0138] There are three generations of CARs. "First-generation" CARs typically consist of an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain, which in turn fuses to an intracellular signaling domain. First-generation CARs can provide de novo antigen recognition and activate CD4+ via the CD3ζ chain signaling domain in a single fusion molecule. + and CD8 + T cells, and are unrelated to HLA-mediated antigen presentation. "Second-generation" CARs add intracellular signaling domains from co-stimulatory molecules (including but not limited to CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail region of the CAR to provide additional signals to the cell. "Second-generation" CARs include those that provide co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third-generation" CARs include those that provide multiple co-stimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ). In some embodiments, a CD19-targeting CAR is a first-generation CAR. In some embodiments, a CD19-targeting CAR does not contain intracellular signaling domains of co-stimulatory molecules. In some embodiments, a CD19-targeting CAR is a second-generation CAR. In some embodiments, a CD19-targeting CAR contains intracellular signaling domains of co-stimulatory molecules.

[0139] In some embodiments, the CAR includes an extracellular antigen-binding domain that specifically binds to CD19, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the extracellular antigen-binding domain is fused with the transmembrane domain, and the transmembrane domain is fused with the intracellular signal transduction domain.

[0140] 5.3.1. Extracellular antigen-binding domain of CAR

[0141] In some embodiments, the extracellular antigen-binding domain of the CAR includes or is an scFv. The scFv may be a human scFv, a humanized scFv, or a mouse scFv. In some embodiments, the scFv is a human scFv. The scFv may be obtained from the variable heavy and light regions of a fusion antibody. Alternatively or additionally, the scFv may be derived from Fab's (rather than from the antibody, for example, from a Fab library).

[0142] In some embodiments, the extracellular antigen-binding domain of the CAR includes Fab. In some embodiments, Fab is cross-linked. In some embodiments, the extracellular antigen-binding domain of the CAR includes F(ab)2.

[0143] Any of the aforementioned molecules may be included in a fusion protein having a heterologous sequence to form the extracellular antigen-binding domain of a CAR.

[0144] In some embodiments, the extracellular antigen-binding domain of the CAR is at least about 1 × 10 -6 M, at least approximately 1×10 -7 M, at least approximately 1×10 -8 M, at least approximately 1×10 -9 M, or at least about 1×10 -10 The dissociation constant of M (K) d The CAR binds to CD19 (e.g., human CD19). In some embodiments, the extracellular antigen-binding domain of the CAR binds at a concentration of at least about 2 × 10⁻⁶. -8 The dissociation constant of M (K) d The CAR binds to CD19 (e.g., human CD19). In some embodiments, the extracellular antigen-binding domain of the CAR binds at a concentration of at least about 2 × 10⁻⁶. -8 M to approximately 8×10 -9 The dissociation constant of M (K) d It binds to CD19 (e.g., human CD19).

[0145] In some embodiments, the extracellular antigen-binding domain of the CAR has a dissociation constant (Ki) between about 1 nM to 50 nM, about 5 nM to 30 nM, about 5 nM to 25 nM, or about 8 nM to 20 nM. dThe CAR binds to CD19 (e.g., human CD19). In some embodiments, the extracellular antigen-binding domain of the CAR has a dissociation constant (Ki) of at least about 50 nM, at least about 40 nM, at least about 35 nM, at least about 30 nM, at least about 25 nM, at least about 20 nM, at least about 19 nM, at least about 18 nM, at least about 17 nM, at least about 16 nM, at least about 15 nM, at least about 14 nM, at least about 13 nM, at least about 12 nM, at least about 11 nM, at least about 10 nM, at least about 9 nM, at least about 8 nM, at least about 7 nM, at least about 6 nM, or at least about 5 nM. d It binds to CD19 (e.g., human CD19).

[0146] Binding of the extracellular antigen-binding domain of a CAR can be determined by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blotting. Each of these assays typically detects the presence of a specific target protein-antibody complex using a labeling reagent (e.g., antibody or scFv) specific to the target complex. For example, scFv can be radiolabeled and used in radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassay, Seventh Training Course on Radioligand Assay Techniques, Endocrine Society, March 1986, incorporated herein by reference). Radioisotopes can be detected by methods such as using a gamma counter or a scintillation counter, or by autoradiography. In some embodiments, the extracellular antigen-binding domain targeting CD127 is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent proteins (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet). In some embodiments, human scFv targeting CD19 is labeled with GFP.

[0147] In some implementations, the extracellular antigen-binding domain of the CAR includes a heavy chain variable region (V... H ) and light chain variable region (V L ).

[0148] In some implementations, V HThis includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or its conservative modifications. In some embodiments, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9. SEQ ID NO:7-9 are provided below.

[0149]

[0150] In some implementations, V H An amino acid sequence comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology with the amino acid sequence shown in SEQ ID NO:10. For example, V H The amino acid sequence comprising about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, V H It contains the amino acid sequence shown in SEQ ID NO:10. SEQ ID NO:10 is provided below.

[0151]

[0152] In some implementations, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conserved modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conserved modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:13 or its conserved modifications. In some embodiments, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:13. SEQ ID NO:11-13 are provided below.

[0153]

[0154] In some implementations, V L An amino acid sequence comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology with the amino acid sequence shown in SEQ ID NO:14. For example, V L The amino acid sequence comprising about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, V L It contains the amino acid sequence shown in SEQ ID NO:14. SEQ ID NO:14 is provided below.

[0155]

[0156] In some implementations, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or its conservative modifications; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conserved modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conserved modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:13 or its conserved modifications. In some embodiments, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:13.

[0157] In some implementations, V H Contains the amino acid sequence shown in SEQ ID NO:10, and V L It contains the amino acid sequence shown in SEQ ID NO:14.

[0158] V H and V LThey can be connected to each other, for example, via connectors. The variable region from the N end to the C end can be V. L -V H or V L -V H In some implementations, V L Located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L The position from the N-terminus to the C-terminus is: V L -V H In some implementations, the extracellular antigen-binding domain of the CAR is scFv, which contains V H The V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L The V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:13. In some embodiments, scFv includes V containing the amino acid sequence shown in SEQ ID NO:10. H and V containing the amino acid sequence shown in SEQ ID NO:14 L In some embodiments, the scFv comprises or consists of the amino acid sequence shown in SEQ ID NO:15. SEQ ID NO:15 is provided below. In some embodiments, the anti-CD19 scFv is designated as "#2 scFv".

[0159]

[0160] In some implementations, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:17 or its conservative modifications. In some embodiments, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:17. SEQ ID NO:16 and 17 are provided below.

[0161]

[0162] In some implementations, V L An amino acid sequence comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology with the amino acid sequence shown in SEQ ID NO:18. For example, V L The amino acid sequence comprising about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, V L It contains the amino acid sequence shown in SEQ ID NO:18. SEQ ID NO:18 is provided below.

[0163]

[0164] In some implementations, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or its conservative modifications; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:17 or its conservative modifications. In some embodiments, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:17.

[0165] In some implementations, V H Contains the amino acid sequence shown in SEQ ID NO:10, and V L It contains the amino acid sequence shown in SEQ ID NO:18.

[0166] In some implementations, V L Located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L The position from the N-terminus to the C-terminus is: V L -V H In some implementations, the extracellular antigen-binding domain of the CAR is scFv, which contains V H The V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L It includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:17. In some embodiments, scFv includes V H and V L V H Contains the amino acid sequence shown in SEQ ID NO:10, V L It comprises the amino acid sequence shown in SEQ ID NO:18. In some embodiments, the scFv comprises or consists of the amino acid sequence shown in SEQ ID NO:19. SEQ ID NO:19 is provided below. In some embodiments, the anti-CD19 scFv is designated as "#8 scFv".

[0167]

[0168] In some implementations, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:20 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:21 or its conservative modifications. In some embodiments, V L This includes CDR1, which contains the amino acid sequence shown in SEQ ID NO:11; CDR2, which contains the amino acid sequence shown in SEQ ID NO:20; and CDR3, which contains the amino acid sequence shown in SEQ ID NO:21. SEQ ID NO:20 and 21 are provided below.

[0169]

[0170] In some implementations, V LAn amino acid sequence comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology with the amino acid sequence shown in SEQ ID NO:22. For example, V L This includes amino acid sequences having approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity or homology with the amino acid sequence shown in SEQ ID NO:22. In some embodiments, V L It contains the amino acid sequence shown in SEQ ID NO:22. SEQ ID NO:22 is provided below.

[0171]

[0172] In some implementations, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or its conservative modifications; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:20 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:21 or its conservative modifications. In some embodiments, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:20, and CDR3 containing the amino acid sequence shown in SEQ ID NO:21.

[0173] In some implementations, V H Contains the amino acid sequence shown in SEQ ID NO:10, and V L It contains the amino acid sequence shown in SEQ ID NO:22.

[0174] In some implementations, V L Located at the N-terminus of the extracellular antigen-binding domain, i.e., VH and V L The position from the N-terminus to the C-terminus is: V L -V H In some embodiments, the extracellular antigen-binding domain of the CAR is scFv, which includes V H The V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L It includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:20, and CDR3 containing the amino acid sequence shown in SEQ ID NO:21. In some embodiments, scFv includes V H and V L V H Contains the amino acid sequence shown in SEQ ID NO:10, V L It comprises the amino acid sequence shown in SEQ ID NO:22. In some embodiments, the scFv comprises or consists of the amino acid sequence shown in SEQ ID NO:23. SEQ ID NO:23 is provided below. In some embodiments, the anti-CD19 scFv is designated as "#15 scFv".

[0175]

[0176] In some implementations, the CDR is identified according to the Kabat numbering system.

[0177] In some implementations, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:57 or its conservative modifications. In some embodiments, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:57. SEQ ID NO:57 is provided below.

[0178]

[0179] In some implementations, V LAn amino acid sequence comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology with the amino acid sequence shown in SEQ ID NO:58. For example, V L The amino acid sequence comprising about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, V L It contains the amino acid sequence shown in SEQ ID NO:58. SEQ ID NO:58 is provided below.

[0180]

[0181] In some implementations, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or its conservative modifications; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:57 or its conservative modifications. In some embodiments, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:57.

[0182] In some implementations, V H Contains the amino acid sequence shown in SEQ ID NO:10, and V L It contains the amino acid sequence shown in SEQ ID NO:58.

[0183] In some implementations, V L Located at the N-terminus of the extracellular antigen-binding domain, i.e., V Hand V L The position from the N-terminus to the C-terminus is: V L -V H In some embodiments, the extracellular antigen-binding domain of the CAR is scFv, which includes V H The V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L It includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:57. In some embodiments, scFv includes V containing the amino acid sequence shown in SEQ ID NO:10. H and V containing the amino acid sequence shown in SEQ ID NO:58 L In some implementations, the anti-CD19 scFv is designated as "#4 scFv".

[0184] In some embodiments, the CDR is identified according to the Kabat numbering system.

[0185] In some implementations, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:59 or its conservative modifications. In some embodiments, V L This includes CDR1, which contains the amino acid sequence shown in SEQ ID NO:11; CDR2, which contains the amino acid sequence shown in SEQ ID NO:12; and CDR3, which contains the amino acid sequence shown in SEQ ID NO:59. SEQ ID NO:59 is provided below.

[0186]

[0187] In some implementations, V L An amino acid sequence comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology with the amino acid sequence shown in SEQ ID NO:60. For example, V LThis includes amino acid sequences having approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, V L It contains the amino acid sequence shown in SEQ ID NO:60. SEQ ID NO:60 is provided below.

[0188]

[0189] In some implementations, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or its conservative modifications; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:59 or its conservative modifications. In some embodiments, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:59.

[0190] In some implementations, V H Contains the amino acid sequence shown in SEQ ID NO:10, and V L It contains the amino acid sequence shown in SEQ ID NO:60.

[0191] In some implementations, V L Located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L The position from the N-terminus to the C-terminus is: V L -V HIn some implementations, the extracellular antigen-binding domain of the CAR is scFv, which contains V H The V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L It includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:59. In some embodiments, scFv includes V containing the amino acid sequence shown in SEQ ID NO:10. H and V containing the amino acid sequence shown in SEQ ID NO:60 L In some implementations, the anti-CD19 scFv is designated as "#5scFv".

[0192] In some embodiments, the CDR is identified according to the Kabat numbering system.

[0193] In some implementations, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:61 or its conservative modifications. In some embodiments, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:61. SEQ ID NO:61 is provided below.

[0194]

[0195] In some implementations, V L An amino acid sequence comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology with the amino acid sequence shown in SEQ ID NO:62. For example, V LThe amino acid sequence comprising about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, V L It contains the amino acid sequence shown in SEQ ID NO:62. SEQ ID NO:62 is provided below.

[0196]

[0197] In some implementations, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or its conservative modifications; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:61 or its conservative modifications. In some embodiments, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:61.

[0198] In some implementations, V H Contains the amino acid sequence shown in SEQ ID NO:10, and V L It contains the amino acid sequence shown in SEQ ID NO:62.

[0199] In some implementations, V L Located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L The position from the N-terminus to the C-terminus is V. L -V HIn some implementations, the extracellular antigen-binding domain of the CAR is scFv, which contains V H The V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L It includes CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:61. In some embodiments, scFv includes V containing the amino acid sequence shown in SEQ ID NO:10. H and V containing the amino acid sequence shown in SEQ ID NO:62 L In some implementations, the anti-CD19 scFv is designated as "#6 scFv".

[0200] In some embodiments, the CDR is identified according to the Kabat numbering system.

[0201] In some implementations, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:63 or its conservative modifications. In some embodiments, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:63. SEQ ID NO:63 is provided below.

[0202]

[0203] In some implementations, V L An amino acid sequence comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology with the amino acid sequence shown in SEQ ID NO:64. For example, V LThe amino acid sequence comprising about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 64. In some embodiments, V L It contains the amino acid sequence shown in SEQ ID NO:64. SEQ ID NO:64 is provided below.

[0204]

[0205] In some implementations, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or its conservative modifications; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:12 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:63 or its conservative modifications. In some embodiments, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:63.

[0206] In some implementations, V H Contains the amino acid sequence shown in SEQ ID NO:10, and V L It contains the amino acid sequence shown in SEQ ID NO:64.

[0207] In some implementations, V L Located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L The position from the N-terminus to the C-terminus is: V L -V HIn some implementations, the extracellular antigen-binding domain of the CAR is scFv, which contains V H The V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L It includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:63. In some embodiments, scFv includes V containing the amino acid sequence shown in SEQ ID NO:10. H and V containing the amino acid sequence shown in SEQ ID NO:64 L In some implementations, the anti-CD19 scFv is designated as "#7 scFv".

[0208] In some embodiments, the CDR is identified according to the Kabat numbering system.

[0209] In some implementations, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:65 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:66 or its conservative modifications. In some embodiments, V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:65, and CDR3 containing the amino acid sequence shown in SEQ ID NO:66. SEQ ID NO:66 and 67 are provided below.

[0210]

[0211] In some implementations, V L An amino acid sequence comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology with the amino acid sequence shown in SEQ ID NO:67. For example, V LThe amino acid sequence comprising about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 67. In some embodiments, V L It contains the amino acid sequence shown in SEQ ID NO:67. SEQ ID NO:67 is provided below.

[0212]

[0213] In some implementations, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:8 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9 or its conservative modifications; and V L This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11 or its conservative modifications, CDR2 containing the amino acid sequence shown in SEQ ID NO:65 or its conservative modifications, and CDR3 containing the amino acid sequence shown in SEQ ID NO:66 or its conservative modifications. In some embodiments, V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and the V L It includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence described in SEQ ID NO:65, and CDR3 containing the nucleic acid sequence described in SEQ ID NO:66.

[0214] In some implementations, V H Contains the amino acid sequence shown in SEQ ID NO:10, and V L It contains the amino acid sequence shown in SEQ ID NO:67.

[0215] In some implementations, V L Located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L The position from the N-terminus to the C-terminus is: V L -V HIn some implementations, the extracellular antigen-binding domain of the CAR is scFv, which contains V H The V H This includes CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9; and V L It includes CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:65, and CDR3 containing the amino acid sequence shown in SEQ ID NO:66. In some embodiments, scFv includes V containing the amino acid sequence shown in SEQ ID NO:10. H and V containing the amino acid sequence shown in SEQ ID NO:67 L In some implementations, the anti-CD19 scFv is designated as "#1 scFv".

[0216] In some embodiments, the CDR is identified according to the Kabat numbering system.

[0217] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:10 is shown in SEQ ID NO:68. SEQ ID NO:68 is provided below.

[0218]

[0219] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:14 is listed in SEQ ID NO:69. SEQ ID NO:69 is provided below.

[0220]

[0221] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:18 is shown in SEQ ID NO:70. SEQ ID NO:70 is provided below.

[0222]

[0223] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:22 is shown in SEQ ID NO:71. SEQ ID NO:71 is provided below.

[0224]

[0225] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:58 is shown in SEQ ID NO:72. SEQ ID NO:72 is provided below.

[0226]

[0227] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:60 is listed in SEQ ID NO:73. SEQ ID NO:73 is provided below.

[0228]

[0229] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:62 is listed in SEQ ID NO:74. SEQ ID NO:74 is provided below.

[0230]

[0231] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:64 is listed in SEQ ID NO:75. SEQ ID NO:75 is provided below.

[0232]

[0233] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:67 is listed in SEQ ID NO:76. SEQ ID NO:76 is provided below.

[0234]

[0235] In some implementations, V H and V L The connection is made via a connector. In some embodiments, the connector comprises or consists of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the connector comprises or consists of the amino acid sequence shown in SEQ ID NO:1.

[0236] V consists of a sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity with a specific sequence (e.g., SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 67). H and / or V L The amino acid sequence may contain substitutions (e.g., conserved substitutions), insertions, or deletions relative to that particular sequence, but retains the ability to bind to the target antigen (e.g., mesothelin). In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in a particular sequence (e.g., SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:67). In some embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR of the extracellular antigen-binding domain (e.g., in the FR). In some embodiments, the extracellular antigen-binding domain includes V selected from SEQ ID NO:10, 14, 18, 22, 59, 61, 63, 65, and 68. H and / or V L Sequences, including post-translational modifications of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64 or SEQ ID NO:67.

[0237] In addition, the extracellular antigen-binding domain may include a leader peptide or signal peptide that guides the nascent protein into the endoplasmic reticulum. The signal peptide or leader peptide may be essential if the CAR is to be glycosylated and anchored in the cell membrane. The signal sequence or leader sequence may be a peptide sequence (approximately 5, 10, 15, 20, 25, or 30 amino acids long) present at the N-terminus of newly synthesized proteins, guiding them into the secretory pathway. In some embodiments, the signal peptide is covalently linked to the 5' end (N-terminus) of the extracellular antigen-binding domain. In some embodiments, the signal peptide includes a CD8 polypeptide; for example, the CAR contains a truncated CD8 signal peptide. In some embodiments, the signal peptide is generated from an antibody derived therefrom. In some embodiments, the signal peptide of #2scFv is shown in SEQ ID NO:54. In some embodiments, the signal peptide of #8scFv is shown in SEQ ID NO:54. In some embodiments, the signal peptide of #15scFv is shown in SEQ ID NO:55.

[0238]

[0239] 5.3.2. Transmembrane domains of CAR

[0240] In some embodiments, the transmembrane domain of the CAR includes at least a portion of a hydrophobic α-helix spanning the membrane. Different transmembrane domains result in different receptor stability. Upon antigen recognition, the receptor aggregates and transmits signals to the cell. In some embodiments, the transmembrane domain of the CAR includes natural or modified transmembrane domains of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CD84, CD166, CD8a, CD8b, ICAM-1, CTLA-4, CD27, CD40, NKGD2, or combinations thereof.

[0241] In some embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof). In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD28 or a portion thereof. The CD28 polypeptide may comprise, or consist of, an amino acid sequence or fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity or homology with, or be composed of, the sequence having NCBI reference number: NP_006130 (SEQ ID NO: 24), and / or may optionally comprise at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the CD28 polypeptide comprises or is composed of an amino acid sequence that is a continuous portion of SEQ ID NO:24, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, and / or at most about 70, at most about 80, at most about 90, at most about 100, at most about 150, at most about 200, or at most about 220 amino acids. In some embodiments, the CD28 polypeptide comprises or is composed of an amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 154-179, 150-200, 153-179, or 200-220 of SEQ ID NO:24. In some embodiments, the transmembrane domain of the CAR comprises the CD28 polypeptide, which comprises or is composed of an amino acid sequence of amino acids 154-179 of SEQ ID NO:24. SEQ ID NO:24 is provided below.

[0242]

[0243] An exemplary nucleotide sequence encoding the amino acid sequence of amino acids 154-179 of SEQ ID NO:24 is shown in SEQ ID NO:25 provided below.

[0244]

[0245] In some embodiments, the transmembrane domain of the CAR includes the transmembrane domain of mouse CD28 or a portion thereof. The CD28 polypeptide may consist of an amino acid sequence or fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity or homology with the sequence having NCBI reference number: NP_031668.3 (SEQ ID NO:26), and / or may optionally include at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence as a continuous portion of SEQ ID NO:26, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, and at most about 218 amino acids. In some embodiments, the CD28 polypeptide comprises or is composed of the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 150-200, 151-177, or 200-218 of SEQ ID NO:26. In some embodiments, the transmembrane domain of the CAR comprises the CD28 polypeptide, which comprises or is composed of the amino acid sequence of amino acids 151-177 of SEQ ID NO:26. SEQ ID NO: 26 is provided below.

[0246]

[0247] In some embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a portion thereof). In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD8 or a portion thereof. In some embodiments, the CD8 polypeptide comprises or contains a fragment of an amino acid sequence or thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the sequence having NCBI reference number: NP_001139345.1 (SEQ ID NO:27), and / or optionally comprises at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence as a continuous portion of SEQ ID NO:27, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, and at most about 235 amino acids. Alternatively or additionally, in some embodiments, the CD8 polypeptide comprises or is composed of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, 137-209, or 200-235 of SEQ ID NO:27. In some embodiments, the transmembrane domain of the CAR comprises the CD8 polypeptide, which comprises or is composed of the amino acid sequence of amino acids 137-209 of SEQ ID NO:27. SEQ ID NO:27 is provided below.

[0248]

[0249] In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of mouse CD8 or a portion thereof. In some embodiments, the CD8 polypeptide comprises or contains an amino acid sequence or fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the sequence having NCBI reference number: AAA92533.1 (SEQ ID NO: 28), and / or may optionally comprise at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the CD8 polypeptide comprises or contains an amino acid sequence as a continuous portion of SEQ ID NO: 28, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200 and at most 247 amino acids. Alternatively or additionally, in some embodiments, the CD8 polypeptide comprises or is composed of the amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 28. In some embodiments, the transmembrane domain of the CAR comprises the CD8 polypeptide, which comprises or is composed of the amino acid sequence of amino acids 151-219 of SEQ ID NO: 28. SEQ ID NO: 28 is provided below.

[0250]

[0251] In some non-limiting embodiments, the CAR further includes a spacer region connecting the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition while maintaining the activation activity of the CAR.

[0252] In some embodiments, the hinge / spacer region of the CAR includes a natural or modified hinge region of CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, CTLA-4, CD27, CD40, and NKGD2, or combinations thereof. The hinge / spacer region may be derived from the following hinge regions: portions of the CH2CH3 region and CD3 of IgG1 or immunoglobulins, portions of CD28 polypeptides (e.g., portions of SEQ ID NO:24 or SEQ ID NO:26), portions of CD8 polypeptides (e.g., portions of SEQ ID NO:27 or SEQ ID NO:28), variants of any of the foregoing, having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% identity or homology with any of the foregoing, or a synthetic spacer sequence.

[0253] In some embodiments, the hinge domain of the CAR includes a native or modified hinge region of CD28. In some embodiments, the hinge domain of the CAR includes a native hinge region of CD28. In some embodiments, the hinge domain of the CAR includes the amino acid sequence of amino acids 114-153 of SEQ ID NO:24. An exemplary nucleotide sequence encoding the amino acid sequence of amino acids 114-153 of SEQ ID NO:24 is shown in SEQ ID NO:56 provided below.

[0254]

[0255] 5.3.3. Intracellular signal transduction domains of CAR

[0256] In some embodiments, the CAR includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain of the CAR includes a CD3ζ polypeptide, which can activate or stimulate cells (e.g., lymphoid lineage cells, such as T cells). Wild-type (“natural”) CD3ζ includes three functional immune receptor tyrosine repressive motifs (ITAMs) and three functional basic-rich stretching (BRS) regions (BRS1, BRS2, ​​and BRS3). Upon antigen binding, CD3ζ transmits activation signals to cells (e.g., lymphoid lineage cells, such as T cells). The intracellular signaling domain of the CD3ζ chain is a major transmitter of signals from the endogenous TCR.

[0257] In some embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ. In some embodiments, native CD3ζ comprises, or consists of, an amino acid sequence or fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the amino acid sequence having NCBI reference number: NP_932170 (SEQ ID NO: 29), and / or may optionally comprise at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the CD3ζ polypeptide comprises, or consists of, an amino acid sequence as a continuous portion of SEQ ID NO: 29, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50 and at most about 164 amino acids. In some embodiments, the native CD3ζ comprises or is composed of the amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 29. In some embodiments, the intracellular signal transduction domain of the CAR comprises or is composed of the native CD3ζ, which comprises or is composed of the amino acid sequence of amino acids 52-164 of SEQ ID NO: 29. SEQ ID NO: 29 is provided below:

[0258]

[0259] In some embodiments, the natural CD3ζ comprises or consists of an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or homology with the amino acid sequence shown in SEQ ID NO:30. SEQ ID NO:30 is provided below:

[0260]

[0261] In some embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide. In some embodiments, the modified CD3ζ polypeptide comprises one, two, or three ITAMs. In some embodiments, the modified CD3ζ polypeptide comprises native ITAM1. In some embodiments, native ITAM1 comprises or consists of the amino acid sequence shown in SEQ ID NO:31. SEQ ID NO:31 is provided below:

[0262]

[0263] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:31 is shown in SEQ ID NO:32 provided below.

[0264]

[0265] In some embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant containing one or more loss-of-function mutations. In some embodiments, the ITAM1 variant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation in a tyrosine residue of ITAM1. In some embodiments, the ITAM1 variant consists of two loss-of-function mutations. In some embodiments, the ITAM1 variant comprises or consists of the amino acid sequence shown in SEQ ID NO:33 provided below.

[0266]

[0267] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:33 is shown in SEQ ID NO:34 provided below.

[0268]

[0269] In some embodiments, the modified CD3ζ polypeptide comprises natural ITAM2. In some embodiments, natural ITAM2 comprises or consists of the amino acid sequence shown in SEQ ID NO:35 provided below.

[0270]

[0271] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:35 is shown in SEQ ID NO:36 provided below.

[0272]

[0273] In some embodiments, the modified CD3ζ polypeptide comprises an ITAM2 variant. In some embodiments, the ITAM2 variant comprises or consists of one or more loss-of-function mutations. In some embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation in a tyrosine residue of ITAM2. In some embodiments, the ITAM1 variant consists of two loss-of-function mutations. In some embodiments, the ITAM2 variant comprises or consists of the amino acid sequence shown in SEQ ID NO:37 provided below.

[0274]

[0275] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:37 is shown in SEQ ID NO:38 provided below.

[0276]

[0277] In some embodiments, the modified CD3ζ polypeptide comprises natural ITAM3. In some embodiments, natural ITAM3 comprises or consists of the amino acid sequence shown in SEQ ID NO:39 provided below.

[0278]

[0279] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:39 is shown in SEQ ID NO:40 provided below.

[0280]

[0281] In some embodiments, the modified CD3ζ polypeptide comprises an ITAM3 variant. In some embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In some embodiments, each of one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM3. In some embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In some embodiments, the ITAM3 variant comprises or consists of the amino acid sequence shown in SEQ ID NO:41 provided below.

[0282]

[0283] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:41 is shown in SEQ ID NO:42 provided below.

[0284]

[0285] Various modified CD3ζ peptides and CARs containing modified CD3ζ peptides are disclosed in International Patent Application Publication No. WO2019 / 133969, the full text of which is incorporated herein by reference.

[0286] In some embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide, the modified CD3ζ polypeptide comprising native ITAM1, an ITAM2 variant comprising one or more (e.g., two) loss-of-function mutations or thereof, and an ITAM3 variant comprising one or more (e.g., two) loss-of-function mutations or thereof. In some embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide, the modified CD3ζ polypeptide comprising native ITAM1 consisting of the amino acid sequence shown in SEQ ID NO:31, an ITAM2 variant consisting of the amino acid sequence shown in SEQ ID NO:37, and an ITAM3 variant consisting of the amino acid sequence shown in SEQ ID NO:41. In some embodiments, the CAR is designated as "1XX". In some embodiments, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:43. SEQ ID NO:43 is provided as follows:

[0287]

[0288] In some embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide containing, or consisting of, an amino acid sequence or fragment thereof having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identity with SEQ ID NO:43, and / or optionally containing at most one, at most two, or at most three conserved amino acid substitutions.

[0289] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:43 is shown in SEQ ID NO:44 provided below.

[0290]

[0291] Another exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:43 is shown in SEQ ID NO:45 provided below.

[0292]

[0293] In some embodiments, the intracellular signaling domain of the CAR further includes at least one co-stimulatory signaling region. In some embodiments, the at least one co-stimulatory region includes a co-stimulatory molecule or a portion thereof. In some embodiments, the at least one co-stimulatory region includes an intracellular domain of at least one co-stimulatory molecule or a portion thereof.

[0294] As used herein, a “co-stimulatory molecule” refers to a cell surface molecule other than an antigen receptor or its ligand that can provide an effective lymphocyte response to an antigen. In some embodiments, a co-stimulatory molecule can provide optimal lymphocyte activation. Non-limiting examples of co-stimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, and combinations thereof. Co-stimulatory molecules can bind to a co-stimulatory ligand, which is a protein expressed on the cell surface that, upon binding to its receptor, produces a co-stimulatory response, i.e., when an antigen recognition receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen, it influences the intracellular response to the provided stimulus. As an example, a 4-1BB ligand (i.e., 4-1BBL) can bind to 4-1BB to provide an intracellular signal, which, in combination with CAR signaling, induces CAR. + Effector cell function of T cells. In some embodiments, at least one co-stimulatory signal transduction region includes an intracellular signal transduction domain or a portion thereof of CD28, an intracellular domain or a portion thereof of 4-1BB, an intracellular domain or a portion thereof of OX40, an intracellular domain or a portion thereof of ICOS, or an intracellular domain or a portion thereof of DAP-10.

[0295] In some embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of CD28 or a portion thereof). In some embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region comprising the intracellular domain of human CD28 or a portion thereof.

[0296] In some embodiments, the CD28 peptide contained in the co-stimulatory signal transduction region of the CAR comprises, or consists of, an amino acid sequence or fragment thereof having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 24, and / or optionally comprises at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the CD28 peptide contained in the co-stimulatory signal transduction region comprises, or consists of, an amino acid sequence that is a continuous portion of SEQ ID NO: 24, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50 and at most about 220 amino acids. Alternatively or additionally, in some embodiments, the CD28 polypeptide contained in the co-stimulatory signal transduction region comprises or is composed of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 180-220, or 200-220 of SEQ ID NO: 24. In some embodiments, the intracellular signal transduction domain of the CAR includes a co-stimulatory signal transduction region containing the CD28 polypeptide, which comprises or is composed of the amino acid sequence of amino acids 180-220 of SEQ ID NO: 24.

[0297] An exemplary nucleic acid sequence encoding amino acid sequence 180-220 of SEQ ID NO: 24 is shown in SEQ ID NO: 46, which is provided below.

[0298]

[0299] In some embodiments, the intracellular signal transduction domain of the CAR includes a co-stimulatory signal transduction region comprising a portion of the mouse CD28 polypeptide or thereof. In some embodiments, the CD28 polypeptide contained in the co-stimulatory signal transduction region comprises, or consists of, an amino acid sequence or fragment thereof having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identity or homology with the amino acid sequence shown in SEQ ID NO: 26, and / or optionally comprises at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the CD28 polypeptide contained in the co-stimulatory signal transduction region comprises, or consists of, an amino acid sequence as a continuous portion of SEQ ID NO: 26, having a length of at least about 20, at least about 30, at least about 40, or at least about 50 and at most 218 amino acids. In some embodiments, the CD28 polypeptide contained in the co-stimulatory signal transduction region comprises or is composed of the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 150-218, 178-218, or 200-218 of SEQ ID NO: 26. In some embodiments, the co-stimulatory signal transduction region of the CAR of this disclosure comprises the CD28 polypeptide, which comprises or is composed of the amino acid sequence of amino acids 178-218 of SEQ ID NO: 26.

[0300] In some embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region containing an intracellular domain of a 4-1BB polypeptide, such as a fragment of the 4-1BB polypeptide or a 4-1BB polypeptide. In some embodiments, the co-stimulatory signaling region comprises an intracellular domain of human 4-1BB or a fragment of the 4-1BB polypeptide. In some embodiments, the 4-1BB polypeptide contained in the co-stimulatory signaling region comprises, or consists of, an amino acid sequence or fragment having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identity or homology with the sequence having NCBI reference number: NP_001552 (SEQ ID NO: 47), and / or optionally comprises at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the 4-1BB polypeptide contained in the co-stimulatory signal transduction region comprises or is composed of an amino acid sequence that is a continuous portion of SEQ ID NO: 47, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, and / or at most about 50, at most about 60, at most about 70, at most about 80, at most about 90, at most about 100, at most about 200, or at most about 255 amino acids. In some embodiments, the 4-1BB polypeptide contained in the co-stimulatory signal transduction region comprises or is composed of an amino acid sequence of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, or 200-255 of SEQ ID NO: 47. In some embodiments, the co-stimulatory signal transduction region comprises a 4-1BB polypeptide that comprises or is composed of an amino acid sequence of amino acids 214-255 of SEQ ID NO: 46. SEQ ID NO:47 is provided below.

[0301]

[0302] In some embodiments, the intracellular signal transduction domain of the CAR includes a co-stimulatory signal transduction region comprising the intracellular domains or portions thereof of two or more co-stimulatory molecules, such as the intracellular domain or portion thereof of CD28 and the intracellular domain or portion thereof of 4-1BB, or the intracellular domain or portion thereof of CD28 and the intracellular domain or portion thereof of OX40.

[0303] 5.3.4. Exemplary CAR

[0304] In some embodiments, the CAR includes (a) an extracellular antigen-binding domain, which includes (i) V HIt comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8; and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9, and (ii) V L The domain comprises: (a) a CD28 polypeptide (e.g., the hinge domain of human CD28 or a portion thereof, e.g., the hinge domain of amino acids 114-153 of SEQ ID NO: 24); (b) a transmembrane domain comprising a CD28 polypeptide (e.g., the hinge domain of human CD28 or a portion thereof, e.g., the CD28 polypeptide comprising the amino acid sequence 114-153 of SEQ ID NO: 24); (c) a transmembrane domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof, e.g., the CD28 polypeptide comprising the amino acid sequence 154-179 of SEQ ID NO: 24); and (d) an intracellular signal transduction domain comprising (i) a CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide, e.g., a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 43) and (ii) a CD28 polypeptide (e.g., the intracellular domain of human CD28 or a portion thereof, e.g., the transmembrane ... The co-stimulatory signal transduction region of the CD28 polypeptide (composed of amino acid sequences 180-220 of IDNO:24). In some embodiments, V H and V L Linked via a connector consisting of the amino acid sequence shown in SEQ ID NO:1. In some embodiments, V H and V L The position from the N-terminus to the C-terminus is: V L -V H In some embodiments, the CAR is designated as "CAR#2". In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:48, which is provided below.

[0305]

[0306] An exemplary nucleic acid sequence of the amino acid sequence of SEQ ID NO: 48 is shown in SEQ ID NO: 49, which is provided below.

[0307]

[0308] In some embodiments, the CAR includes (a) an extracellular antigen-binding domain, which includes (i) V HIt comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8; and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9, and (ii) V L The SEQ ID NO: 16 comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 12, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 17; (b) a hinge domain comprising a CD28 polypeptide (e.g., the hinge domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of the amino acid sequence 114-153 of SEQ ID NO: 24); (c) a transmembrane domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of the amino acid sequence 114-179 of SEQ ID NO: 24); and (c) an intracellular signal transduction domain comprising (i) a CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide, e.g., a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 43) and (ii) a CD28 polypeptide (e.g., the intracellular domain of human CD28 or a portion thereof, e.g., a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 17). The co-stimulatory signal transduction region of the CD28 polypeptide (composed of amino acid sequences 180-220 of IDNO:24). In some embodiments, V H and V L Linked via a connector consisting of the amino acid sequence shown in SEQ ID NO:1. In some embodiments, V H and V L The position from the N-terminus to the C-terminus is: V L -V H In some embodiments, CAR is designated as "CAR#8". In some embodiments, CAR comprises the amino acid sequence shown in SEQ ID NO:50, which is provided below.

[0309]

[0310] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50 is shown in SEQ ID NO: 51, which is provided below.

[0311]

[0312] In some embodiments, the CAR includes (a) an extracellular antigen-binding domain, which includes (i) V HIt comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8; and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9, and (ii) V L The SEQ ID NO: 11 comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 20, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 21; (b) a hinge domain comprising a CD28 polypeptide (e.g., the hinge domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of the amino acid sequence 114-153 of SEQ ID NO: 24); (c) a transmembrane domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of the amino acid sequence 114-179 of SEQ ID NO: 24); and (c) an intracellular signal transduction domain comprising (i) a CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide, e.g., a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 43) and (ii) a CD28 polypeptide (e.g., the intracellular domain of human CD28 or a portion thereof, e.g., a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 21). The co-stimulatory signal transduction region of the CD28 polypeptide (composed of amino acid sequences 180-220 of IDNO:24). In some embodiments, V H and V L Linked via a connector consisting of the amino acid sequence shown in SEQ ID NO:1. In some embodiments, V H and V L The position from the N-terminus to the C-terminus is: V L -V H In some embodiments, the CAR is designated as "CAR#15". In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:51, which is provided below.

[0313]

[0314] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 52 is shown in SEQ ID NO: 53, which is provided below.

[0315]

[0316] 5.4. Cells

[0317] The subject matter of this disclosure provides cells comprising a CD19-targeting CAR of this disclosure (e.g., one disclosed in Section 5.3). In some embodiments, the cells are selected from lymphoid lineage cells and bone marrow lineage cells. In some embodiments, the cells are immune-responding cells. In some embodiments, the immune-responding cells are lymphoid lineage cells.

[0318] In some embodiments, the cells are lymphoid lineage cells. Lymphoid lineage cells can provide antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, and detection of foreign cells in the host. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can differentiate. In some embodiments, the stem cells are pluripotent stem cells (e.g., induced pluripotent stem cells).

[0319] In some embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of this disclosure can be any type of T cell, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. The patient's own T cells can be genetically modified to target specific antigens through the introduction of a CAR. In some embodiments, the immune response cell is a T cell. T cells can be CD4+. + T cells or CD8 + T cells. In some implementations, T cells are CD4+ cells. + T cells. In some implementations, the T cells are CD8+ cells. + T cells. In some embodiments, the cell is a T cell, and the CD19-targeting CAR of this disclosure is integrated into a site within the T cell genome. Non-limiting examples of sites include TRAC sites, TRBC sites, TRDC sites, and TRGC sites. In some embodiments, the site is a TRAC site or a TRBC site. Methods for targeting CARs to sites within the T cell genome are disclosed in WO2017180989 and Eyquem et al., Nature. (2017 Mar 2); 543(7643): 113–117, both of which are incorporated herein by reference in their entirety.

[0320] In some embodiments, the cell is an NK cell. Natural killer (NK) cells can be lymphocytes, which are part of cell-mediated immunity and play a role in the innate immune response. NK cells do not require prior activation to perform cytotoxic effects on target cells. In some embodiments, the cell is a genetically modified NK cell. In some embodiments, the cell is an edited NK cell. In some embodiments, the cell is an NK cell derived from stem cells. In some embodiments, the cell is an NK cell derived from pluripotent stem cells. In some embodiments, the cell is an NK cell derived from induced pluripotent stem cells (iPSCs).

[0321] Cells (e.g., T cells or NK cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor cells or stem cells.

[0322] The cells in this disclosure can be cells of the bone marrow lineage. Non-limiting examples of bone marrow lineage cells include monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and stem cells from which myeloid cells can differentiate. In some embodiments, the stem cells are pluripotent stem cells (e.g., induced pluripotent stem cells).

[0323] In some embodiments, cells can be transduced with the CD19-targeting CAR of this disclosure, so that the cells express the CD19-targeting CAR.

[0324] 5.5. Nucleic Acid Molecules and Vectors

[0325] The subject matter of this disclosure provides nucleic acid molecules encoding CARs targeting CD19 (e.g., those disclosed in Section 5.3). Cells containing such nucleic acid molecules are also provided.

[0326] In some embodiments, the nucleic acid molecule also includes a promoter operatively linked to the CD19-targeting CAR of this disclosure.

[0327] In some embodiments, the promoter is endogenous or exogenous. In some embodiments, the exogenous promoter is selected from the elongation factor (EF)-1 promoter, the cytomegalovirus immediate early promoter (CMV) promoter, the simian virus 40 early promoter (SV40) promoter, the phosphoglycerate kinase (PGK) promoter, the metallothionein promoter, and the ubiquitin C promoter. In some embodiments, the endogenous promoter is selected from the TCR α promoter, the TCR β promoter, and the β2-microglobulin promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, the IL-2 promoter, the 4-1BB promoter, the PD1 promoter, and the LAG3 promoter.

[0328] The subject matter of this disclosure also provides vectors containing nucleic acid molecules of this disclosure.

[0329] Nucleic acid molecules can be delivered into cells by methods known in the art or as described herein. Genetic modification of cells can be accomplished by transducing a substantially homogeneous cellular composition with a recombinant DNA construct. In some embodiments, retroviral vectors (e.g., gamma-retroviral vectors or lentiviral vectors) are used to introduce the DNA construct into cells. For example, a polynucleotide encoding a CAR can be cloned into a retroviral vector and its expression can be driven from its endogenous promoter, a retroviral long terminal repeat sequence, or a promoter specific to the target cell type. Non-viral vectors can also be used.

[0330] For the initial genetic modification of cells to include the CD19-targeting CAR of this disclosure, retroviral vectors can be used for transduction; however, any other suitable viral vector or non-viral delivery system can be used. CARs can be constructed in a single polycistronic expression cassette, multiple expression cassettes of a single vector, or multiple vectors. Examples of elements that generate polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, poliovirus IRES, foot-and-mouth disease virus IRES, microRNA virus IRES, poliovirus IRES, and encephalocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, such as P2A, T2A, E2A, and F2A peptides). Combinations of retroviral vectors and suitable packaging lines are also suitable, wherein the capsid protein will have the function of infecting human cells. Various cell lines that produce amphoteric viruses are known, including but not limited to PA12 (Miller et al., (1985) Mol Cell Biol (1985);5:431-437); PA317 (Miller et al., Mol Cell Biol (1986);6:2895-2902); and CRIP (Danos et al., Proc Natl Acad Sci USA (1988);85:6460-6464). Non-amphibious particles are also suitable, for example, those coated with VSVG, RD114, or GALV and any other pseudomorphic particles known in the art.

[0331] Possible transduction methods also include direct co-culture of cells with producing cells (Bregni et al. Blood (1992); 80:1418-1422), or culture alone with viral supernatant or concentrated vector stock with or without appropriate growth factors and polycations (Xu et al. Exp. Hemat. (1994); 22:223-230); and Hughes et al. J. Clin. Invest. (1992); 89: 1817).

[0332] Other transducing viral vectors can be used to modify cells. In some implementations, the selected vectors exhibit high infection efficiency and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94: 10319, 1997). Other viral vectors that can be used include, for example, adenoviruses, lentiviruses and adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, or herpesviruses such as Epstein-Barr virus (see also Miller, Human Gene Thera (1990); 15-14; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques (1988); 6:608-614; Tolstoshev et al., Cur Opin Biotechnol (1990); 1:55-61; Sharp, The Lancet (1991); 337:1277-78; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-22, 1987; Anderson, Science (1984); 226:401-409; Moen, Blood Cells 17:407-16, 1991; Miller et al., Biotechnol (1989); 7:980-90; LeGal La Salle et al., Science (1993); 259:988-90; and Johnson, Chest (1995) 107:77S-83S vectors). Retroviral vectors have been developed particularly well and have been used clinically (Rosenberg et al., N. Engl. J. Med (1990); 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0333] Non-viral methods can also be used for genetic modification of cells. For example, nucleic acid molecules can be introduced into cells by administering nucleic acids via lipid transfection (Feigner et al., Proc Natl Acad Sci USA (1987); 84:7413; Ono et al., Neurosci Lett (1990); 17:259; Brigham et al., Am J Med Sci (1989); 298:278; Staubinger et al., Methods in Enzymol (1983); 101:512; Wu et al., J Biol Chem (1988); 263:14621; Wu et al., J Biol Chem (1989); 264:16985), or via microinjection under surgical conditions (Wolff et al., Science (1990); 247:1465). Other non-viral gene transfer methods include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be beneficial for delivering DNA into cells. Normal gene transplantation into a subject's affected tissue can also be accomplished by transferring normal nucleic acids into a cultureable cell type (e.g., autologous or allogeneic primary cells or their progeny), followed by injection of the cells (or their progeny) into the target tissue or systemic injection. Recombinant receptors can also be derived or obtained using transposases or targeting nucleases (e.g., zinc finger nucleases, broad-spectrum nucleases, or TALEN, CRISPR). Transient expression can be obtained via RNA electroporation.

[0334] Any targeted genome editing method can also be used to deliver the CD19-targeting CAR of this disclosure into cells. In some embodiments, a CRISPR system is used to deliver the CD19-targeting CAR of this disclosure. In some embodiments, a zinc finger nuclease is used to deliver the CD19-targeting CAR of this disclosure. In some embodiments, a TALEN system is used to deliver the CD19-targeting CAR of this disclosure.

[0335] The CRISPR system of regularly spaced short palindromic repeats is a genome editing tool found in prokaryotic cells. When used for genome editing, the system comprises Cas9 (a protein that uses crRNA as its guide to modify DNA), CRISPR RNA (crRNA, containing the RNA that Cas9 uses to guide it to the correct segment of host DNA, and a region that binds to tracrRNA (usually in the form of a hairpin loop), forming an active complex with Cas9), trans-activating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and optional fragments of the DNA repair template (DNA that guides the cell repair process to allow the insertion of specific DNA sequences). CRISPR / Cas9 is typically used with plasmids transfected into target cells. The crRNA needs to be designed for each application because it is the sequence that Cas9 uses to recognize and bind directly to the target DNA in the cell. The repair template carrying the CAR expression cassette also needs to be designed for each application because it must overlap the sequences on both sides of the nick and encode the inserted sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be linked to the Cas9 gene and made into a plasmid for transfection into cells.

[0336] Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by combining a zinc finger DNA-binding domain with a DNA-cleaving domain. The zinc finger domain can be engineered to target specific DNA sequences, allowing the zinc finger nuclease to target desired sequences within the genome. The DNA-binding domain of each ZFN typically contains multiple independent zinc finger repeat sequences, each capable of recognizing multiple base pairs. The most common method for generating novel zinc finger domains is by binding smaller zinc finger “modules” with known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain derived from the type II restriction endonuclease FokI. Using an endogenous homologous recombination (HR) machine and a homologous DNA template with a CAR expression cassette, ZFNs can be used to insert CAR expression cassettes into the genome. After the target sequence is cleaved by the ZFN, the HR machine searches for homology between the damaged chromosome and the homologous DNA template, then replicates the template sequence between the two broken ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0337] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. TALEN systems function almost identically to ZFNs. They are generated by combining a transcription activator-like effector DNA-binding domain with a DNA-cleaving domain. A transcription activator-like effector (TALE) consists of a 33-34 amino acid repeat motif with two variable positions that strongly recognize specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind the desired DNA sequence, thereby directing the nuclease to cleave specific locations in the genome. cDNA expression for polynucleotide therapies can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), metallothionein promoter, or ubiquitin C promoter) and regulated by any appropriate mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, enhancers known to preferentially direct gene expression in specific cell types can be used to direct nucleic acid expression if desired. The enhancers used may include, but are not limited to, those characterized as tissue- or cell-specific enhancers. Alternatively, if the genome clone is used as a therapeutic construct, regulation may be mediated by homologous regulatory sequences or, if desired, by heterologous regulatory sequences derived from any of the promoters or regulatory elements described above.

[0338] The methods used to deliver genome editing agents / systems can vary as needed. In some embodiments, components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In some embodiments, components are delivered via viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impale infection, hydrostatic pressure, continuous infusion, sonication, magnetic transfection, adeno-associated virus, envelope protein pseudotypes of viral vectors, reproducible vector cis and trans elements, herpes simplex virus, and chemical vectors (e.g., oligonucleotides, lipid complexes, polymer bodies, multi-complexes, dendritic polymers, inorganic nanoparticles, and cell-penetrating peptides).

[0339] 5.6. Dosage Form and Administration

[0340] The subject matter of this disclosure also provides compositions comprising cells of the present disclosure, said cells comprising the CD19-targeting CAR of the present disclosure. In some embodiments, said compositions are pharmaceutical compositions further comprising a pharmaceutically acceptable carrier.

[0341] The compositions comprising the cells of this disclosure can be readily provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersants, or viscous compositions, which can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within appropriate viscosity ranges to provide longer contact time with a particular tissue. Liquid or viscous compositions may contain a carrier, which can be a solvent or dispersion medium comprising, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0342] Compositions containing the cells of this disclosure can be systematically or directly administered to a subject to induce and / or enhance an immune response to an antigen and / or treat and / or prevent vegetations. In some embodiments, the immune-responding cells of this disclosure or compositions containing them are injected directly into a target organ (e.g., an organ affected by vegetations). Alternatively, the cells of this disclosure or compositions containing them may be administered indirectly to a target organ, for example, by administration to a circulatory system (e.g., a tumor vascular system). Expansion and differentiation agents may be provided before, during, or after administration of the cells or compositions to increase the production of cells in vitro or in vivo.

[0343] The number of cells to be administered may vary depending on the subject being treated. In some embodiments, approximately 10 cells of this disclosure are administered to the subject (e.g., a human subject). 4 To about 10 10 Between, about 10 4 To about 10 7 Between, about 10 5 To about 10 7 Approximately 10 5 To about 10 9 Between, or about 10 6 To about 10 8 Between. In some implementations, approximately 1 × 10⁻⁶ is administered to the subject. 6 Approximately 5×10 8 This invention discloses a cell method. More effective cells can be applied in smaller quantities. Typically, at least approximately 1 × 10⁻⁶ cells are applied. 5 10 cells, eventually reaching approximately 1 × 10⁻⁶ cells. 10 Or more. In some implementations, at least about 1×10 5 5×10 5 1×10 6 Approximately 5×10 6 Approximately 1×10 7 Approximately 2.5 × 10 7Approximately 5×10 7 Approximately 1×10 8 Approximately 1.5 × 10 8 Approximately 2×10 8 Or approximately 5×10 8 The cells disclosed herein are administered to a subject. In some embodiments, approximately 1 × 10⁻⁶ cells are used. 6 The cells disclosed herein are administered to a subject. The precise determination of the effective dose can be made based on each subject's individual factors, including their size, age, sex, weight, and specific condition. Those skilled in the art can readily determine the dose from this disclosure and their knowledge in the art.

[0344] The cells and compositions of this disclosure may be administered by any method known in the art, including but not limited to intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to a subject. The cells of this disclosure in any physiologically acceptable carrier may generally be administered intravascularly; however, they may also be introduced into the bone or other convenient sites where the cells can find suitable sites for regeneration and differentiation (e.g., the thymus).

[0345] 5.7. Treatment Methods

[0346] The subject matter of this disclosure provides various methods of using the cells of this disclosure or compositions comprising thereof. The cells of this disclosure and compositions comprising thereof can be used in therapies or pharmaceuticals. For example, the subject matter of this disclosure provides methods for inducing and / or increasing an immune response in a subject in need. The cells of this disclosure and compositions comprising thereof can be used to reduce tumor burden in a subject. The cells of this disclosure and compositions comprising thereof can reduce the number of tumor cells in a subject, reduce the size of a tumor in a subject, and / or eradicate a tumor in a subject. The cells of this disclosure and compositions comprising thereof can be used to treat and / or prevent vegetations in a subject. The cells of this disclosure and compositions comprising thereof can be used to prolong the survival of a subject with vegetations. In some embodiments, each of the above methods includes administering the cells of this disclosure or compositions comprising thereof (e.g., pharmaceutical compositions) to achieve a desired effect, such as alleviating an existing condition or preventing recurrence. For treatment, the dosage is the amount that effectively produces the desired effect. The effective amount can be provided in a single or series of administrations. The effective amount can be provided by bolus injection or continuous infusion.

[0347] In some embodiments, the tumor and / or vegetation is associated with CD19. In some embodiments, the tumor and / or vegetation express CD19. In some embodiments, the tumor and / or vegetation overexpress CD19. In some embodiments, the tumor and / or vegetation that can be treated with the cells and compositions of this disclosure are blood cancers. Non-limiting examples of blood cancers include multiple myeloma, leukemia, and lymphoma. Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. The lymphoma may be Hodgkin lymphoma or non-Hodgkin lymphoma. In some embodiments, the lymphoma is B-cell lymphoma (BCL).

[0348] In some embodiments, the tumor and / or vegetation is a B-cell malignancy. Non-limiting examples of B-cell malignancies include B-cell lymphoma (BCL), B-cell acute lymphoblastic leukemia (ALL), B-cell chronic lymphoblastic leukemia (CLL), multiple myeloma (MM), CLL with Richter transformation, and CNS lymphoma. B-cell lymphomas include B-cell non-Hodgkin lymphoma (NHL) and B-cell Hodgkin lymphoma. In some embodiments, the tumor and / or vegetation is a B-cell lymphoma. In some embodiments, the B-cell lymphoma is a relapsed or refractory (R / R) B-cell lymphoma.

[0349] In some implementations, the subject is a human subject. The subject may have an advanced form of the disease, in which case treatment goals may include alleviating or reversing disease progression and / or reducing side effects. The subject may have a history of having previously received treatment, in which case treatment goals typically include reducing or delaying the risk of relapse.

[0350] Due to the surface expression of the CD19-targeting CAR of this disclosure, adoptive transfer cells are endowed with enhanced and selective cytolytic activity at the tumor site. Furthermore, after localization to the tumor and its proliferation, the cells transform the tumor site into a highly conductive environment for use by a wide range of cells involved in physiological antitumor responses.

[0351] The cells of this disclosure can be further modified to avoid or minimize immune complications (known as “malignant T-cell transformation”), such as graft-versus-host disease (GvHD), or the risk of GvHD-like outcomes when healthy tissue expresses the same target antigen as tumor cells. A potential solution to this problem is to engineer a suicide gene into the cells of this disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible Caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) peptides. In some embodiments, the suicide gene is an EGFRt peptide. The EGFRt peptide can be eliminated by administration of an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently linked upstream of a CD19-targeting CAR. The suicide gene can be contained within a vector comprising nucleic acid encoding the CD19-targeting CAR of this disclosure. In this manner, administration of a prodrug designed to activate a suicide gene (e.g., a prodrug such as AP1903 that can activate iCasp-9) during malignant T-cell transformation (e.g., GVHD) can trigger apoptosis in cells expressing a CD19-targeting CAR activated by the suicide gene. Incorporating the suicide gene into the CD19-targeting CAR of this disclosure improves safety and enables the elimination of a large proportion of receptor-expressing cells within a short timeframe. Cells of this disclosure incorporating the suicide gene can be preemptively eliminated at a given time point after cell infusion, or at the earliest signs of toxicity.

[0352] 7. Examples

[0353] The subject matter of this disclosure will be better understood by referring to the following embodiments, which are provided as examples of the subject matter of this disclosure and not as limitations.

[0354] Example 1 – Identification and Characterization of Anti-CD19 Antibody

[0355] This embodiment demonstrates V including the scFv disclosed herein. H and V L The anti-CD19 antibody was characterized. The human anti-CD19 antibody was derived from and produced in Adimab yeast. The antigen was biotinylated using the EZ-Link Sulfo-NHS Biotinylation Kit (ThermoScientific, Cat#21425). The antigen was concentrated to approximately 1 mg / mL, and the buffer was exchanged into PBS before adding the biotinylation reagent at a 1:7.5 molar ratio. The mixture was incubated overnight at 4°C before performing another buffer exchange to remove free biotin from solution. Biotinylation was confirmed by binding to the streptavidin sensor of the labeled protein on a ForteBio.

[0356] As mentioned earlier, eight juvenile human synthetic yeast libraries were bred, each containing approximately 10... 9 See, for example, Y. Xu et al., PEDS 26(10), 663-70 (2013); WO2009036379; WO2010105256; and WO2012009568.)

[0357] For the first two rounds of selection, magnetic bead sorting was performed using the Miltenyi MACS system, as previously described (see, for example, Siegel et al., J Immunol Methods 286(1-2), 141-153 (2004)). Briefly, yeast cells (approximately 10¹⁰ cells / library) were incubated with biotinylated antigens at 30°C for 30 min in wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)). After washing once with 40 mL of ice-cold wash buffer, the cell pellet was resuspended in 20 mL of wash buffer, and 500 μl of Streptavidin MicroBeads were added to the yeast. The pellet was then incubated at 4°C for 15 min. Next, the yeast pellet was resuspended in 5 mL of wash buffer and loaded onto a Miltenyi LS column. After loading 5 mL, the column was washed three times with 3 mL of wash buffer. The column was then removed from the magnetic field, the yeast was eluted with 5 mL of growth medium, and growth was initiated overnight.

[0358] A third round of selection was performed using flow cytometry (FACS). Approximately 2 × 10⁻⁶ cells were used. 7 Yeast pellets were washed three times with washing buffer and incubated at 30°C with 100–200 nM biotinylated antigen under equilibration conditions. The fourth and fifth rounds of selection were performed by incubating biotinylated NALM-6 and Raji cells with yeast products selected from the third round of FACS. After incubation, pre-washed M-280 Strepavidin Dynabeads (Cat#60210) were added to the yeast / mammalian cell complex and incubated. Next, the complex was separated using a DynaMag-2 magnet, and unbound supernatant was removed. The bead / cell complex was washed three times with 1 mL of selection buffer. The captured complex was then transferred to flasks containing yeast growth medium for propagation. In the sixth round of selection, the propagated yeast was subjected to either additional round of NALM-6 / Raji cell selection with 100 nM recombinant CD19 antigen or negative selection with a multispecific reagent (PSR) to remove nonspecific antibodies.

[0359] For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent as previously described (see, e.g., Y. Xu et al., PEDS 26(10), 663-70 (2013)). The yeast was then washed twice with wash buffer and stained at 4°C for 15 min with a secondary reagent of 1:100 dilution of goat F(ab')2 anti-human kappa-FITC (LC-FITC) (Southern Biotech, Cat#2062-02) and 1:500 dilution of Streptavidin-AF633 (SA-633) (Life Technologies, Cat#S21375) or 1:50 dilution of Extravidin-phycoethyrin (EA-PE) (Sigma Aldrich, Cat#E4011). After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a strainer-capped sort tube. Sorting was performed using a FACS ARIA sorter (BDBiosciences), and sorting gates were determined to select antibodies with the desired characteristics. The selection rounds were repeated until a population with all desired characteristics was obtained. After the final round of sorting, yeast cells were plate-inoculated, and individual colonies were selected for characterization.

[0360] Light chain diversification

[0361] Heavy chains derived from juvenile yeast were used to prepare a diverse library of light chains for additional selection rounds. Heavy chain plasmids were extracted from yeast, propagated in *E. coli*, and subsequently purified and converted to plasmids with a density of 5 × 10⁻⁶. 6 Diverse light chain libraries. As described above, these libraries were selected using a four-round FACS process: one round of MACS, two rounds of cell selection using Raji or NALM-6 cells, followed by a fourth round of FACS selection using recombinant CD19 antigen. For light chain diversity, the Raji and NALM-6 cell selection involved initial negative selection using engineered Raji and NALM-6 cells that had undergone targeted gene knockout of the CD19 gene. Positive selection was performed using engineered Raji and NALM-6 cells expressing and overexpressing endogenous CD19 after depletion of CD19 knockout cells. In each round of FACS selection, the libraries' (multispecific reagent) PSR binding, species cross-reactivity, and affinity pressure were assessed by antigen titration. Sorting was performed to obtain populations with the desired characteristics. Individual colonies were selected from each round of FACS selection for sequencing and identification.

[0362] Antibody production and purification

[0363] Yeast clones were grown to saturation and then induced with shaking at 30°C for 48 hours. After induction, the yeast cells were precipitated and the supernatant was harvested for purification. IgG was purified using a Protein A column and eluted with acetic acid at pH 2.0.

[0364] ForteBio K D Measurement

[0365] The binding affinity of these anti-CD19 antibodies to soluble CD19 was tested in the ForteBio Octet system (Octet RED384, generally as previously described (see, e.g., Estep et al., Mabs 5(2), 270-278 (2013)). Briefly, the ForteBio affinity assay was performed by loading IgG online onto an AHC sensor. The antibody was immobilized on an anti-human IgG needle and bound to a soluble CD19-HSA fusion protein (a CD19 extracellular domain fused to human serum albumin). The sensor was equilibrated offline in assay buffer for 30 minutes, then monitored online for 60 seconds to establish a baseline. The sensor with the loaded IgG was exposed to 100 nM of soluble CD19-HSA fusion protein antigen for 3 minutes, then transferred to assay buffer for 3 minutes for off-rate measurement. All kinetics were analyzed using a 1:1 binding model. A summary of the binding kinetics is provided in Table 1. The affinity ranges of exemplary antibodies are in the ranges of 8 nM and 20 nM. Between nM. All anti-CD19 antibodies shown in Table 1 include V containing the amino acid sequence shown in SEQ ID NO:10. H .

[0366] Table 1. Binding kinetics of anti-CD19 antibody

[0367]

[0368] Example 2 - CAR targeting CD19 generate and CAR expression

[0369] Three CD19-targeting CARs, “CAR#2”, “CAR#8”, and “CAR#15”, were generated. CAR expression on cell surfaces was examined using a PE-conjugated anti-human LNGFR antibody, followed by flow cytometry analysis. LNGFR is co-expressed with CARs via a self-cleaving adapter 2A sequence; therefore, LNGFR expression was used as an indicator of CAR expression. The results are shown in Table 2.

[0370] Table 2

[0371]

[0372] UTD: Untransduced T cells

[0373] The positive control is a CAR that targets CD19, which contains mouse scFv SJ25c1 and CD3ζ1XX, and is called "1928z1XXCAR".

[0374] Example 3 – In vitro activity of CD19-targeting CAR

[0375] To examine the cytotoxicity of these CD19-targeting CARs against CD19-expressing Raji cancer cell lines and CD19 knockout Raji cells, each target cell line was seeded in 384-well plates, with CD19CAR-T or untransduced T cells (UTDs) added at an effector cell to target cell (E:T) ratio. Wells containing only target cells and wells containing only effector cells served as controls. Cell viability was measured using a CellTiter Glo® One Solution Assay (Promega, G8462) after 24 hours. The percentage of target cells surviving was calculated by first subtracting the signal from the effector cell-only wells from the chemiluminescence signal of the co-culture wells, and then dividing by the signal from the target cell-only wells. The killing percentage was calculated by subtracting the percentage of target cell survival from 100%. Figure 1A and 1B As shown, compared with CD19 knockout Raji cells, T cells containing CAR#2, T cells containing CAR#8, and T cells containing CAR#15 all showed cytotoxic activity against Raji cells, indicating that the cytotoxic activity of T cells containing these CD19-targeting CARs is antigen-dependent.

[0376] To confirm antigen-dependent cytotoxicity, T cells containing these CD19-targeting CARs were co-cultured with either CD19-expressing Raji cancer cell lines or CD19-knockout Raji cells. Each target cell line was labeled with a small amount of purple dye and then seeded in 96-well plates, with CD19 CAR-T or untransduced T cells (UTDs) added at an effector cell to target cell (E:T) ratio. Wells containing only target cells and wells containing only effector cells served as controls. After 20 hours, cell viability was measured by staining with a live / dead dye and then analyzed by flow cytometry. The percentage of viable target cells was calculated by measuring viable cells by flow cytometry and then dividing by the signal from the well containing only target cells. The killing percentage was calculated by subtracting the percentage of viable target cells from 100%. Figure 2A and 2B As shown, compared with CD19 knockout Raji cells, T cells containing CAR#2, T cells containing CAR#8, and T cells containing CAR#15 all showed cytotoxic activity against Raji cells, confirming that the cytotoxic activity of T cells containing these CD19-targeting CARs is antigen-dependent.

[0377] T cells containing CAR#2, CAR#8, and CAR#15 were co-cultured with CD19-expressing Raji cancer cell lines and CD19 knockout Raji cells to assess the secretion of antigen-independent cytokines (IL-2 and IFN-γ). Each target cell line was seeded in 384-well plates, with CD19CAR-T or untransduced T cells added at an effector cell to target cell (E:T) ratio. Wells containing only target cells and wells containing only effector cells served as controls. After 24 hours, the supernatant was harvested, and the secreted IFN-γ and IL-2 in the supernatant were detected using the Intellicyt QBeads Human PlexScreen kit (Sartorius, 90702). IL-2 secretion results are shown below. Figure 3A and 3B The results of IFN-γ secretion are shown in [the original text]. Figure 4A and 4B Compared to CD19 knockout Raji cells, T cells containing these CD19-targeting CARs exhibited IL-2 and IFN-γ secretion when co-cultured with Raji cells, indicating antigen-dependent cytokine release from T cells containing these CD19-targeting CARs.

[0378] Example 4 – In vivo activity of CD19-targeting CAR

[0379] The next step is to investigate the in vivo activity of T cells containing these CD19-targeting CARs.

[0380] NOD-scid IL2Rg null (NSG) mice were intravenously injected (iv) with 0.5 × 10⁻⁶ 6 NALM6 cells (B-ALL cell line) expressing firefly luciferase, with wild-type CD19 levels (CD19-WT) or reduced CD19 levels (CD19-low, NALM6 clones generated by CRISPR-Cas9 editing of the CD19 gene, subsequently re-expressed CD19 using a lentiviral vector). Four days later, fresh 5 × 10⁵ cells were injected intravenously. 4 One (red) or 2×10 5 Mice were treated with one (blue, green) #17, #8, #15, or #2 CAR-T cells to carry CD19-WT (blue, red) and CD19-low (green) NALM6. Repeated imaging of mice treated with CAR-T cells was performed within 60–100 days to assess tumor response and potential recurrence, thus rigorously evaluating CAR T-cell efficacy. Tumor burden was analyzed by bioluminescence imaging on day 30 post-T-cell infusion. Figure 5The time points shown are presented for each mouse. #2 CAR T cells exhibited rapid and effective anti-tumor activity, leading to complete tumor elimination by NALM6 WT and NALM6 CD19-low cells at all tested T cell doses. #17 showed inadequate tumor control, resulting in rapid tumor progression, and served as a control.

[0381] Example 5-#2 Binding epitope of scFv

[0382] Anti-CD19 antibodies binding to the CD19-expressing NALM6 cell line were determined by serial dilution and flow cytometry. Competitive binding of CD19-T2 conjugates, SJ25c1 (from the 19-28z CAR), and FMC63 (from Kymriah's "Tisagenleleucel" and Yescarta's "Axicabtagene ciloleucel") scFv to CD19 was assessed by Biacore SPR. A mixture of 100 nM CD19-HSA-His (Takeda, Cambridge) and increasing concentrations of soluble CD19-T2 SJ25c1 or FMC63-scFv (0, 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM) was flowed through a Biacore CMS chip with immobilized CD19-T2 scFv for 3 min. The dissociation of the complex bound to the chip was assessed by infusing HBS-EP buffer (300 mM NaCl) for 5 minutes. CD19-HSA-His was purified from HEK293, which was transiently transfected with pcDNA3.4 plasmid (ThermoFisher) containing a gene expressing the CD19 extracellular domain fused with a 10-decahistine tag, a TEV cleavage site, a GS adapter, and HAS. Recombinant CD19-HSA-His10 was purified using nickel affinity chromatography. The amino acid sequence of CD19-HSA-His10 is shown below. Figure 6 As shown.

[0383] Epitope combination data for SJ25c, FMC63, and #2 scFv are as follows: Figure 7 As shown. Figure 7 As shown, #2 scFv competes with FMC63 and SJ25C1 for binding to CD19. These findings confirm that #2 scFv, FMC63, and SJ25C1 scFv bind to overlapping epitopes on CD19. Unrelated antibodies (anti-CTLA4 VHH) that do not bind to CD19 failed to compete with #2 scFv for binding to CD19.

[0384] Example 6 – Pharmacological Study

[0385] Summarize

[0386] To support the first human clinical trial, a series of studies were conducted to demonstrate that #2 CAR T cells (referred to in this example as "19(T2)28z1XX CAR") improved efficacy compared to 1928z CAR T cells (which have been widely used in previous clinical trials), measured by increased antitumor potency and improved response durability at low T cell doses. CAR T cells engineered to express CAR showed comparable cytolytic activity, cytokine secretion, and proliferation in vitro, but #2 CAR T cells achieved greater potency in vivo due to their stronger functional durability (Feucht et al., Nature Medicine (2019);25(1):82-88).

[0387] result

[0388] In vitro cytotoxic activity of #2 CAR T cells and 1928z-1XX CAR T cells was measured using an 18-hour bioluminescence assay. The two CARs are identical, but their scFvs differ (#2 vs. SJ25c1). Retroviral vectors containing #2 CARs are shown below. Figure 8 As shown. As previously described (Przybylowski et al., (2006);13(1):95-100), recombinant retroviral particles were produced by HEK293 GalV9 packaging cells. NALM6 cells expressing firefly luciferase (FFL) were used as target cells. T cells were isolated from human PMBCs by negative selection, activated for 48 h with a CD3 / CD28 antibody bound to magnetic beads, and transduced with a γ-retroviral vector expressing CAR after the magnetic beads were removed. The CAR+ population was determined by LNGFR assay co-expressing the CAR gene. CAR+ T cells were incubated with target NALM6 cells for 18 h at different effector (E) to target (T) ratios, and then FFL activity was quantified. The cytotoxic activity of #2 CAR T cells was compared with that of 1928z-1xx CAR T cells, which were previously shown to effectively kill CD19 in vitro. +NALM6 cells (Feucht et al., Nature Medicine (2019);25(1):82-88). Untransduced (UT) T cells were used as a negative control. NALM6-CD19KO is a cell line generated by CRISPR-Cas9 editing of the CD19 gene (Hamieh et al., Nature (2019);568(7750):112-116) and was used to demonstrate the specificity of CAR T cell cytotoxic activity. A representative experiment (n=2 independent experiments on 2 healthy donors) is shown. Figure 10 As shown, #2 CAR T cells and 1928z-1XX CAR T cells exhibited comparable cytotoxicity.

[0389] Next, the in vivo activity of #2 CAR T cells (denoted as "19(T2)28z1XX CAR") and 1928z-1XX CAR T cells was measured. NALM6, a tumor cell line from pre-B acute lymphoblastic leukemia patients expressing normal (wt) or low (low) levels of CD19 antigen, was transduced using firefly luciferase and green fluorescent protein (GFP) (Zhao et al., Cancer Cell (2015);28:415-428). CD19-carrying cells... + wt (blue) and CD19 + Low (red) NALM6 mice were administered a single intravenous injection of 2 × 10⁻⁶ fresh mice. 5 Mice were treated with untransduced T cells, #2 CAR T cells, or 1928z-1XX CAR T cells. Tumor burden was monitored in each mouse for 85 days using bioluminescence imaging (BLI). Each group consisted of 5 mice, and the response of each mouse was as follows: Figure 11 As shown. Figure 11 As shown, #2 CAR T cells and 1928z1XX CAR T cells exhibit similar tumor elimination kinetics. CAR cDNA is co-expressed with the LNGFR reporter (to monitor T cell transduction). “19(T2)28z1XX CAR” represents “#2 CAR”.

[0390] The activity of #2 CAR T cells and 1928z CAR T cells was studied and compared. First, CAR expression and phenotype were measured in 1928z CAR T cells and #2 CAR T cells (denoted as "19(T2)28z1XX CAR T"). Vectors containing 1928z CAR were used... Figure 12 As shown. CD4 was transduced using a γ-retroviral vector. + and CD8 +T cells expressing either the 1928z CAR or the #2 CAR (denoted as "19(T2)28z1XX CAR") were produced using a clinically representative manufacturing process. Since the SJ25c1 scFv in SGF-1928z is mouse-derived, while the #2 scFv (denoted as "19(T2)scFv") is humanized, two distinct goat antibodies specific to the mouse (GAM) or human (GAH) Fc fraction of the IgG heavy chain were used to detect CAR surface expression. Figure 13 As shown, over 50% of CD4 + and CD8 + T cells express CAR.

[0391] Next, the phenotype of CAR T cells was determined by flow cytometry using antibodies that identify naive, central memory, effector memory, and effector T cells, as well as exhaustion markers (LAG3, PD1, and Tim3). "SFG-T2-1XX-GAH" refers to CD4 / CD8 T cells transduced with a gamma-retroviral vector expressing 19(T2)28z1XX CAR (representing "#2 CAR"), while "SFG-1928z-GAM" is derived from T cells transduced with a gamma-retroviral vector expressing 1928z CAR. Figure 14 As shown, the phenotypes of 1928z CAR T cells and #2 CAR T cells are similar. The CD4 / CD8 T cell ratio is similar in both 1928z and #2 CAR T cells, and the T cell differentiation status (CD4 and CD8 T cells) is comparable in both CAR groups, as determined by CD62L / CD45RA and CD45RA / CCR7 staining. The expression of exhaustion markers PD1, LAG3, and TIM3 did not show any major differences between the two CAR T cell groups.

[0392] Subsequently, the in vivo activity of #2 CAR T cells and 1928z CAR T cells was evaluated and compared. Mice were injected via tail vein with 5 × 10⁵ cells. 5 CD19 + Four days after NALM6-FFLuc / GFP cells were introduced, they were given thawed, cryopreserved, untransduced or CAR T cells. Figure 15 As shown, #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") provided superior tumor control compared to 1928z CAR cells. CD19-carrying patients treated with untransduced or un-CAR T cells... + The survival rate of NALM6 mice was plotted on Figure 16 The Kaplan-Meier curve diagram is shown below. (As shown in the diagram...) Figure 16As shown, CD19-carrying cells were treated with #2 CAR T cells (represented as "19(T2)28z1XX CAR T cells"). + NALM6 mice had longer survival times than mice treated with 1928z CAR T cells (all CAR T cell doses were less than 1 × 10⁻⁶ per mouse). 6 (1928z CAR T cell). All recipients of 1928z CAR T cells died on day 25, while only one mouse died in the group receiving #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells"). At the highest dose (1×10⁻⁶ cells), 6 At that time, one mouse died in both the control group and the experimental group.

[0393] CD19 was also measured. + In vivo persistence of #2 CAR T cells (denoted as "19(T2)28z1XXCAR T cells") and 1928z CAR T cells in a NALM6 leukemia mouse model. Persistence of CAR T cells and number of CAR T cells in bone marrow at 17 days post-treatment (n=5 per group) are shown in the figure. Figure 17 As shown. Figure 17 The figure shows the percentages of PD1, LAG3, and TIM3 expression in CAR T cells, as well as the T expression levels of CAR T cells. n T cm T eff and T em percentage.

[0394] like Figure 17 As shown, by day 17, #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells"), including CD4, were readily detectable in the bone marrow. + and CD8 + CAR T cells. 1928z CAR T cells were scarce, consistent with their limited persistence (Zhao et al., Cancer Cell (2015);28:415-428). The individual phenotypes of persistent T cells detected on day 17 were generally similar (Tn, Tcm, and Tem), showing a slightly higher proportion of effector T cells (Teff) in the #2 CAR group (denoted as "19(T2)28z1XX CAR"). The latter expressed more PD1 and comparable levels of LAG3 and TIM3. This observation confirms that CAR T cells expressing the CD28 / CD3z-1XX signaling motif are more persistent (Feucht et al., Nature Medicine (2019);25(1):82-88).

[0395] These results confirm the greater potency of #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") compared to 1928z CAR T cells (higher tumor eradication rates at four dose levels). This study further confirms that the phenotype of #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") is generally similar to that of 1928z CAR T cells and consistent with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells"), achieving better tumor control due to their greater persistence compared to the shorter-lived 1928z CAR T cells.

[0396] Methods and Materials

[0397] Cell lysis assay

[0398] Luciferase-based assays were used to measure the cytotoxicity of T cells transduced with different CAR constructs. NALM6 cells expressing FFLuc-GFP were used as target cells. Effector cells and tumor target cells were co-cultured in black-walled 96-well plates at a specified effector / target cell ratio, in triplicate. X-VIVO15 was seeded at a total volume of 100 μL per well with 5% human AB serum (Gemini), 10 U / ml IL-7, and 10 U / ml IL-15 (PeproTech, Inc.) added to 5 × 10⁶ human AB serum (Gemini), 10 U / ml IL-7, and 10 U / ml IL-15 (PeproTech, Inc.). 4 Target cells were used. Maximum luciferase expression (relative light units (RLU)) was determined using target cells seeded individually at the same cell density. After co-culturing for 18 hours, 100 µL of luciferase substrate (Bright Glo; Promega) was added directly to each well. Emission was measured using a luminescent plate reader. Tumor cell lysis was calculated as (1 - (RLU sample) / (RLU maximum)) × 100.

[0399] Isolation, transduction, and expansion of genetically modified human T cells

[0400] Erythrocyte sedimentation rate (ESR) amber layers from unidentified healthy donors were purchased from the New York Blood Center (not subject to institutional review). All blood samples were processed according to established ethical and safety procedures. Peripheral blood mononuclear cells were isolated by density gradient centrifugation as previously described (Zhao et al., Cancer Cell (2015);28:415-428) and activated with Dynabeads® ClinExVivo™ CD3 / CD28 antibody beads, followed by transduction with a γ-retroviral vector via centrifugation on RetroNectin-coated plates (Takara). Two days later, activated T cells were debeaded and transduced via spinoculation as previously described (Hollyman et al., J. Immunotherapy (2009);32(2):160-180). The transduced cells were then amplified in G-Rex 6M well plates according to the manufacturer's instructions (Wilson Wolf Corporation). Transduction efficiency was determined 5 days post-transduction. CAR-T cells can be used fresh or frozen in Plasmalyte in 50% CS10 and 5% HSA and then thawed for use.

[0401] Mouse whole-body tumor model

[0402] In accordance with protocols approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center (MSKCC), 6- to 12-week-old male NOD / SCID / IL-2Rγnull mice were used. All relevant animal use guidelines and ethical standards were followed. Typically, mice were inoculated via tail vein injection of 0.5 × 10⁻⁶ oz. 6 NALM6 (CD19) + NALM6 cells (wt or low expression)-FFLuc-GFP tumor cells were injected into mice, and each mouse received a specific dose of CAR-T cell therapy 4 days after tumor injection. NALM6 cells provided a homogeneous tumor burden, and no mice were excluded prior to CAR-T cell therapy. Randomization and blinding were not used.

[0403] Bioluminescence imaging

[0404] Tumor burden was assessed as previously described (Gade et al., Cancer Research (2005);65(16):9080-9088). In short, mice were intraperitoneally injected with D-fluorescein (Xenogen, 3 mg per mouse) resuspended in PBS for 10 minutes under 2% isoflurane anesthesia, and imaging was performed. The IVIS imaging system (PerkinElmer) was used to image the bioluminescence of the injected tumors. Data were analyzed using in vivo imaging software (PerkinElmer).

[0405] Antibodies for flow cytometry

[0406] The following fluorophore-conjugated antibodies were used to detect in vitro generated antibodies or at 1×10⁻⁶. 6 Expression of surface markers of CAR T cells isolated from bone marrow and spleen of mice 17 days after CAR T treatment: APC-Cy7 anti-human CD8 (SK1), APC-Cy7 mouse anti-human CD45 (2D1), BUV395 mouse anti-human CD4 (SK3), BV421 mouse anti-human CD62L (DREG-56), BV650 mouse anti-human CD45RA (HI100), BV480 or BV510 mouse anti-human PD-1 (EH12.1) and BUV737 mouse anti-human CD19 (SJ25C1) (BD Bioscience); PE-Cy7 anti-human CD8 (SK1) and PerCP-eFluor 710 anti-human LAG-3 (3DS223H) (eBioscience); PerCP anti-human CD45RA (HI100), Brilliant Violet 785 anti-human Tim-3 (F38-2E2) and PE anti-human CD127 (IL7Ra) (Biolegend). Expression of the 1928z CAR was detected using Alexa Fluor 647 goat anti-mouse AffiniPure IgG, F(ab')2 fragment. For detection of #2 CAR (denoted as "19(T2)28z1XX CAR"), Alexa Fluor 647 goat anti-human IgG, F(ab')2 fragment was used. 7-AAD (Beckman Coulter) was used as the reactive dye. Flow cytometry was performed on a Cytek Aurora instrument (Cytek Biosciences). Data were analyzed using FlowJo software v.10.1 (FlowJo LLC).

[0407] Statistical analysis

[0408] All statistical analyses were performed using Prism 8 (GraphPad) software. Statistical comparisons between the two groups were determined using a two-tailed unpaired t-test. For in vivo studies, overall survival was displayed using Kaplan-Meier curves. Statistical significance was defined as p-value < 0.05.

[0409] Example 7 – Toxicity Assessment

[0410] Summarize

[0411] The toxicity of #2 CAR T cells (denoted as "19(T2)28z1XXCAR T cells") was evaluated in NSG mice carrying NALM6 leukemia. Mice were treated with the same dose of 1928z CAR T cells intravenously as a control. Recorded parameters included body weight, tumor burden, and survival time for all mice. More extensive toxicology studies focused on the highest dose (1×10⁻⁶). 6 Mice were treated with CAR T cells per recipient at a dose exceeding the lowest therapeutic (cured) dose in this mouse model (10x) and also exceeding the clinically used CAR T cell doses proportional to recipient body weight (10-100x). These studies included T cell counts and phenotypes at the primary tumor site (bone marrow), serum cytokine assays (human, i.e., T cell-derived, and mouse, i.e., host-derived), baseline serum biochemistry (including liver enzymes, peripheral blood cell counts, and organ weight), and comprehensive tissue immunohistochemistry at the end of the study (days 27-28). The infused CAR T cells were produced in a clinically relevant manner and were comparable between the two CAR T cell products.

[0412] There were no significant differences in body weight and survival time between the experimental and control groups. Serum cytokine levels were also comparable, and no CRS was observed in either group. On day 10, ALT and AST enzyme levels were comparable between the two groups. On days 27 / 28, mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") had lower enzyme levels than mice treated with 1928z CAR T cells.

[0413] As expected, #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") had a longer lifespan than 1928z CAR T cells. Mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") had higher lymphocyte and reticulocyte counts than those treated with 1928z CAR T cells. Mice receiving 1928z CAR T cells exhibited diffuse tumor infiltration, but this was not observed in mice receiving #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells"). Mice with persistent #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") showed mild to moderate xenogeneic GVHD and hepatocellular necrosis.

[0414] Although GVHD is expected in NSG mice treated with high doses of human CAR T cells, the mechanism of the latter remains uncertain. Given the xenogeneic nature of the study (human T cells readily elicit xenogeneic responses in mouse tissues) and its use of a single donor (an unknown potential repertoire of T cell receptors), the significance of this adverse event for human autologous therapy remains unclear.

[0415] The rationale for animal models

[0416] The NALM6 model in NSG mice has been widely used to assess the activity of CD19-specific CAR T cells in Sadelain's laboratory (Zhao et al., Cancer Cell (2015);28:415-428, Feucht et al., Nature Medicine (2019);25(1):82-88, Hamieh et al., Nature (2019);568(7750):112-116, Eyquem et al., Nature (2017);543(7643):113-117) and others. NALM6 is a human pre-B acute lymphoblastic leukemia (ALL) that expresses CD19 but does not express CD80, CD86, 4-1BBL, or other major co-stimulatory ligands.

[0417] Reasonableness of dosage and schedule

[0418] Based on % transduction efficiency and total viable cells, a single intravenous injection of 1×10⁶ cells was recommended. 6 One live CAR T cell was selected as the dose, which corresponds to the lowest effective therapeutic dose in this animal model (1 × 10⁻⁶). 5 10 times that of CAR T cells (see 10 times) (see Figure 15 For adults, when converted from mouse weight to adult weight, the CAR T-cell dose is approximately 10-100 times higher than the approved CD19 CAR T-cell dose (e.g., the approved dose is 0.1-1.5 × 10⁻⁶). 8 (One live CAR T cell). #2 The recommended starting unit dose for human clinical trials of CAR T cells is 25 × 10⁻⁶ cells. 6 CAR T cells.

[0419] method

[0420] Test sample and carrier

[0421] The test samples were 1928z CAR T cells, #2 CAR T cells (referred to as "19(T2)28z1XXCAR T cells" in this embodiment), and untransduced T cells prepared by MSKCC's Cell Therapy and Cell Engineering Facility (CTCEF). Cells were frozen in liquid nitrogen prior to the start of the study. On the day of T cell injection, the cells were thawed at 37°C and washed with Plasmalyte containing 1% HSA. The cells were then formulated into 1×10⁶ cells per mouse in a 200 μL volume using Plasmalyte containing 1% HSA. 6 5×10 5 2×10 5 and 1×10 5 The dose of CAR-T cells. The number of CAR T cells is calculated by multiplying the total number of cells by the CAR dose. + Determined by percentage, CAR + The percentages were predetermined using flow cytometry.

[0422] Test System

[0423] Use injection 0.5×10 6 Six- to 12-week-old male NOD / SCID / IL-2Rγnull mice with FFLuc-GFP NALM6 cancer cells were used in the experiment. Four days after tumor cell infusion, thawed cryopreserved 1×10⁻⁶ cells were... 6 A CD4 / CD8-selected T cell was infused into mice, which was transduced with a γ-retroviral vector of 1928z CAR or #2 CAR (denoted as "19(T2)28z1XX CAR"). Untreated mice were expected to develop hindlimb paralysis due to myeloinfiltrative leukemia on days 16–18.

[0424] The wild-type NALM6 human tumor cell line is an excellent model of pre-B acute lymphoblastic leukemia. The Sadelain laboratory has used this model in many peer-reviewed publications (Zhao et al., Cancer Cell (2015);28:415-428, Feucht et al., Nature Medicine (2019);25(1):82-88, Hamieh et al., Nature (2019);568(7750):112-116, Eyquem et al., Nature (2017);543(7643):113-117).

[0425] Research Design

[0426] 115 mice were randomly assigned to a 1928z CAR group, a #2 CAR group (denoted as "19(T2)28z1XX CAR"), and a non-transduced group. All mice received 0.5 × 10⁻⁶ CAR via tail vein injection 4 days prior to T-cell therapy. 6 FFLuc-GFP NALM6 tumor cells. On day 0 of the study, mice were assigned to the treatment group. Each group received a single intravenous dose (200 µl) of the test substance. Table 3 lists the group assignments.

[0427] Table 3 – Experimental Design and Group Assignment

[0428]

[0429] *BLI – Perform bioluminescence imaging (CAR T cell infusion) weekly, starting from day 0.

[0430] ‡ - Untransduced CD4 / CD8 T cells.

[0431] ‡‡ - All mice were weighed on days 7, 11, 21, and 27, until the animals were interim or finally euthanized.

[0432] Toxicological parameters

[0433] Toxicity was monitored by changes in body weight following administration of the test product. Ten and twenty-eight days after administration, 1×10⁻⁶ 1928z CAR T cells and #2 CAR T cells (denoted as "19(T2)28z1XX CART cells") were collected from the orbital venous plexus after anesthesia with 2% isoflurane. 6 Blood samples from the dosage groups. Blood samples were collected in test tubes containing a serum separator and used to measure cytokine and liver enzyme levels. On days 27 and 28, 1×10⁻⁶ serum samples were taken from 1928z CAR and #2 CAR (denoted as "19(T2)28z1XX CAR"). 6 Mice in the dosage group underwent full autopsy analysis on each designated day.

[0434] Cytokine analysis

[0435] On days 10 and 28 following CAR T therapy, patients receiving 1×10⁻⁶ t / v were observed. 6Mouse serum was collected from the CAR T-cell doses of 1928z CAR and #2 CAR (denoted as "19(T2)28z1XX CAR"). Samples were stored at -80°C until analysis. ProcartaPlex assays were used to quantify levels of mouse cytokines (MCP-1, IL-6, and G-CSF) and human cytokines (IL2, IL3, IFN-γ, GM-CSF, granzyme B, and TNF-α). All ProcartaPlex-related reagents used in this study were purchased from ThermoFisher Scientific. Serum collected from a mouse model with severe cytokine release syndrome was used as a positive control (Giavridis et al., Nature Medicine (2018);24(6):731-738). 60 μL beads from a single-cytokine simplex kit were pooled, and 50 μL of the bead mixture was dispensed into each well of a 96-well plate according to the manufacturer's protocol. Standards, controls, and samples diluted 1:4 with universal analytical buffer were added to the wells. Seal the plate, shake at 500 rpm for 30 minutes at room temperature, and transfer to 4°C. After incubation overnight, shake the plate again at 500 rpm for 30 minutes at room temperature. Wash the plate and add the detection antibody to each well. Seal the plate and shake at 500 rpm for 30 minutes at room temperature. After washing, add Streptavidin RPE solution to each well. Seal the plate and shake at 500 rpm for 30 minutes at room temperature. After the final washing step, resuspend the beads in reading buffer and obtain the assay signal on a Luminex 200 instrument. Standard curves for individual cytokines were generated using a 5-parameter logistic equation, and the concentration of individual cytokines in each sample was calculated using xPONENT software (version 4.2.1509.0).

[0436] statistics

[0437] All statistical analyses were performed using Prism 8 (GraphPad) software. Statistical comparisons between the two groups were determined using a two-tailed unpaired t-test. Statistical significance was defined as p-value < 0.05.

[0438] result

[0439] Changes in mouse body weight were measured during treatment with 1928z CAR T cells and #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells"). NALM6 tumor-bearing mice were treated with 1×10 6 5×10 5 2×10 5 Or 1×10 5Treatment with either 1928z CAR T cells or #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") (n=10 per group) was administered, while untransduced T cells served as the control group (n=5). Results were as follows: Figure 18 As shown. Figure 18 As shown, the weight changes were similar between the two CAR T-cell therapies.

[0440] Next, liver enzymes in the serum of mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") and 1928z CART cells were measured. Figure 19 Shown on days 10 and 27 / 28 (dose level = 1×10⁻⁶) 6 Quantification of liver enzyme levels in mice treated with two CAR T receptors (n=10 per group) using 1×10⁻⁶ CAR T receptors. Assessment was performed on days 10 and 27 / 28 using 1×10⁻⁶ CAR T receptors. 6 ALT and AST levels in NALM6 tumor-bearing mice treated with 1928z CAR T cells or #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells"). Figure 19 As shown, on day 10, the ALT and AST enzyme levels were comparable in both study groups. On days 27 / 28, mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") had lower enzyme levels than mice treated with 1928z CAR T cells.

[0441] Measurements were taken on days 10 and 27 / 29 following infusion, using 1×10⁻⁶ units. 6 Human and mouse cytokine levels in mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") or 1928z CAR T cells. Figure 20 This shows the quantitative levels of human cytokines in serum obtained from two different CAR T-treated mice on days 10 and 27 / 28 (dose level = 1 × 10⁻⁶). 6 CAR T cells, n=10 per group. Serum collected from a mouse model with severe cytokine release syndrome was used as a positive control (n=2) (Giavridis et al., Nature Medicine (2018);24(6):731-738). IL2, IL3, GM-CSF, granzyme B, and TNF-α levels were comparable between the two CAR T cell groups on day 10 and day 27 / 28. On day 27 / 28, IFN-γ levels in the 19(T2)28z1XX CAR T cell treatment group were higher than those in mice receiving 1928z CAR T cells.

[0442] Figure 21 The results showed that 1928z CAR T cells or #2 CAR T cells (represented as "19(T2)28z1XX CAR T cells") were administered on days 10 and 27 / 28 (dose level = 1×10⁻⁶). 6 Quantitative results of mouse cytokine levels in serum of mice treated with #2 CAR T cells (n=10 per group). Serum collected from a mouse model with severe cytokine release syndrome was used as a positive control (n=2) (Giavridis et al., Nature Medicine (2018);24(6):731-738). On day 10, mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX-CAR T cells") had slightly higher levels of MCP-1 and G-CSF than mice infused with 1928z CAR T cells, while IL-6 levels were comparable in both CAR T cell groups. On days 27 / 28, MCP-1, IL-6, and G-CSF levels in mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX-CAR T cells") were similar to those in the 1928z CAR T group. These observations suggest no evidence of cytokine release syndrome (CRS) in either group.

[0443] Blood cell counts were measured in mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") and 1928z CAR T cells 27 / 28 days after infusion. Figure 22 The images show two different CAR T treatment groups on day 27 / 28 (dose level = 1 × 10⁻⁶). 6 Basic hematologic quantification (CAR T, n=10 per group). Comparisons of neutrophil, lymphocyte, and monocyte counts, as well as reticulocyte counts, are shown. Figure 22 As shown, neutrophils and monocytes in the two CART groups (1×10⁻⁶) 6 The dose levels were comparable. The lymphocyte and reticulocyte counts in mice treated with 19(T2)28z1XX CAR T cells were higher than those in the 1928z CAR T cell group.

[0444] In addition, the body weight and organ weight of mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") and 1928z CAR T cells were measured at necropsy. The two different CAR T treatment groups (dose level = 1 × 10⁻⁶) were compared on day 27 / 28. 6 The body weight and major organ weight of each CAR T (cartilaginous tissue sample). Results as follows Figure 23 As shown. Figure 23As shown, the body weight, spleen, and kidney weights of the two treatment groups 1 and 2 were comparable. The liver weight of mice in the 19(T2)28z1XX CAR T treatment group (labeled "SFG-19(T2)28z-1XX") was lighter than that of mice in the 1928z CAR T group (labeled "SFG-1928z"). The difference in heart weight was attributed to uneven technical treatment.

[0445] The CD3+ levels in the liver, bone marrow, and spleen of mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") and 1928z CAR T cells were measured. + and CD19 + Immunohistochemistry. CAR T therapy (dose level = 1 × 10⁻⁶) 6 Assessment of lymphocyte and tumor infiltration levels in the liver, spleen, and bone marrow of mice 27 / 28 days after CAR T (n=6) administration: Figure 24 As shown. Figure 24 As shown, mice in the 19(T2)28z1XX CAR T group (labeled "SFG-19(T2)28z-1XX") had higher CD3+ cell levels in all assessed organs than animals treated with 1928z CAR T cells (labeled "SFG-1928z"). Tumor infiltration was observed in all tested organs in the 1928z CAR T group, but not in the 19(T2)28z1XX CAR T group.

[0446] in conclusion

[0447] The high dose for each recipient is 1×10 6The toxicity curves of #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") were compared with those of 1928z CAR T cells. No differences in whole body weight or survival were found. Serum cytokine levels measured on day 10 were comparable between the two groups, indicating that #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") were less likely to induce cytokine release syndrome (CRS) than 1928z CAR T cells. Liver enzymes increased in both groups on days 10 and 17, but the increase was less pronounced in recipients of #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") than in recipients of 1928z CAR T cells. Peripheral blood cell counts in recipients of #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") showed elevated lymphocyte counts, consistent with the stronger persistence of the latter as recorded in FACs analysis (day 17) and tissue IHC (days 27 / 28). Isolated reticulocytosis was observed without anemia, polycythemia, or erythropoiesis, and therefore was not considered an adverse event.

[0448] Organ weights were similar between the two groups, except for the liver; mice treated with 1928z CAR T cells had heavier livers due to substantial tumor progression (BLI and IHC). Conversely, mice treated with #2 CAR T cells (denoted as "19(T2)28z1XXCAR T cells") showed no tumors by IHC, confirming the BLI results in these same mice. Multi-organ lymphocytic infiltration was observed in #2 CAR T cell (denoted as "19(T2)28z1XX CAR T cells") recipient mice, consistent with xenogeneic GVHD. The latter was confirmed by the presence of epithelial necrosis in these tissues. Mild to moderate hepatocellular necrosis was also observed in mice infused with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells"). This microscopic result is generally unrelated to GVHD and therefore represents a possible adverse event. Its effect on liver function is limited, given the modest elevation of liver enzymes (AST only, not ALT) and normal bilirubin levels. Other pathological findings were minor and identical between the two treatment groups, and therefore not considered representative of important outcomes. The T-cell flow cytometry atlases were similar between the two treatment groups, consistent with the expected outcome that #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") demonstrated stronger therapeutic efficacy due to the greater persistence of CAR T cells.

[0449] A toxicity study evaluating #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") compared with clinically tested 1928z CART cells (Park et al., N. Engl. J. Med. (2018);378(5):449-459) found mild to moderate GVHD and mild to moderate hepatocellular necrosis in mice treated with #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells").

[0450] The data shown in this example and Example 6 lead to the conclusion that #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") are more effective because they have greater persistence than 1928z CAR T cells without posing a greater risk. The relatively enhanced potency of #2 CAR T cells (denoted as "19(T2)28z1XX CAR T cells") supports the efficacy of 25 × 10 6 This is a human clinical trial design that starts with a dose of CAR T cells, which is a relatively low dose compared to most CAR trials targeting CD19.

[0451] Example 8 – Pharmacological Study

[0452] On-cell binding of anti-CD19 antibody

[0453] The scFv containing this disclosure was evaluated by flow cytometry in Raji and NALM-6 cell lines expressing endogenous CD19. H and V L The anti-CD19 antibody was tested on cells to confirm target-specific binding. Each antibody was tested on CD19-positive parental lines and the corresponding CD19 knockout Raji and NALM-6 lines to confirm target-specific binding on cells. Figure 25 The flow cytometry chromatograms shown indicate that CD19-positive cell lines exhibit a 1-2 log shift compared to the background binding of the corresponding knockout cell lines.

[0454] Figures 26A-26C The serial dilution flow cytometry results shown demonstrate saturated binding on NALM-6 cells. EC50 values ​​calculated from the curves are provided in Table 4. Exemplary anti-CD19 antibodies bind to CD19-positive NALM-6 cells with a high affinity of at least 0.2 nM. The higher affinity observed on CD19-positive cells compared to soluble proteins suggests that the epitopes bound by these antibodies may be more naturally present on cells than in the CD19-HSA fusion protein. All anti-CD19 antibodies shown in Table 4 include V containing the amino acid sequence shown in SEQ ID NO:10. H .

[0455] Table 4. EC50 of anti-CD19 antibody on NALM6 cells

[0456]

[0457] Example 9 – CD19-targeting 19(T2)28z1XX CAR T cells in relapsed or refractory B-cell malignancies Phase I study in 20-year patients

[0458] This embodiment describes a phase I study of CD19-targeting 19(T2)28z1XX CAR T cells in adult patients with relapsed or refractory B-cell malignancies. "19(T2)28z1XX" and "19(2)28z1XX" are used interchangeably here. Autologous CAR T cell therapy targeting the B-cell-specific surface antigen CD19 has shown promising clinical responses in relapsed or refractory (R / R) B-cell lymphoma (BCL). However, despite initial complete response (CR) rates of 40–60%, only a subset of patients experience durable remission, necessitating further enhancement of CAR therapy efficacy through relapse prevention and achieving deeper clinical responses (CR).

[0459] Research Design and Methods This study is a single-center phase I clinical trial of 19(T2)28z1XX in patients with recurrent or recurrent large B-cell malignancies. Key disease eligibility criteria included recurrent or recurrent diffuse large B-cell lymphoma (DLBCL), high-grade BCL, primary mediastinal BCL, indolent BCL, and chronic lymphocytic leukemia (CLL). Patients who had previously received CD19 CAR therapy were eligible, provided CD19 expression was confirmed. Key exclusion criteria included persistent immunosuppression, such as systemic GvHD therapy, and active CNS disease.

[0460] This study used a 3+3 dose escalation design to determine the maximum tolerated dose for BCL. There were five planned average dose levels: 25 × 10⁻⁶. 6 50×10 6 100×10 6 150×10 6 and 200×10 6Patients received 3 days of conditioning chemotherapy with fludarabine and cyclophosphamide, followed by a single infusion of 19(T2)28z1XX CAR T cells. During the dose-escalation phase, patients with DLBCL, high-grade BCL, and primary mediastinal BCL were eligible to participate. Once the recommended phase 2 dose (RP2D) was determined, the study entered a dose-expansion phase in two cohorts. Cohort 1 included patients with DLBCL, high-grade BCL, and primary mediastinal BCL (i.e., the same eligibility criteria as the dose-escalation phase). Cohort 2 included patients with indolent BCL, CLL, and Richter transformation. The dose-expansion portion of the trial aimed to further characterize the safety, efficacy, and pharmacokinetics of 19(T2)28z1XX CAR in multiple indications. Up to 60 patients were enrolled.

[0461] The primary objective of the trial was to assess safety and tolerability and to determine the recommended phase 2 dose of 19(T2)28z1XX. Key secondary objectives included evaluating the efficacy and pharmacokinetics of 19(T2)28z1XX. Exploratory objectives included assessing B-cell aplasia and measurable residual disease (MRD), characterizing the 19(T2)28z1XX CAR T-cell phenotype before and after infusion, and analyzing serum cytokines.

[0462] Although the subject matter and certain advantages of this disclosure have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of this disclosure. Furthermore, the scope of this application is not limited to the specific embodiments of the processes, machines, manufactures, and material compositions and methods described in the specification. Those skilled in the art will readily understand from the disclosure of the subject matter, processes, machines, manufactures, material compositions, or methods of this disclosure that existing or future developments can be used to perform substantially the same functions or achieve substantially the same results as the corresponding embodiments described herein. Therefore, the appended claims are intended to encompass such processes, machines, manufactures, material compositions, or methods within their scope.

[0463] This application incorporates various patents, patent applications, publications, product descriptions, agreements, and serial numbers, the disclosures of which are incorporated herein by reference in their entirety for all purposes. sequence list <110> Memorial Sloan-Katherine Cancer Center Millennium Pharmaceutical Company <120> Chimeric antigen receptors targeting CD19 and their applications <130> 087108.0107 <150> US 63 / 073,133 <151> 2020-09-01 <150> US 63 / 015,362 <151> 2020 - 04 - 24 <160> 77 <170> PatentIn 3.5 version <210> 1 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 1 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 2 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 2 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 1 5 10 15 Gly Gly Ser <210> 3 <211> 24 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Ser Gly Gly Gly Gly Ser 20 <210> 4 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> 5 <211> 557 <212> PRT <213> Homo sapiens <400> 5 Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met 1 5 10 15 Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp 20 25 30 Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln 35 40 45 Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu 50 55 60 Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile 65 70 75 80 Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu 85 90 95 Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110 Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125 Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140 Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala 145 150 155 160 Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro 165 170 175 Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro 180 185 190 Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser 195 200 205 Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser 210 215 220 Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp 225 230 235 240 Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255 Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265 270 Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly 275 280 285 Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu 290 295 300 Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg 305 310 315 320 Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val 325 330 335 Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu 340 345 350 Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala 355 360 365 Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp 370 375 380 Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly 385 390 395 400 Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu 405 410 415 Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu 420 425 430 Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly 435 440 445 Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu 450 455 460 Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser 465 470 475 480 Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Ala 485 490 495 Gly Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro 500 505 510 Gln Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp 515 520 525 Ala Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala 530 535 540 Trp Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg 545 550 555 <210> 6 <211> 556 <212> PRT <213> Homo sapiens <400> 6 Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met 1 5 10 15 Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp 20 25 30 Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln 35 40 45 Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu 50 55 60 Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile 65 70 75 80 Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu 85 90 95 Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110 Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125 Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140 Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala 145 150 155 160 Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro 165 170 175 Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro 180 185 190 Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser 195 200 205 Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser 210 215 220 Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp 225 230 235 240 Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255 Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265 270 Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly 275 280 285 Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu 290 295 300 Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg 305 310 315 320 Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val 325 330 335 Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu 340 345 350 Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala 355 360 365 Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp 370 375 380 Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly 385 390 395 400 Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu 405 410 415 Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu 420 425 430 Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly 435 440 445 Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu 450 455 460 Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser 465 470 475 480 Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly 485 490 495 Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln 500 505 510 Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala 515 520 525 Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp 530 535 540 Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg 545 550 555 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 Ser Tyr Gly Met His 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 11 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 9 Pro Val Glu Gly Leu Leu Arg Gly Phe Asp Tyr 1 5 10 <210> 10 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 10 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Val Glu Gly Leu Leu Arg Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 11 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 11 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 12 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 12 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 13 Gln Gln Ala Gly Ala Val Pro Ile Thr 1 5 <210> 14 <211> 108 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ala Gly Ala Val Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 15 <211> 243 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ala Gly Ala Val Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Trp Tyr 165 170 175 Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Pro Val Glu 210 215 220 Gly Leu Leu Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser <210> 16 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 16 Arg Ala Ser Gln Ser Val Arg Ser Ser Tyr Leu Ala 1 5 10 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 17 Gln Gln Leu Phe Asp Ser Pro Tyr Thr 1 5 <210> 18 <211> 108 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Leu Phe Asp Ser Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 19 <211> 242 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 19 Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu 1 5 10 15 Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Leu Phe Asp Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Trp Tyr Asp 165 170 175 Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Pro Val Glu Gly 210 215 220 Leu Leu Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 Ser Ser <210> 20 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 20 Gly Ala Ser Arg Arg Ala Thr 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> twenty one Gln Gln Ala Gly Ile Pro Pro Tyr Thr 1 5 <210> twenty two <211> 108 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> twenty two Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ala Gly Ile Pro Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 243 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 23 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Val Glu Gly Leu Leu Arg Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly 130 135 140 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala 145 150 155 160 Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Arg Arg Ala Thr 180 185 190 Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Ala Gly Ile Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val 225 230 235 240 Glu Ile Lys <210> 24 <211> 220 <212> PRT <213> Homo sapiens <400> 24 Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80 Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140 Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 145 150 155 160 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 165 170 175 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 180 185 190 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 195 200 205 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 210 215 220 <210> 25 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 25 tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 60 tttattattt tctgggtg 78 <210> 26 <211> 218 <212> PRT <213> Mus musculus <400> 26 Met Thr Leu Arg Leu Leu Phe Leu Ala Leu Asn Phe Phe Ser Val Gln 1 5 10 15 Val Thr Glu Asn Lys Ile Leu Val Lys Gln Ser Pro Leu Leu Val Val 20 25 30 Asp Ser Asn Glu Val Ser Leu Ser Cys Arg Tyr Ser Tyr Asn Leu Leu 35 40 45 Ala Lys Glu Phe Arg Ala Ser Leu Tyr Lys Gly Val Asn Ser Asp Val 50 55 60 Glu Val Cys Val Gly Asn Gly Asn Phe Thr Tyr Gln Pro Gln Phe Arg 65 70 75 80 Ser Asn Ala Glu Phe Asn Cys Asp Gly Asp Phe Asp Asn Glu Thr Val 85 90 95 Thr Phe Arg Leu Trp Asn Leu His Val Asn His Thr Asp Ile Tyr Phe 100 105 110 Cys Lys Ile Glu Phe Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Arg 115 120 125 Ser Asn Gly Thr Ile Ile His Ile Lys Glu Lys His Leu Cys His Thr 130 135 140 Gln Ser Ser Pro Lys Leu Phe Trp Ala Leu Val Val Val Ala Gly Val 145 150 155 160 Leu Phe Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp 165 170 175 Thr Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met 180 185 190 Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala 195 200 205 Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro 210 215 <210> 27 <211> 235 <212> PRT <213> Homo sapiens <400> 27 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr 20 25 30 Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser �5 40 45 Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala 50 55 60 Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala 六十五 七十 七十五 八十 Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp 85 90 95 Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr 100 105 110 Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe 115 120 125 Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg 130 135 140 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 145 150 155 160 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 165 170 175 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 180 185 190 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His 195 200 205 Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser 210 215 220 Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val 225 230 235 <210> 28 <211> 247 <212> PRT <213> Mus musculus <400> 28 Met Ala Ser Pro Leu Thr Arg Phe Leu Ser Leu Asn Leu Leu Leu Met 1 5 10 15 Gly Glu Ser Ile Ile Leu Gly Ser Gly Glu Ala Lys Pro Gln Ala Pro 20 25 30 Glu Leu Arg Ile Phe Pro Lys Lys Met Asp Ala Glu Leu Gly Gln Lys 35 40 45 Val Asp Leu Val Cys Glu Val Leu Gly Ser Val Ser Gln Gly Cys Ser 50 55 60 Trp Leu Phe Gln Asn Ser Ser Ser Lys Leu Pro Gln Pro Thr Phe Val 65 70 75 80 Val Tyr Met Ala Ser Ser His Asn Lys Ile Thr Trp Asp Glu Lys Leu 85 90 95 Asn Ser Ser Lys Leu Phe Ser Ala Val Arg Asp Thr Asn Asn Lys Tyr 100 105 110 Val Leu Thr Leu Asn Lys Phe Ser Lys Glu Asn Glu Gly Tyr Tyr Phe 115 120 125 Cys Ser Val Ile Ser Asn Ser Val Met Tyr Phe Ser Ser Val Val Pro 130 135 140 Val Leu Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val Leu Arg Thr 145 150 155 160 Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu 165 170 175 Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu Asp Phe Ala 180 185 190 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Pro 195 200 205 Leu Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr His Arg Ser Arg Lys 210 215 220 Arg Val Cys Lys Cys Pro Arg Pro Leu Val Arg Gln Glu Gly Lys Pro 225 230 235 240 Arg Pro Ser Glu Lys Ile Val 245 <210> 29 <211> 164 <212> PRT <213> Homo sapiens <400> 29 Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu 1 5 10 15 Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys 20 25 30 Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala 35 40 45 Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 50 55 60 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 65 70 75 80 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 85 90 95 Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 100 105 110 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 115 120 125 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 130 135 140 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 145 150 155 160 Leu Pro Pro Arg <210> 30 <211> 112 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 30 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 31 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 31 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 1 5 10 15 Asp Val Leu Asp Lys Arg 20 <210> 32 <211> 66 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 32 cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 60 aagaga 66 <210> 33 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 33 Gln Asn Gln Leu Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Phe 1 5 10 15 Asp Val Leu Asp Lys Arg 20 <210> 34 <211> 66 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 34 cagaaccagc tctttaacga gctcaatcta ggacgaagag aggagttcga tgttttggac 60 aagaga 66 <210> 35 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 35 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 1 5 10 15 Tyr Ser Glu Ile Gly Met Lys 20 <210> 36 <211> 69 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 36 caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 60 gggatgaaa 69 <210> 37 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 37 Gln Glu Gly Leu Phe Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 1 5 10 15 Phe Ser Glu Ile Gly Met Lys 20 <210> 38 <211> 69 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 38 caggaaggcc tgttcaatga actgcagaaa gataagatgg cggaggcctt cagtgagatt 60 gggatgaaa 69 <210> 39 <211> 22 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 39 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 1 5 10 15 Asp Ala Leu His Met Gln 20 <210> 40 <211> 66 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 40 cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 60 atgcag 66 <210> 41 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 41 His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Phe 1 5 10 15 Asp Ala Leu His Met Gln 20 <210> 42 <211> 66 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 42 cacgatggcc ttttccaggg gctcagtaca gccaccaagg acaccttcga cgcccttcac 60 atgcag 66 <210> 43 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 43 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Phe Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Phe Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 44 <211> 336 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 44 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgttcaat 180 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgttcaat 180 gaactgcaga aagataagat ggcggaggcc ttcagtgaga ttgggatgaa aggcgagcgc 240 gaactgcaga aagataagat ggcggaggcc ttcagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggccttttc caggggctca gtacagccac caaggacacc 300 cggaggggca aggggcacga tggccttttc caggggctca gtacagccac caaggacacc 300 ttcgacgccc ttcacatgca ggccctgccc cctcgc 336 ttcgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 45<210> 45 <211> 336<211> 336 <212> DNA<212> DNA <213> 人工序列<213> Artificial sequence <220> <220> <223> 合成 <223> Synthetic <400> 45 <400> 45 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgttcaat 180 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgttcaat 180 gaactgcaga aagataagat ggcggaggcc ttcagtgaga ttgggatgaa aggcgagcgc 240 gaactgcaga aagataagat ggcggaggcc ttcagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggccttttc cagggtctca gtacagccac caaggacacc 300 ttcgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 46 <211> 123 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 46 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123 <210> 47 <211> 255 <212> PRT <213> Homo sapiens <400> 47 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 85 90 95 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln 115 120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 <210> 48 <211> 488 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 48 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Asp Thr Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Gly Thr 20 25 30 Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 35 40 45 Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 50 55 60 Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly 65 70 75 80 Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 85 90 95 Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln 100 105 110 Gln Ala Gly Ala Val Pro Ile Thr Phe Gly Gly Gly Thr Lys Val Glu 115 120 125 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly 145 150 155 160 Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 165 170 175 Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 180 185 190 Val Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser 195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 210 215 220 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Ala Lys Pro Val Glu Gly Leu Leu Arg Gly Phe Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Leu Val Thr Val Ser Ser Arg Ala Ala Ala Ile Glu Val 260 265 270 Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile 275 280 285 Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly 290 295 300 Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 305 310 315 320 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 325 330 335 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 340 345 350 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 355 360 365 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Phe Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Phe Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Phe Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Phe Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <210> 49 <211> 1464 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 49 atggatatga gagtaccagc tcagctgctg ggcctgctgc ttttgtggtt gccggacaca 60 cgctgtgaga ttgtcctgac tcagtctccc gggactctgt ccctcagccc cggtgaacgc 120 gctacccttt catgcagagc ctctcagtct gtgtccagca gctacctcgc atggtatcag 180 cagaagcccg gacaggctcc caggctgttg atctatggag ctagtagtcg agcaacaggc 240 atcccagatc gcttctcagg gagcggttca ggtacagact tcacgctgac gatttcaagg 300 ctggaacccg aagattttgc cgtctattat tgtcaacagg caggggctgt gccaatcact 360 ttcgggggcg ggaccaaggt ggaaatcaaa ggaggcggag gaagtggagg aggagggagc 420 ggtggaggag ggtcacaggt gcagctggta gaatctggcg gaggggtcgt tcaaccaggg 480 aggtcattgc ggttgagctg cgcagcgagt ggttttacct tcagcagtta tggaatgcat 540 tgggtgagac aagcaccagg aaaaggtctg gagtgggtgg ctttgatttg gtacgacggc 600 agtaataaat actacgccga ttctgttaag ggcagattta ctatttctcg cgacaacagc 660 aagaacacgc tgtacctgca gatgaactct ctgagagccg aagatacagc agtgtactat 720 tgtgctaagc ccgtagaagg gctcctgagg ggattcgatt attggggcca gggtacgctt 780 gtgacagtgt ctagtcgggc ggccgcaatt gaagttatgt atcctcctcc ttacctagac 840 aatgagaaga gcaatggaac cattatccat gtgaaaggga aacacctttg tccaagtccc 900 ctatttcccg gaccttctaa gcccttttgg gtgctggtgg tggttggtgg agtcctggct 960 tgctatagct tgctagtaac agtggccttt attattttct gggtgaggag taagaggagc 1020 aggctcctgc acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag 1080 cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctccag agtgaagttc 1140 agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200 aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260 atggggggaa agccgagaag gaagaaccct caggaaggcc tgttcaatga actgcagaaa 1320 gataagatgg cggaggcctt cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380 gggcacgatg gccttttcca gggtctcagt acagccacca aggacacctt cgacgccctt 1440 cacatgcagg ccctgccccc tcgc 1464 <210> 50 <211> 488 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 50 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Asp Thr Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Gly Thr 20 25 30 Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 35 40 45 Gln Ser Val Arg Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 50 55 60 Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly 65 70 75 80 Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 85 90 95 Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln 100 105 110 Gln Leu Phe Asp Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu 115 120 125 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly 145 150 155 160 Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 165 170 175 Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 180 185 190 Val Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser 195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 210 215 220 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Ala Lys Pro Val Glu Gly Leu Leu Arg Gly Phe Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Leu Val Thr Val Ser Ser Arg Ala Ala Ala Ile Glu Val 260 265 270 Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile 275 280 285 Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly 290 295 300 Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 305 310 315 320 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 325 330 335 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 340 345 350 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 355 360 365 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Phe Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Phe Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Phe Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Phe Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <210> 51 <211> 1464 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 51 atggatatga gagtaccagc tcagctgctg ggcctgctgc ttttgtggtt gccggacaca 60 cgctgtgaga ttgtgctgac acagtctcca ggcacactct cccttagccc gggcgagagg 120 gccactctga gctgtcgggc tagtcagtca gtaaggagct cttatctggc ctggtatcag 180 cagaaaccag ggcaggctcc aaggctgctt atctacggtg caagttcccg ggcaacaggc 240 atcccagatc gctttagcgg tagcgggagt gggaccgatt tcactctgac catctcccgc 300 cttgagcccg aggatttcgc tgtctattat tgccagcaac tgtttgactc accctatacg 360 ttcggtggag ggaccaaagt ggagatcaag ggaggcggag gaagtggagg aggagggagc 420 ggtggaggag ggtcacaggt gcagctggta gaatctggcg gaggggtcgt tcaaccaggg 480 aggtcattgc ggttgagctg cgcagcgagt ggttttacct tcagcagtta tggaatgcat 540 tgggtgagac aagcaccagg aaaaggtctg gagtgggtgg ctttgatttg gtacgacggc 600 agtaataaat actacgccga ttctgttaag ggcagattta ctattctcg cgacaacagc 660 aagaacacgc tgtacctgca gatgaactct ctgagagccg aagatacagc agtgtactat 720 tgtgctaagc ccgtagaagg gctcctgagg ggattcgatt attggggcca gggtacgctt 780 gtgacagtgt ctagtcgggc ggccgcaatt gaagttatgt atcctcctcc ttacctagac 840 aatgagaga gcaatggaac cattatccat gtgaaaggga aacacctttg tccaagtccc 900 ctatttcccg gaccttctaa gccctttgg gtgctggtgg tggttggtgg agtcctggct 960 tgctatagct tgctagtaac agtggccttt attattct gggtgaggag taagaggagc 1020 aggctcctgc acagtgacta catgaacatg actccccgcc gccccggcc cacccgcaag 1080 cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctccag agtgaagttc 1140 agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200 aatctaggac gagagagga gtacgatgtt tggacaga gacgtggccg ggaccctgag 1260 atgggggaa agccgagaag gagaaccct caggaaggcc tgttcaatga actgcagaaa 1320 gataagatgg cggaggcctt cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380 gggcacgatg gccttttcca ggggctcagt acagccacca aggacacctt cgacgccctt 1440 cacatgcagg ccctgccccc tcgc 1464 <210> 52 <211> 485 <212> PRT <213>Synthetic Sequence <220> <223> Synthetic <400> 52 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Lys Pro Val Glu Gly Leu Leu Arg Gly Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln 145 150 155 160 Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser 165 170 175 Cys Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln 180 185 190 Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Arg 195 200 205 Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 210 215 220 Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val 225 230 235 240 Tyr Tyr Cys Gln Gln Ala Gly Ile Pro Pro Tyr Thr Phe Gly Gly Gly 245 250 255 Thr Lys Val Glu Ile Lys Arg Ala Ala Ala Ile Glu Val Met Tyr Pro 260 265 270 Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val 275 280 285 Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys 290 295 300 Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 305 310 315 320 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 325 330 335 Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 340 345 350 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 355 360 365 Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Phe 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Phe Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 53 <211> 1455 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 53 atggaattcg gcttgtcatg ggtgttcctc gtcgcgctgc tgcgcggcgt tcagtgccag 60 gtgcagctgg tagaatctgg cggaggggtc gttcaaccag ggaggtcatt gcggttgagc 120 tgcgcagcga gtggttttac cttcagcagt tatggaatgc attgggtgag acaagcacca 180 ggaaaaggtc tggagtgggt ggctttgatt tggtacgacg gcagtaataa atactacgcc 240 gattctgtta agggcagatt tactatttct cgcgacaaca gcaagaacac gctgtacctg 300 cagatgaact ctctgagagc cgaagataca gcagtgtact attgtgctaa gccgtagaa 360 gggctcctga ggggattcga ttattggggc cagggtacgc ttgtgacagt gtctagtgga 420 ggcggaggaa gtggaggagg agggagcggt ggaggagggt cagaaatcgt gctcacccag 480 tccccgggaa cactgagtct ctctccaggg gaaagagcaa cattgtcctg cagagcatcc 540 cagagcgtga gctccagcta cctcgcctgg tatcagcaga aaccaggcca ggcaccccgc 600 ctgcttatct acggtgcatc caggagagcc actgggatcc ccgatcgatt ctctggatca 660 gggtctggca ctgactttac attgacgatc tcacggctgg aacccgagga ttcgccgtg 720 tattactgcc aacaggccgg aattccaccg tataccttcg gaggaggtac taaagtagag 780 attaaacggg cggccgcaat tgaagttatg tatcctcctc cttacctaga caatgagaag 840 agcaatggaa ccattatcca tgtgaaaggg aaacacctttt gtccaagtcc cctatttccc 900 ggaccttcta agccctttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 960 ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg 1020 cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 1080 ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1140 gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1200 cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1260 aagccgagaa ggaagaaccc tcaggaaggc ctgttcaatg aactgcagaa agataagatg 1320 gcggaggcct tcagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380 ggccttttcc aggggctcag tacagccacc aaggacacct tcgacgccct tcacatgcag 1440 gccctgcccc ctcgc 1455 <210> 54 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 54 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Asp Thr Arg Cys 20 <210> 55 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 55 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys <210> 56 <211> 120 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 56 attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60 catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120 <210> 57 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 57 Gln Gln Val Asp Ser Leu His Pro Phe Thr 1 5 10 <210> 58 <211> 109 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 58 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Val Asp Ser Leu His 85 90 95 Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 59 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 59 Gln Gln Ala Gly Gly Val Pro Pro Leu Thr 1 5 10 <210> 60 <211> 109 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 60 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ala Gly Gly Val Pro 85 90 95 Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 61 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 61 Gln Gln Ala Gly Val Pro Pro Leu Thr 1 5 <210> 62 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 62 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ala Gly Val Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 63 <211> 10 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 63 Gln Gln Ala Gly Gly Val Pro Pro Phe Thr 1 5 10 <210> 64 <211> 109 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 64 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ala Gly Gly Val Pro 85 90 95 Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 65 <211> 7 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 65 Gly Ala Ser Asn Arg Ala Thr 1 5 <210> 66 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 66 Gln Gln Ala Gly Val Phe Pro Phe Thr 1 5 <210> 67 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 67 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ala Gly Val Phe Pro 85 90 95 Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 68 <211> 360 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 68 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcactg atatggtatg atggaagtaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggcggtgt actactgcgc caagccagtg 300 gaaggactat taagaggatt cgattactgg ggacagggta cattggtcac cgtctcctca 360 <210> 69 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 69 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag caggccggag ccgtccctat cacttttggc 300 ggagggacca aggttgagat caaa 324 <210> 70 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 70 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagg agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagctcttcg acagtcctta cacttttggc 300 ggagggacca aggttgagat caaa 324 <210> 71 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthesis <400> 71 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gaagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag caggccggca tcccccctta cacttttggc 300 ggagggacca aggttgagat caaa 324 <210> 72 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> Synthesis <400> 72 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag caggtcgaca gtctccatcc tttcactttt 300 ggcggaggga ccaaggttga gatcaaa 327 <210> 73 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 73 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag caggccggag gcgtccctcc tctcactttt 300 ggcggaggga ccaaggttga gatcaaa 327 <210> 74 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 74 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagg agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag caggccggag tcccccctct cacttttggc 300 ggagggacca aggttgagat caaa 324 <210> 75 <211> 327 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 75 gaaattgtga tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag caggccggag gcgtccctcc tttcactttt 300 ggcggaggga ccaaggttga gatcaaa 327 <210> 76 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 76 gaaattgtga tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca acagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag caggccggag tcttcccttt cacttttggc 300 ggagggacca aggttgagat caaa 324 <210> 77 [[ID='26']]<211> 920 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 77 Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met 1 5 10 15 Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp 20 25 30 Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln 35 40 45 Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu 50 55 60 Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile 65 70 75 80 Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu 85 90 95 Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110 Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125 Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140 Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala 145 150 155 160 Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro 165 170 175 Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro 180 185 190 Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser 195 200 205 Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser 210 215 220 Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp 225 230 235 240 Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255 Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265 270 Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly 275 280 285 Gly Trp Lys His His His His His His His His His His Asp Tyr Asp 290 295 300 Ile Pro Thr Thr Glu Asn Leu Tyr Phe Gln Gly Gly Gly Gly Gly Ser 305 310 315 320 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 325 330 335 Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu 340 345 350 Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln 355 360 365 Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe 370 375 380 Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser 385 390 395 400 Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg 405 410 415 Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu 420 425 430 Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro 435 440 445 Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp 450 455 460 Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg 465 470 475 480 His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr 485 490 495 Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys 500 505 510 Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser 515,520,525 Only Lys Gln Arg With Lys Cys Only Ser Leu Gln Lys Phe Gly Glu Arg 530 535 540 Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys 545 550 555 560 Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val 565,570,575 His Thr Glu Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg 580,585,590 Only Asp Leu Only Only Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser 595,600,605 Leo Lys Glu Cys Glu Lys Pro Leo Leo Glu Lys Ser His Cys 610 615 620 Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu 625 630 635 640 Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu 645,650,655 Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg 660,665,670 His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr 675 680 685 Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys 690 695 700 Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln 705 710 715 720 Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr 725 730 735 Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln 740 745 750 Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val 755 760 765 Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala 770 775 780 Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu 785 790 795 800 Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu 805 810 815 Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr 820 825 830 Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile 835 840 845 Cys Thr Leu Serves as Glu Lys Glu Arg Gln and Lys Gln Thr Ala Leu 850 855 860 Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys 865 870 875 880 Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Lys Ala 885,890,895 Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Leu Val Ala 900 905 910 Ser Gln Wing Wing Leu Wing Gly Leu 915,920

Claims

1. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain specifically binding to CD19, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises: a) Heavy chain variable region, which includes CDR1 of the amino acid sequence shown in SEQ ID NO:7, CDR2 of the amino acid sequence shown in SEQ ID NO:8, and CDR3 of the amino acid sequence shown in SEQ ID NO:

9. and b) Light chain variable region, which includes: i) CDR1 of the amino acid sequence shown in SEQ ID NO:11, CDR2 of the amino acid sequence shown in SEQ ID NO:12, and CDR3 of the amino acid sequence shown in SEQ ID NO:13; ii) CDR1 of the amino acid sequence shown in SEQ ID NO:16, CDR2 of the amino acid sequence shown in SEQ ID NO:12, and CDR3 of the amino acid sequence shown in SEQ ID NO:17; or iii) CDR1 of the amino acid sequence shown in SEQ ID NO:11, CDR2 of the amino acid sequence shown in SEQ ID NO:20, and CDR3 of the amino acid sequence shown in SEQ ID NO:

21.

2. The CAR according to claim 1, wherein the extracellular antigen-binding domain comprises a single-stranded variable fragment (scFv), Fab, or F(ab)2.

3. The CAR according to claim 2, wherein the extracellular antigen-binding domain includes scFv.

4. The CAR of claim 3, wherein the scFv is a human scFv.

5. The CAR according to any one of claims 1-4, wherein: (a) The heavy chain variable region includes CDR1 of the amino acid sequence shown in SEQ ID NO:7, CDR2 of the amino acid sequence shown in SEQ ID NO:8, and CDR3 of the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 of the amino acid sequence shown in SEQ ID NO:11, CDR2 of the amino acid sequence shown in SEQ ID NO:12, and CDR3 of the amino acid sequence shown in SEQ ID NO:13; (b) The heavy chain variable region comprises CDR1 of the amino acid sequence shown in SEQ ID NO:7, CDR2 of the amino acid sequence shown in SEQ ID NO:8, and CDR3 of the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region comprises CDR1 of the amino acid sequence shown in SEQ ID NO:16, CDR2 of the amino acid sequence shown in SEQ ID NO:12, and CDR3 of the amino acid sequence shown in SEQ ID NO:17; or (c) The heavy chain variable region includes CDR1 of the amino acid sequence shown in SEQ ID NO:7, CDR2 of the amino acid sequence shown in SEQ ID NO:8, and CDR3 of the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region includes CDR1 of the amino acid sequence shown in SEQ ID NO:11, CDR2 of the amino acid sequence shown in SEQ ID NO:20, and CDR3 of the amino acid sequence shown in SEQ ID NO:

21.

6. The CAR according to any one of claims 1-4, wherein the amino acid sequence of the heavy chain variable region has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or homology with the amino acid sequence shown in SEQ ID NO:

10.

7. The CAR according to any one of claims 1-4, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

10.

8. The CAR according to any one of claims 1-4, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, SEQ ID NO:18 or SEQ ID NO:

22.

9. The CAR according to any one of claims 1-4, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:10; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, SEQ ID NO:18 or SEQ ID NO:

22.

10. The CAR according to any one of claims 1-4, wherein: (a) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14; (b) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:18; or (c) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

22.

11. The CAR according to any one of claims 1-4, wherein the extracellular antigen-binding domain includes a linker between the heavy chain variable region and the light chain variable region.

12. The CAR according to claim 11, wherein the adapter is composed of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

13. The CAR according to any one of claims 1-4, wherein the positions of the heavy chain variable region and the light chain variable region from the N-terminus to the C-terminus are: V L -V H .

14. The CAR according to any one of claims 1-4, wherein the extracellular antigen-binding domain comprises or scFv, the amino acid sequence of which is shown in SEQ ID NO:15, SEQ ID NO:19 or SEQ ID NO:

23.

15. The CAR according to any one of claims 1-4, wherein the transmembrane domain comprises a CD8 peptide, a CD28 peptide, a CD3ζ peptide, a CD4 peptide, a 4-1BB peptide, an OX40 peptide, an ICOS peptide, a CTLA-4 peptide, a PD-1 peptide, a LAG-3 peptide, a 2B4 peptide, or a BTLA peptide.

16. The CAR of claim 15, wherein the transmembrane domain comprises a CD28 polypeptide.

17. The CAR according to any one of claims 1-4, wherein the intracellular signal transduction domain comprises a CD3ζ polypeptide.

18. The CAR according to claim 17, wherein the CD3ζ polypeptide is a modified CD3ζ polypeptide, the amino acid sequence of which is shown in SEQ ID NO:

43.

19. The CAR according to any one of claims 1-4, wherein the intracellular signal transduction domain further comprises at least one co-stimulatory signal transduction region.

20. The CAR of claim 19, wherein the at least one co-stimulatory signal transduction region comprises a CD28 peptide, a 4-1BB peptide, an OX40 peptide, an ICOS peptide, a DAP-10 peptide, or a combination thereof.

21. The CAR of claim 20, wherein the at least one co-stimulatory signal transduction region comprises a CD28 polypeptide.

22. The CAR according to any one of claims 1-4, wherein the CAR is expressed by a vector.

23. The CAR according to claim 22, wherein the vector is a retroviral vector.

24. A cell comprising the CAR according to any one of claims 1-23.

25. The cell of claim 24, wherein the cell is transduced by CAR.

26. The cell of claim 24 or 25, wherein the CAR is constitutively expressed on the surface of the cell.

27. The cell according to claim 24 or 25, wherein the cell is an immune response cell.

28. The cell according to claim 24 or 25, wherein the cell is a lymphoid lineage cell or a bone marrow lineage cell.

29. The cell of claim 24, wherein the cell is selected from T cells, natural killer (NK) cells, stem cells from which lymphoid cells can differentiate, and stem cells from which myeloid cells can differentiate.

30. The cell according to any one of claims 24 or 25, wherein the cell is a T cell.

31. The cell of claim 30, wherein the T cell is selected from helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells.

32. The cell according to claim 24 or 25, wherein the cell is an NK cell.

33. The cell of claim 32, wherein the NK cell is derived from stem cells.

34. The cell according to claim 29 or 33, wherein the stem cell is a pluripotent stem cell.

35. The cell of claim 34, wherein the pluripotent stem cell is an induced pluripotent stem cell.

36. A nucleic acid molecule encoding a CAR according to any one of claims 1-23.

37. The nucleic acid molecule of claim 36, further comprising a promoter operatively linked to the CAR.

38. The nucleic acid molecule of claim 37, wherein the promoter is endogenous or exogenous.

39. The nucleic acid molecule of claim 38, wherein the exogenous promoter is selected from the elongation factor (EF)-1 promoter, cytomegalovirus immediate early (CMV) promoter, simian virus 40 early (SV40) promoter, phosphoglycerate kinase (PGK) promoter, metallothionein promoter, and ubiquitin C promoter.

40. The nucleic acid molecule according to claim 37, wherein the promoter is an inducible promoter.

41. The nucleic acid molecule of claim 40, wherein the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, IL-2 promoter, 4-1BB promoter, PD1 promoter, and LAG3 promoter.

42. The nucleic acid molecule according to claim 38, wherein the promoter is an endogenous promoter.

43. The nucleic acid molecule according to claim 42, wherein the endogenous promoter is selected from the TCR α promoter, the TCR β promoter, and the β2-microglobulin promoter.

44. A vector comprising a nucleic acid molecule according to any one of claims 36-43.

45. The vector according to claim 44, wherein the vector is a retroviral vector.

46. ​​A cell that expresses a nucleic acid molecule according to any one of claims 36-43.

47. The cell of claim 46, wherein the cell is a T cell or a natural killer (NK) cell.

48. A composition comprising cells according to any one of claims 24-35, 46 and 47.

49. The composition according to claim 48, wherein it is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

50. The composition according to claim 48 or 49, comprising 1 × 10 6 Up to 5×10 8 Each cell.

51. The composition according to claim 48 or 49, comprising 1 × 10 6 Up to 1×10 8 Each cell.

52. The composition according to claim 48 or 49, comprising 1 × 10 6 Up to 5×10 7 Each cell.

53. The composition according to any one of claims 48 or 49, comprising 2.5 × 10 7 Each cell.

54. Use of the cell according to any one of claims 24-35, 46 and 47 in the preparation of a medicament for reducing tumor burden in a subject, wherein the tumor is selected from acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), hairy cell leukemia, and B-cell prelymphoblastic leukemia.

55. Use of the cell according to any one of claims 24-35, 46 and 47 in the preparation of a medicament for reducing tumor burden in a subject, wherein the tumor is selected from B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia, B-cell chronic lymphoblastic leukemia, and CLL with Richter transformation.

56. The use according to claim 54 or 55, wherein the use reduces the number of tumor cells in the subject, reduces the size of the tumor in the subject, and / or eradicates the tumor in the subject.

57. Use of the cell according to any one of claims 24-35, 46 and 47 in the preparation of a medicament for increasing or prolonging the survival of a subject suffering from a tumor, wherein the tumor is selected from acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), hairy cell leukemia, and B-cell prelymphoblastic leukemia.

58. Use of the cell according to any one of claims 24-35, 46 and 47 in the preparation of a medicament for increasing or prolonging the survival of a subject suffering from a tumor, wherein the tumor is selected from B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia, B-cell chronic lymphoblastic leukemia, and CLL with Richter transformation.

59. Use of the cell according to any one of claims 24-35, 46 and 47 in the preparation of a medicament for treating a tumor in a subject, wherein the tumor is selected from acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), hairy cell leukemia, and B-cell prelymphoblastic leukemia.

60. Use of the cell according to any one of claims 24-35, 46 and 47 in the preparation of a medicament for treating a tumor in a subject, wherein the tumor is selected from B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia, B-cell chronic lymphoblastic leukemia, and CLL with Richter transformation.

61. The use according to any one of claims 54-55 and 57-60, wherein the subject is a human subject.

62. A method for producing cells comprising a CAR according to any one of claims 1-23, comprising introducing a nucleic acid molecule encoding the CAR according to any one of claims 36-43 into the cells.

63. A kit for reducing tumor burden in a subject, treating and / or preventing vegetations in a subject and / or increasing or prolonging the survival of a subject with vegetations, comprising cells according to any one of claims 24-35, 46 and 47.

64. The kit of claim 63, wherein the kit further comprises written instructions for using the cells to reduce tumor burden in a subject, treat and / or prevent vegetations in a subject and / or increase or prolong the survival of a subject with vegetations.

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