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WO2022089644A1 - 靶向cd5的全人源抗体、全人源嵌合抗原受体(car)及其应用 - Google Patents

靶向cd5的全人源抗体、全人源嵌合抗原受体(car)及其应用 Download PDF

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WO2022089644A1
WO2022089644A1 PCT/CN2021/127995 CN2021127995W WO2022089644A1 WO 2022089644 A1 WO2022089644 A1 WO 2022089644A1 CN 2021127995 W CN2021127995 W CN 2021127995W WO 2022089644 A1 WO2022089644 A1 WO 2022089644A1
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seq
amino acid
acid sequence
sequence
car
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PCT/CN2021/127995
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English (en)
French (fr)
Inventor
谭涛超
戴振宇
周剑锋
魏巧娥
贾向印
赵雅
谢萌
Original Assignee
南京驯鹿医疗技术有限公司
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Priority to CN202180073309.1A priority Critical patent/CN116568709A/zh
Priority to EP21885370.3A priority patent/EP4238990A4/en
Priority to US18/251,294 priority patent/US20240002505A1/en
Publication of WO2022089644A1 publication Critical patent/WO2022089644A1/zh

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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
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    • C07K2319/00—Fusion polypeptide
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    • C—CHEMISTRY; METALLURGY
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    • C07K2319/00—Fusion polypeptide
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    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia

Definitions

  • This application includes a Sequence Listing in .txt format submitted electronically.
  • This .txt file contains a sequence listing named "CD5 CAR_ST25.txt" created on November 1, 2020 and is approximately 126 kilobytes in size.
  • the Sequence Listing contained in the .txt file is part of the specification, which is incorporated herein by reference in its entirety.
  • the present application relates to the field of biomedicine, in particular to fully human antibodies, fully human chimeric antigen receptors that can specifically bind to CD5 protein, and applications thereof.
  • CAR-T chimeric antigen receptor T cell
  • CD19CAR-T in the treatment of relapsed/refractory ALL can be as high as 90%, and the effective rate in CLL and some B-cell lymphomas is greater than 50%.
  • CAR-T therapy has achieved great success in the field of B-cell malignancies, its research and application in T-cell malignancies is very limited.
  • T-cell malignancies include acute T-lymphocytic leukemia (T-ALL) and T-cell lymphoma (TCL).
  • T-ALL is a blood disorder caused by abnormal proliferation of T lymphocytes, which is aggressive and progresses rapidly.
  • T-cell lymphoma is a malignant tumor of T cells that can develop in lymphoid tissues (eg, lymph nodes and spleen) or outside of lymphoid tissues (eg, gastrointestinal tract, liver, nasal cavity, skin, etc.) Golden lymphoma is 10% to 15%, and the proportion is higher in my country.
  • CD5 is constitutively expressed on lymphocyte precursors, mature T cells and a subset of mature B cells (Bl cells) [10][11] .
  • CD5 is highly expressed in about 85% of T-ALL and about 75% of peripheral T-cell lymphomas. In addition, CD5 is also frequently expressed in mantle cell lymphoma, chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia cells (HCL). At present, T-cell malignant tumor has a high recurrence rate and poor prognosis after radiotherapy and chemotherapy, and it is a clinically incurable hematological malignancy. There is an urgent need to develop cell therapy drugs for this disease.
  • CD5 expression is restricted to a subset of mature T cells and B cells, and the biological properties of the CD5 antigen allow CD5 CAR T cells to generate potent antibodies against T-ALL and T lymphoma cells in vitro and in vivo tumor activity [12] . Therefore, CD5 can serve as a safe and reliable target for T-cell tumors.
  • CD5 CAR-T cells were effective in r/r CD5+T-ALL and T-ALL treated with previous multi-line therapy. It is safe in patients with cellular non-Hodgkin's lymphoma (T-NHL), produces clinical efficacy, and does not result in complete T-cell depletion. More importantly, depletion of malignant T cells by CD5 CAR-T cells may enable HSCT (hematopoietic stem cell transplantation) in patients who were previously ineligible for transplantation.
  • HSCT hematopoietic stem cell transplantation
  • the mouse-derived scFv CAR-T may recur CD5+ malignant disease after infusion.
  • the limited duration of CAR-T in patients may be related to the production of anti-mouse antibodies, while the fully human scFv may address the short duration of CD5 CAR-T in patients.
  • the development of fully human CD5 antibodies is of great significance for the development of next-generation CAR-T products with longer in vivo duration and better long-term efficacy.
  • the present application provides fully human antibodies that can specifically bind to CD5, fully human chimeric antigen receptors and applications thereof.
  • the application provides a fully human antibody or single chain antibody or fragment thereof targeting CD5, wherein the fully human antibody comprises a heavy chain variable region (HCVR) and/or a light chain variable region (LCVR) ), the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, the light chain variable region includes LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3 and/or LCDR1, LCDR2 and LCDR3 are selected from one of the following combinations:
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • LCDR1 The amino acid sequence of LCDR1 is SGSIARNY (SEQ ID NO: 64);
  • the amino acid sequence of LCDR2 is EDN (SEQ ID NO: 65);
  • LCDR3 The amino acid sequence of LCDR3 is QSYDDNTSWV (SEQ ID NO: 66);
  • HCDR1 The amino acid sequence of HCDR1 is GGTFSSNA (SEQ ID NO: 61);
  • amino acid sequence of HCDR2 is IIPMFGTT (SEQ ID NO: 62);
  • amino acid sequence of HCDR3 is ARDGGGRHPYYYYGMDV (SEQ ID NO: 63);
  • LCDR2 The amino acid sequence of LCDR2 is DND (SEQ ID NO: 72);
  • LCDR3 The amino acid sequence of LCDR3 is AVWDSSLSAAV (SEQ ID NO: 73);
  • amino acid sequence of HCDR1 is GYSFTSYW (SEQ ID NO: 67);
  • the amino acid sequence of HCDR2 is IYPDDSDT (SEQ ID NO: 68);
  • HCDR3 The amino acid sequence of HCDR3 is ARMSLTSYLATDG (SEQ ID NO: 69);
  • LCDR1 The amino acid sequence of LCDR1 is SDDIGNYKR (SEQ ID NO: 76);
  • LCDR2 The amino acid sequence of LCDR2 is EVT (SEQ ID NO: 77);
  • LCDR3 The amino acid sequence of LCDR3 is SSYASGDTYV (SEQ ID NO: 78);
  • HCDR1 The amino acid sequence of HCDR1 is TYTFTNYY (SEQ ID NO: 73);
  • amino acid sequence of HCDR2 is INPSGDTT (SEQ ID NO: 74);
  • amino acid sequence of HCDR3 is ARLSWYWGGSFDD (SEQ ID NO: 75);
  • LCDR1 The amino acid sequence of LCDR1 is SSNIGAGYD (SEQ ID NO: 46);
  • the amino acid sequence of LCDR2 is GNI (SEQ ID NO: 47);
  • amino acid sequence of LCDR3 is GTWDNSLSAHYV (SEQ ID NO: 48);
  • amino acid sequence of HCDR1 is GFTFNNYT (SEQ ID NO: 43);
  • amino acid sequence of HCDR2 is ISSSSSYI (SEQ ID NO: 44);
  • the amino acid sequence of HCDR3 is ARYFSGSAGDY (SEQ ID NO: 45);
  • LCDR1 The amino acid sequence of LCDR1 is NSNIGNNY (SEQ ID NO: 52);
  • the amino acid sequence of LCDR2 is DNN (SEQ ID NO: 53);
  • LCDR3 The amino acid sequence of LCDR3 is GIWDSSLDAYV (SEQ ID NO: 54);
  • HCDR1 The amino acid sequence of HCDR1 is GFTFSSYS (SEQ ID NO: 49);
  • amino acid sequence of HCDR2 is ISSSSSYI (SEQ ID NO: 50);
  • amino acid sequence of HCDR3 is ARGNPWYGVDY (SEQ ID NO: 51);
  • LCDR1 The amino acid sequence of LCDR1 is SSNIGNNY (SEQ ID NO: 58);
  • LCDR2 The amino acid sequence of LCDR2 is DNN (SEQ ID NO: 59);
  • amino acid sequence of LCDR3 is GTWDSSLSAVV (SEQ ID NO: 60);
  • HCDR1 The amino acid sequence of HCDR1 is GFTFSSYA (SEQ ID NO: 55);
  • HCDR2 The amino acid sequence of HCDR2 is ITDSGDIT (SEQ ID NO: 56);
  • amino acid sequence of HCDR3 is ARMSSHWYFSADY (SEQ ID NO: 57); or
  • the fully human antibody includes a variant of the CDR sequence combination in any one of (1)-(6), wherein the CDR sequence in any one of the variants (1)-(6) has at least 90% sequence identity, or a total of at least 1 and no more than 10, or no more than 5, 4, 3, 2, or 1 amino acid change in the CDR sequence.
  • amino acid sequence of the heavy chain variable region and/or light chain variable region is selected from any combination of the following:
  • sequence shown in SEQ ID NO: 30 or a heavy chain variable region sequence with at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 29 or a light chain with at least 90% sequence identity therewith may be variable region sequence;
  • sequence shown in SEQ ID NO: 36 or a heavy chain variable region sequence with at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 35 or a light chain with at least 90% sequence identity therewith can be variable region sequence;
  • sequence shown in SEQ ID NO: 42 or a heavy chain variable region sequence with at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 41 or a light chain with at least 90% sequence identity therewith can be variable region sequence;
  • sequence shown in SEQ ID NO: 90 or the heavy chain variable region sequence with at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 89 or the light chain with at least 90% sequence identity therewith can be variable region sequence;
  • sequence shown in SEQ ID NO: 93 or a heavy chain variable region sequence with at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 92 or a light chain with at least 90% sequence identity therewith can be variable region sequence;
  • sequence shown in SEQ ID NO: 96 or a heavy chain variable region sequence with at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 95 or a light chain with at least 90% sequence identity therewith can be variable region sequence.
  • amino acid sequence of the heavy chain variable region and/or light chain variable region is selected from any combination of the following:
  • the fully human antibody comprises the amino acid sequence set forth in SEQ ID NO: 28, 34, 40, 88, 91 or 94.
  • the present application provides nucleic acid molecules encoding the above-mentioned fully human antibodies or single-chain antibodies or fragments thereof.
  • the nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 25-27, 31-33, 37-39, and 79-87.
  • the present application provides an expression vector comprising the above-mentioned nucleic acid molecule.
  • the present application provides a host cell comprising the above-mentioned expression vector.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned fully human antibody or single-chain antibody or fragment thereof, and a pharmaceutically acceptable carrier or diluent.
  • the application provides a method of treating a disease or disorder, comprising administering to a patient in need thereof a therapeutically effective amount of the above-described fully human antibody or single-chain antibody or fragment thereof, host cell, or pharmaceutical composition , to eliminate, inhibit or reduce CD5 activity, thereby preventing, alleviating, ameliorating or inhibiting a disease or condition.
  • the disease or disorder is cancer.
  • the present application provides antibodies or fragments of the above fully human antibodies or single-chain antibodies or fragments thereof that compete for the same epitope.
  • the present application provides a kit for detecting CD5 protein in a sample, wherein the kit includes the above-mentioned fully human antibody or single-chain antibody or fragment thereof.
  • the application provides the above-mentioned fully human antibody or its single-chain antibody or fragment or host cell in the preparation of a medicament for eliminating, inhibiting or reducing CD5 activity, thereby preventing, alleviating, improving or inhibiting diseases or conditions. use.
  • the disease or disorder is cancer.
  • the cancer is a malignant T cell tumor or a malignant B cell tumor.
  • the malignant T-cell tumor is selected from T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma (TCL), and the malignant B-cell tumor is selected from chronic lymphocytic leukemia (B- CLL) and mantle cell lymphoma (B-MCL).
  • T-ALL T-cell acute lymphoblastic leukemia
  • T-cell lymphoma TCL
  • B- CLL chronic lymphocytic leukemia
  • B-MCL mantle cell lymphoma
  • the application includes a chimeric antigen receptor (CAR), wherein the CAR comprises a CD5 binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain, the HCDR1, HCDR2 , the amino acid sequence of HCDR3 is selected from the following combinations: (1) HCDR1 as the amino acid sequence of SEQ ID NO:43, HCDR2 as the amino acid sequence of SEQ ID NO:44, and HCDR3 as the amino acid sequence of SEQ ID NO:45; (2) HCDR1 as the amino acid sequence of SEQ ID NO:49, HCDR2 as the amino acid sequence of SEQ ID NO:50, and HCDR3 as the amino acid sequence of SEQ ID NO:51; (3) as SEQ ID NO:55 HCDR1 of the amino acid sequence, such as HCDR2 of the amino acid sequence of SEQ ID NO:56, and HCDR3 of the amino acid sequence of SEQ ID NO:57; (4) HCDR1 of the amino acid sequence
  • the antibody further comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3)
  • the amino acid sequence is selected from the following combinations: (1) LCDR1 of the amino acid sequence of SEQ ID NO:46, LCDR2 of the amino acid sequence of SEQ ID NO:47, and LCDR3 of the amino acid sequence of SEQ ID NO:48; (2) LCDR1 as the amino acid sequence of SEQ ID NO:52, LCDR2 as the amino acid sequence of SEQ ID NO:53, and LCDR3 as the amino acid sequence of SEQ ID NO:54; (3) as the amino acid sequence of SEQ ID NO:58 LCDR1, such as LCDR2 of the amino acid sequence of SEQ ID NO:59, and LCDR3 of the amino acid sequence of SEQ ID NO:60; (4) LCDR1 of the amino acid sequence of SEQ ID NO:64, such as the amino acid of SEQ ID NO:65 LCDR2 of the sequence, and LCDR3
  • the antibody comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 selected from any one of the following combinations: (1) HCDR1 as the amino acid sequence of SEQ ID NO: 43, as in SEQ ID HCDR2 of the amino acid sequence of NO:44, and HCDR3 of the amino acid sequence of SEQ ID NO:45, LCDR1 of the amino acid sequence of SEQ ID NO:46, LCDR2 of the amino acid sequence of SEQ ID NO:47, and LCDR1 of the amino acid sequence of SEQ ID NO:47 LCDR3 of the amino acid sequence of NO:48; (2) HCDR1 of the amino acid sequence of SEQ ID NO:49, HCDR2 of the amino acid sequence of SEQ ID NO:50, and HCDR3 of the amino acid sequence of SEQ ID NO:51, such as LCDR1 of the amino acid sequence of SEQ ID NO:52, LCDR2 of the amino acid sequence of SEQ ID NO:53, and LCDR3 of the amino acid sequence of SEQ ID
  • the antibody comprises a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 30, 36, 42, 90, 93, or 96. In certain embodiments, the antibody comprises a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 29, 35, 41, 89, 92, or 95. In certain embodiments, the antibody is a single chain antibody. In certain embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID No: 28, 34, 40, 88, 91 or 94 or a functional variant thereof.
  • the transmembrane domain of the CAR comprises a polypeptide from a protein selected from the group consisting of alpha, beta or zeta chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5, CD8a, CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain comprises the amino acid sequence set forth in SEQ ID No: 6 or a functional variant thereof.
  • the costimulatory domain of the CAR comprises a polypeptide selected from the group consisting of CD28, 4-1BB, OX-40, and ICOS. In certain embodiments, the costimulatory domain comprises the amino acid sequence set forth in SEQ ID No: 8 or a functional variant thereof.
  • the intracellular signaling domain of the CAR comprises a signaling domain from CD3 ⁇ . In certain embodiments, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID No: 10 or a functional variant thereof.
  • the CAR further comprises a hinge region connecting the antibody and the transmembrane domain.
  • the hinge region comprises the amino acid sequence set forth in SEQ ID No: 4 or a functional variant thereof.
  • the CAR is further linked to CD8 ⁇ to a signal peptide.
  • the signal peptide comprises the amino acid sequence set forth in SEQ ID No: 2 or a functional variant thereof.
  • the CAR is also linked to a cleavage peptide.
  • the cleavage peptide comprises an amino acid sequence from a T2A peptide.
  • the cleavage peptide comprises the amino acid sequence set forth in SEQ ID No: 12 or a functional variant thereof.
  • the CAR is further linked to a truncated form of the EGFR molecule (tEGFR) via a cleavage peptide and a CSF2RA signal peptide.
  • the truncated form of the EGFR molecule comprises the amino acid sequence set forth in SEQ ID No: 16 or a functional variant thereof.
  • the CSF2RA signal peptide comprises the amino acid sequence shown in SEQ ID No: 14 or a functional variant thereof.
  • the CAR comprises an amino acid sequence selected from SEQ ID Nos: 22-24, 98, 100, or 102, or a functional variant thereof.
  • the present application also includes isolated nucleic acid molecules encoding the CARs described herein.
  • the application also includes an isolated nucleic acid molecule encoding a CAR comprising a nucleic acid molecule selected from the group consisting of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19-21, 25-27, A nucleic acid sequence of 31-33, 37-39, 79-87, 97, 99 or 101 or a functional variant thereof.
  • the present application also includes a vector comprising the nucleic acid molecule described herein.
  • the vector is selected from the group consisting of plasmids, retroviral vectors and lentiviral vectors.
  • the present application also includes an immune effector cell, which comprises the CAR described in the present application, the nucleic acid molecule described in the present application, or the vector described in the present application.
  • the immune effector cells are selected from T lymphocytes and natural killer (NK) cells.
  • NK natural killer
  • CD5 is not expressed on the immune effector cells.
  • the present application also includes a method for preparing immune effector cells, comprising knocking out the CD5 gene of the immune effector cells, and introducing the vector described in the present application into the immune effector cells.
  • the knockout is performed using the sgRNA of any one of SEQ ID NOs: 103-106.
  • the present application also includes a pharmaceutical composition comprising the immune effector cells described in the present application and a pharmaceutically acceptable adjuvant.
  • the present application also includes the use of the CAR, the nucleic acid molecule, the vector, or the immune effector cell in the preparation of a drug, wherein the drug is used for treatment related to the expression of CD5 disease or condition.
  • the disease or disorder associated with the expression of CD5 is cancer or malignancy.
  • the present application also relates to the CAR, the nucleic acid molecule, the vector, or the immune effector cell or the pharmaceutical composition for treating a disease or condition related to the expression of CD5 .
  • the disease or disorder associated with the expression of CD5 is cancer or malignancy.
  • the application also includes a method of treating a disease or disorder associated with the expression of CD5, comprising administering to a patient in need thereof a therapeutically effective amount of the immune effector described herein, or the pharmaceutical composition.
  • an EGFR antibody such as Cetuximab can be further administered to a patient in need to inhibit the effect of the immune effector cells or the pharmaceutical composition, wherein the patient in need comprises Those with severe adverse reactions to immune effector cells or pharmaceutical compositions described herein.
  • Figure 1 Screening of CD5ko sgRNA with jurkat cell line, showing the results of flow cytometry analysis of cell clones obtained by knocking out jurkat cells with different sgRNAs.
  • Figure 2 shows the results of the characterization of CD5 expression in the monoclonal jurkat-CD5ko cell line by flow cytometry.
  • Figure 3 shows the CAR structure used in this experiment (Figure 3A) and the detection of CD5 and EGFRt expression in CAR-T cells after transfection ( Figure 3B).
  • Figure 4 shows the results of CD107a degranulation of CAR-T cells by different target cells.
  • results of each CAR molecule from left to right are the results after co-incubation with Jurkat, Jurkat CD5ko, CCRF-CEM, MOLT4, K562-CD5, K562, RAJI and CAR-T cells alone.
  • Figure 5 shows the killing results of CAR-T cells on various target cells.
  • Figure 6 shows the experimental results of repeated stimulation of CAR-T cell proliferation with mitomycin-treated CCRF-CEM cells.
  • Figure 7 shows the kinetic binding curves and KD, kon, kdis parameters of the affinity determination of H65, Clone 10, Clone 32 and Clone 35 with CD5 antigen.
  • Figure 8 shows the general flow of the present invention for screening specific antibodies targeting CD5 from a phage antibody library.
  • Figure 9 shows the results of an enzyme-linked immunosorbent assay (ELISA) of some of the panned phage monoclones with target and control antigens.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 10 shows the results of flow cytometric analysis of the binding of some phage monoclones to Raji and Jurkat cells.
  • Figures 11A-F show the results of flow cytometric analysis (peak graphs and MFI values) of the binding of screened phage monoclones #1-64 to various different CD5 positive and negative cell lines.
  • Negative Control is a negative control phage antibody clone.
  • Figures 12A-F show the results of ELISA analysis of screened phage monoclones #1-64 with CD5 antigen proteins and non-related antigens from various companies.
  • Negative control is a negative control phage antibody clone
  • anti-M13 phage mouse Ab/anti-mouse HRP Ab is a negative antibody control with only primary antibody and secondary antibody added
  • anti-mouse HRP Ab is a negative antibody with only secondary antibody added Control
  • Mouse anti-human CD5 Ab/anti-mouse HRP Ab is the positive antibody control for the target antigen (CD5-Fc-Bio)
  • anti-human IgG-HRP Ab/anti-his-HRP Ab is the positive antibody control for detecting the antigen label .
  • the bar graphs corresponding to each test antibody and the control group from left to right represent the protein Kactus-CD5-Fc-Bio, SB-CD5-his-Bio, Acro-CD5-his, Test results for Kactus-BAFFR-his-Bio, Kactus-CD19-FC-Bio, SA.
  • the CAR described in this application can specifically bind to CD5, the CAR-T cells prepared by using the CAR can stably express the CAR, and the CAR-T cells prepared by using the CAR have a high CAR positive rate.
  • the CAR can promote the release of cytokines and can be used to treat diseases or conditions associated with the expression of CD5.
  • chimeric antigen receptor Chimeric Antigen Receptor, CAR
  • CAR Chimeric Antigen Receptor
  • TAA tumor-associated antigen
  • transmembrane domain transmembrane domain
  • costimulatory domain intracellular signaling domain
  • the CAR may be a genetically engineered chimeric protein capable of redirecting the cytotoxicity of immune effector cells to B cells, which specifically associates an antibody-based antigen (eg, CD5) with T cell receptor activation
  • an antibody-based antigen eg, CD5
  • the intracellular domains are grouped together.
  • Genetically modified T cells expressing CAR can specifically recognize and eliminate malignant cells expressing the target antigen.
  • Sadelain M, Brentjens R, Rivi'ere I The basic principles of chimeric antigen receptor design. Cancer Discov. 2013; 3(4): 388-398; Turtle CJ, Hudecek M , Jensen MC, Riddell SR.
  • CD5 is a type I transmembrane glycosylated protein that plays an important role in the negative regulation of T cell receptor signaling and promotes the survival of normal and malignant lymphocytes.
  • CD5 is one of the characteristic surface markers of malignant T-cell tumors, and 80% of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas express CD5.
  • T-ALL T-cell acute lymphoblastic leukemia
  • the CD5 can be human CD5, whose GenBank accession number is NM_014207.4.
  • CD5 proteins can also include fragments of CD5, such as the extracellular domain and fragments thereof.
  • CD5 binding domain generally refers to the extracellular domain of a CD5 CAR, which can specifically bind to an antigen.
  • the CD5 extracellular binding domain may comprise a chimeric antigen receptor, an anti-CD5 antibody or an antigen-binding fragment thereof capable of specifically binding to CD5 polypeptide expressed on human cells.
  • binding domain "binding domain”, “extracellular domain”, “extracellular binding domain”, “antigen-specific binding domain” and “extracellular antigen-specific binding domain” are used interchangeably in this application use, and provide CARs with the ability to specifically bind to the target antigen of interest (eg, CD5).
  • the CD5 binding domain can be of natural, synthetic, semi-synthetic or recombinant origin.
  • an antibody generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (eg, dAbs), single chain antibodies (eg, scFvs).
  • a "fragment" of an antibody may refer to an antigen-binding fragment of an antibody, eg, Fab, Fab' and (Fab') 2 fragments, and the like.
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof as described.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • the VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL can consist of three CDRs and four FR regions, which can be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • single-chain antibody may also be referred to as scFv, which refers to an antibody composed of one chain composed of a heavy chain variable region and a light chain variable region linked by a linking peptide.
  • a "murine antibody” is an antibody produced by a mouse against a specific antigen, usually referring to an antibody produced by mouse B lymphocytes. In most cases, the murine antibody is a monoclonal antibody produced by hybridoma cells.
  • the fully human antibody of the present application is obtained by screening a human-derived phage antibody library, which reduces the immunogenicity compared with the mouse-derived antibody, and is more beneficial to the therapeutic use of the human body.
  • the "fully human antibody or single-chain antibody or fragment thereof" described in this application generally refers to any form of antigen-binding molecule that can bind to a target antigen, for example, the antigen-binding molecule can be a protein or polypeptide, including, for example, antibodies and their Antigen-binding fragments, single chain scFv antibodies, single domain antibodies, various fusions and conjugates based on scFv constructions such as scFv-Fc antibodies, immunoconjugates, antibody drug conjugates (ADC), multi/bispecific Antibody, Chimeric Antigen Receptor (CAR).
  • ADC antibody drug conjugates
  • CAR Chimeric Antigen Receptor
  • epitope refers to a portion of a molecule that is bound by an antigen binding protein (eg, an antibody). Epitopes may comprise non-adjacent portions of the molecule (eg, in a polypeptide that are not adjacent in the main sequence of the polypeptide, but are sufficiently close to each other in the trivalent and tetravalent structure of the polypeptide to be bound by the antigen binding protein amino acid residues).
  • an antigen binding protein eg, an antibody
  • transmembrane domain generally refers to the domain in the CAR that passes through the cell membrane, which is linked to the intracellular signal transduction domain and plays a role in transmitting signals.
  • the transmembrane domain may be the CD28 transmembrane domain.
  • costimulatory domain generally refers to an intracellular domain that can provide immune costimulatory molecules, which are cell surface molecules required for effective lymphocyte responses to antigens.
  • the costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, eg, the costimulatory domains of OX40 and 4-1BB.
  • the term "hinge region” generally refers to the connecting region between the antigen binding region and the immune cell Fc receptor (FcR) binding region.
  • the hinge region may be the CD28 hinge region.
  • intracellular signaling domain generally refers to the component of the CAR located in intracellular signaling, which comprises a signaling domain and a domain that specifically binds to the receptor component, for example: Can be selected from CD3 ⁇ intracellular domain, CD28 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain and OX40 intracellular domain.
  • CD8 ⁇ signal peptide (Signal peptide) generally refers to short (5-30 amino acids in length) peptide chains that direct the transfer of newly synthesized proteins to the secretory pathway.
  • cleavage peptide refers to a self-cleaving 2A peptide, which can perform the function of a cleavage protein by ribosome hopping rather than proteolysis, which may include T2A, F2A and P2A, etc.
  • the term "marker detection signal” generally refers to a gene, protein or other molecule of known function or sequence that can function as a specific marker and emit a signal that can be detected.
  • the labeled detection signal can be a fluorescent protein, such as: GFP, RFP, YFP, and the like.
  • the labeled detection signal may be EGFRt.
  • EGFRt or “tEGFR” are used interchangeably herein and refer to a gene encoding a truncated human epidermal growth factor receptor polypeptide that lacks the distal membrane EGF binding domain and cytoplasmic signaling tail, but retains the Extracellular epitopes recognized by anti-EGFR antibodies.
  • EGFRt can be used as a non-immunogenic selection tool as well as a tracking marker with genetically modified cellular functions.
  • it can be used as a marker molecule for CAR-T cells to clear the CAR-T cell EGFR antibody (eg, cetuximab) mediated ADCC pathway in vivo if necessary (cetuximab mediated ADCC pathway) ( See US8802374B2), which is used as a safety switch in clinical translation.
  • cetuximab cetuximab mediated ADCC pathway
  • CSF2RA signal peptide namely the colony stimulating factor 2 receptor alpha subunit (colony stimulating factor 2 receptor subunit alpha) signal peptide
  • colony stimulating factor 2 receptor alpha subunit colony stimulating factor 2 receptor subunit alpha
  • EGFR antibody refers to an antibody-dependent cell-mediated cytotoxicity that causes immune cells to attack CAR-T cells with truncated epidermal growth factor receptor (EGFRt) , antibodies that assist in the removal of CAR-T cells.
  • EGFR antibody can be used when a patient has severe adverse reactions after infusion of CAR-T or other conditions that require the removal of CAR-T cells, which can assist in the removal of CAR-T cells and relieve symptoms associated with CAR-T therapy.
  • the EGFR antibodies include, but are not limited to, cetuximab, panitumumab, nexituzumab, and nimotuzumab.
  • sequence identity or “sequence identity” generally refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison. "Percent sequence identity” can be calculated by comparing two optimally aligned sequences in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences.
  • the same amino acid residue eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met
  • Optimal alignment to determine percent sequence identity can be achieved in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest.
  • the fully human antibodies provided herein also include at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 28, 34, 40, 88, 91 or 94 (eg, at least 95% sequence identity). %, at least 98%, at least 99%, or even 100% sequence identity) amino acid sequence.
  • the fully human antibody or single-chain antibody or fragment thereof of the present application may have at least 1 and no more than 10, or no more than 5, 4, 3, 2, or 1 amino acid changes in the full-length or CDR sequence. .
  • an antibody light chain library (such as a human phage light chain library) can be screened by using CD5 as an antigen, so as to obtain the same
  • the heavy chain variable region matches and maintains the CD5 binding capacity of the light chain variable region.
  • Anti-CD5 antibody molecules obtainable in this manner are also included within the scope of the present invention.
  • the antigen-binding molecules of the present application may further comprise post-translational modifications.
  • post-translational protein modifications include: phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitination, biotinylation, or addition of polypeptide side chains or hydrophobic groups group.
  • modified soluble polypeptides may contain non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates.
  • a preferred form of glycosylation is the sialylation modification, which binds one or more sialic acid groups to the polypeptide. The sialic acid group improves the solubility and serum half-life of the protein, while also reducing the possible immunoheritability of the protein. See Raju et al. Biochemistry. 2001 31;40(30):8868-76.
  • the term "functional variant" of a protein or polypeptide sequence in this application means, by 1 or more, eg 1-30, or 1-20 or 1-10, eg 1 or 2 or 3 or 4 or 5 A sequence in which amino acid substitutions, deletions and/or insertions have amino acid changes compared to the parent. Functional variants substantially retain the biological properties of the protein or polypeptide sequence before the alteration. In one aspect, the application encompasses variants of any protein or polypeptide sequence described in the application. In certain embodiments, a functional variant of a protein or polypeptide sequence retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity of the parent before the alteration.
  • the functional variants of the present invention can be functional variants of signal peptides, antibodies, hinge regions, transmembrane domains, co-stimulatory domains, intracellular signaling domains, cleavage peptides, CSF2RA signal peptides, EGFRt.
  • antibody variable regions eg VH or VL
  • antibody constant regions eg CH or CL
  • heavy chain CDR regions HCDR1, HCDR2 or HCDR3
  • light chain CDR regions are also included Functional variants of (LCDR1, LCDR2 or LCDR3) etc.
  • Amino acid substitutions, deletions and/or insertions can occur in the heavy chain CDR region or the light chain CDR region, or the heavy chain FR region or the light chain FR region, or the heavy chain constant region or the light chain constant region, wherein the variant substantially remains Change the biological properties of the previous antibody molecule.
  • their biological activities include, for example, antigen-binding ability.
  • functional variants of antibodies contain amino acid changes that do not cause the antibody variant to lose binding to the antigen, but optionally can confer properties such as increased antigen affinity and different effector functions. It will be appreciated that the variable heavy or light chain regions of an antibody, or each CDR region, may be altered individually or in combination.
  • the amino acid change can be an amino acid substitution, eg, can be a conservative substitution.
  • the functional variant has at least 80%, 85%, 90% or 95% or 99% or more amino acid identity to the parent.
  • a "functional variant" of a nucleic acid molecule refers to a nucleic acid molecule that encodes the same amino acid sequence as the parent nucleic acid molecule.
  • the term "isolated” generally refers to an antibody that has been separated from components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed-phase HPLC).
  • electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography eg, ion exchange or reversed-phase HPLC
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, isolated from their natural environment or artificially synthesized, or analogs thereof.
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • nucleic acids encoding the antibodies or antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including, but not limited to, manipulation using restriction fragments or using synthetic oligonucleotides.
  • methods known in the art including, but not limited to, manipulation using restriction fragments or using synthetic oligonucleotides.
  • overlap extension PCR see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host for the transfer of an inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
  • one or more of the nucleic acid molecules may be contained in the vector.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and can include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary depending on species or cell type function, but typically comprises 5' untranslated and 5' and 3' untranslated sequences involved in transcription and translation initiation, respectively, such as the TATA box, plus Cap sequences, CAAT sequences, etc.
  • a 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid.
  • the vectors described herein can be selected from plasmids, retroviral vectors and lentiviral vectors.
  • the plasmids, retroviral vectors, and lentiviral vectors described herein may comprise a CAR.
  • Plasmid generally refers to DNA molecules other than chromosomes or nucleoids in organisms such as bacteria and yeast, which exist in the cytoplasm and have the ability to autonomously replicate, enabling them to maintain a constant copy in daughter cells number and express the genetic information it carries. Plasmids are used as carriers of genes in genetic engineering research.
  • retroviral vector generally refers to a kind of RNA virus, whose genetic information is stored on ribonucleic acid, and most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, the genes that contain the proteins that make up the center and structure of the virus; pol, the genes that make up the reverse transcriptase, and env, the genes that make up the viral coat. Through retroviral transfection, the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome, for example, CAR molecules can be integrated into the host cell.
  • lentiviral vector generally refers to a diploid RNA viral vector that is a retrovirus.
  • the lentiviral vector is based on the genome of the lentivirus, and many sequence structures related to the virus activity are removed to make it biologically safe, and then the sequence of the target gene required for the experiment is introduced into the genome backbone. and the vector prepared from the expression construct.
  • lentiviral vectors Compared with other retroviruses, lentiviral vectors have a wider range of hosts and have the ability to infect both dividing and non-dividing cells. For some cells that are difficult to transfect, such as primary cells, stem cells, undifferentiated cells, etc.
  • Retroviral vectors can randomly and stably integrate their own genome and the foreign genes they carry into the host cell genome, for example, CAR molecules can be integrated into the host cell.
  • transposon generally refers to a discrete DNA segment containing a transposase gene, flanked by terminal inverted repeats (TIRs) containing transposase binding sites.
  • TIRs terminal inverted repeats
  • the transposase can bind to the TIR and transfer the transposon to a new site.
  • the transposon described in this application is a two-component system consisting of a plasmid carrying a CAR (transposon) and another plasmid carrying a transposase.
  • the transposon can be introduced into target cells by means of electrotransduction or the like.
  • the two components are electroporated into peripheral blood mononuclear cells (PBMC), and the expressed transposase acts on the terminal inverted repeats (TIRs) flanking the CAR.
  • TIRs terminal inverted repeats
  • CAR proteins can be expressed on the surface of target cells (see Cheng Zhang, Jun Liu, Jiang F Zhong, et al. Engineering CAR-T cells. Biomarker Research. 2017, 5:22).
  • the term "gene editing” generally refers to the technology of site-directed modification of the genome, which may include zinc finger nucleases (zinc finger nucleases, ZFNs), transcription activator like effector nucleases (transcription activator like effector nucleases) , TALENs), clusters of regularly repeated short palindromic sequences (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (Cas9), CRISPR/Cas9) and other technologies. It enables efficient targeted modification of the genome by adding, removing or changing genetic material at specific locations in the genome.
  • Gene editing as described herein may include introducing a CAR molecule into the genome of a recipient cell through gene editing techniques (eg, CRISPR-Cas9).
  • immune effector cells generally refers to immune cells that participate in the clearance of foreign antigens and perform effector functions in an immune response.
  • immune effector cells For example, plasma cells, cytotoxic T cells, NK cells, APSC pluripotent cells, mast cells, etc.
  • pharmaceutically acceptable adjuvant generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation.
  • additives are well known to those skilled in the art.
  • cancer generally refers to or describes a physiological condition in mammals that is typically characterized by dysregulated cell proliferation or survival.
  • hyperproliferative diseases referred to as cancer include, but are not limited to, solid tumors, such as those occurring in the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid adenocarcinomas, and their distant metastases.
  • diseases also include lymphomas, sarcomas, and leukemias.
  • breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
  • respiratory cancers include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenomas and pleuropulmonary blastomas.
  • brain cancers include, but are not limited to, brainstem and hypothalamic keratinoma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and neuroectodermal and pineal tumors.
  • Male genital tumors include, but are not limited to, prostate and testicular cancer.
  • Female genital tumors include, but are not limited to, endometrial, cervical, ovarian, vaginal and vulvar cancers, and uterine tumors.
  • Gastrointestinal tumors include, but are not limited to, anal, colon, colorectal, esophagus, gallbladder, stomach, pancreas, rectal, small bowel, and salivary gland cancers.
  • Urethral tumors include, but are not limited to, bladder, penis, kidney, renal pelvis, ureter, and urethral cancers.
  • Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.
  • liver cancer examples include, but are not limited to, hepatocellular carcinoma (hepatoma with or without fibrolamellar variation), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma.
  • Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
  • Head and neck cancers include, but are not limited to, laryngeal/hypopharyngeal/nasopharyngeal/oropharyngeal cancers, and lip and oral cavity cancers.
  • Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma.
  • Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.
  • Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.
  • pharmaceutically acceptable carrier is used to refer to substances such as solid or liquid diluents, fillers, antioxidants, stabilizers, etc., which are safe for administration, and which are suitable for humans and/or animals Administration without undue adverse side effects, while being suitable for maintaining the viability of the drug or active agent located therein.
  • a “therapeutically effective amount” refers to an amount of an active compound or cell sufficient to elicit the biological or medical response desired by a clinician in a subject.
  • the “therapeutically effective amount” can be determined by those skilled in the art according to the administration route, the subject's body weight, age, condition and other factors. For example, for antibodies, a typical daily dosage range may be from 0.01 mg to 100 mg of active ingredient per kg of body weight.
  • Modes of administration of the antibodies of the present application include, but are not limited to, injection, eg, by intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal, and the like.
  • the term “comprising” or “comprising” means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.
  • the term “comprising” or “comprising” when used, unless otherwise indicated, it also covers situations consisting of the stated elements, integers or steps.
  • reference to an antibody variable region that "comprises” a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present invention uses fully human phage for antibody screening, and directly obtains fully human monoclonal antibodies. Compared with traditional hybridoma technology, the difficult step of humanization of murine antibodies is omitted, and fully human antibodies have lower immunogenicity than humanized murine antibodies.
  • Antibodies, ADC, etc.), cell therapy drugs (including CAR-T, CAR-NK, etc.), detection reagents and other applications have better potential.
  • the present invention uses the method of antigen protein panning, which can efficiently enrich the antibody that binds to recombinant CD5 and the natural structure CD5 on the cell membrane, greatly reduces the difficulty of later antibody screening, and improves the efficiency.
  • the CAR may comprise an extracellular domain, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain that specifically bind to CD5.
  • the extracellular domain of the CAR may comprise the single-chain scFv antibody of the present invention.
  • the single chain antibody can be linked to the transmembrane domain through a hinge region, such as the CD8 hinge.
  • the CAR can be used to transduce immune effector cells (eg T cells) and express on the cell surface. Therefore, the present application can also provide T cells expressing the chimeric antigen receptor, and the use of the T cells and/or the CAR for preparing a drug for treating CD5-related diseases.
  • the chimeric antigen receptor may comprise a CD5 binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain.
  • the CD5 binding domain may comprise an antibody or fragment thereof that specifically binds to CD5
  • the antibody may comprise a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain Complementarity determining region 3 (HCDR3)
  • the amino acid sequence of the HCDR1-3 is shown in SEQ ID NO: 43-45, 49-51, 55-57, 61-63, 67-69 or 73-75
  • the antibody Can also comprise light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3)
  • the amino acid sequence of said LCDR1-3 is as SEQ ID No:46-48, 52-54, 58-60, 64-66, 70-72 or
  • the antibody may comprise a heavy chain variable region whose amino acid sequence is shown in SEQ ID No: 30, 36, 42, 90, 93 or 96.
  • the antibody may comprise a light chain variable region whose amino acid sequence is shown in SEQ ID No: 29, 35, 41, 89, 92 or 95.
  • the antibody may be a single chain antibody.
  • the antibody may comprise the amino acid sequence set forth in SEQ ID No: 28, 3440, 88, 91 or 94 or a functional variant thereof.
  • the single chain antibody can include scFv10, whose sequence is shown in SEQ ID No: 28; the single chain antibody can include scFv32, whose sequence is shown in SEQ ID No: 34; the single chain antibody can include scFv35 , whose sequence is shown in SEQ ID No: 40; the single-chain antibody can include scFv6, whose sequence is shown in SEQ ID No: 88; the single-chain antibody can include scFv7, whose sequence is shown in SEQ ID No: 91
  • the single-chain antibody can include scFv9, the sequence of which is shown in SEQ ID No: 94;.
  • the single-chain antibody described in this application can be scFv10 (original numbering is clone 10), and its sequence is shown in SEQ ID No: 28.
  • the amino acid sequences of LCDR1-3 of the single-chain antibody scFv10 are shown in SEQ ID No: 46, SEQ ID No: 47 and SEQ ID No: 48 respectively; the amino acid sequence of VL is shown in SEQ ID No: 29; the amino acid sequences of HCDR1-3
  • the amino acid sequences are shown in SEQ ID No: 43, SEQ ID No: 44 and SEQ ID No: 45 respectively; the amino acid sequence of VH is shown in SEQ ID No: 30.
  • the single-chain antibody described in this application can be scFv32 (original numbering is clone 32), and its sequence is shown in SEQ ID No:34.
  • the amino acid sequences of LCDR1-3 of the single-chain antibody scFv32 are shown in SEQ ID No: 52, SEQ ID No: 53 and SEQ ID No: 54 respectively; the amino acid sequence of VL is shown in SEQ ID No: 35; the amino acid sequences of HCDR1-3
  • the amino acid sequences are shown in SEQ ID No: 49, SEQ ID No: 50 and SEQ ID No: 51 respectively; the amino acid sequence of VH is shown in SEQ ID No: 36.
  • the single-chain antibody described in this application can be scFv35 (original numbering is clone 35), and its sequence is shown in SEQ ID No: 40.
  • the amino acid sequences of LCDR1-3 of the single-chain antibody scFv35 are shown in SEQ ID No: 58, SEQ ID No: 59 and SEQ ID No: 60 respectively; the amino acid sequence of VL is shown in SEQ ID No: 41; the amino acid sequences of HCDR1-3
  • the amino acid sequences are shown in SEQ ID No: 55, SEQ ID No: 56 and SEQ ID No: 57 respectively; the amino acid sequence of VH is shown in SEQ ID No: 42.
  • the single-chain antibody described in this application can be scFv6 (original numbering is clone 6), and its sequence is shown in SEQ ID No: 88.
  • the amino acid sequences of LCDR1-3 of single-chain antibody scFv6 are shown in SEQ ID No: 64, SEQ ID No: 65 and SEQ ID No: 66 respectively; the amino acid sequence of VL is shown in SEQ ID No: 89; The amino acid sequences are shown in SEQ ID No: 61, SEQ ID No: 62 and SEQ ID No: 63 respectively; the amino acid sequence of VH is shown in SEQ ID No: 90.
  • the single-chain antibody described in the present application can be scFv7 (original numbering is clone7), and its sequence is shown in SEQ ID No: 91.
  • the amino acid sequences of LCDR1-3 of the single-chain antibody scFv7 are shown in SEQ ID No: 70, SEQ ID No: 71 and SEQ ID No: 72 respectively; the amino acid sequence of VL is shown in SEQ ID No: 92; the amino acid sequences of HCDR1-3
  • the amino acid sequences are shown in SEQ ID No: 67, SEQ ID No: 68 and SEQ ID No: 69 respectively; the amino acid sequence of VH is shown in SEQ ID No: 93.
  • the single-chain antibody described in this application can be scFv9 (original numbering is clone9), and its sequence is shown in SEQ ID No: 94.
  • the amino acid sequences of LCDR1-3 of the single-chain antibody scFv9 are shown in SEQ ID No: 76, SEQ ID No: 77 and SEQ ID No: 78 respectively; the amino acid sequence of VL is shown in SEQ ID No: 95; The amino acid sequences are shown in SEQ ID No: 73, SEQ ID No: 74 and SEQ ID No: 75 respectively; the amino acid sequence of VH is shown in SEQ ID No: 96.
  • the CAR described herein may comprise a transmembrane domain, which may comprise a polypeptide from a protein selected from the group consisting of alpha, beta or zeta chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5 , CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID No: 6 or a functional variant thereof.
  • the transmembrane domain of the present application may include CD8a, the sequence of which is set forth in SEQ ID No:6.
  • the costimulatory domain may comprise a polypeptide from a protein selected from the group consisting of CD28, 4-1BB, OX40 and ICOS.
  • the costimulatory domain may comprise the amino acid sequence shown in SEQ ID No: 8 or a functional variant thereof.
  • the CAR described herein can include an intracellular signaling domain, which can include a signaling domain from CD3 ⁇ .
  • the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID No: 10 or a functional variant thereof.
  • the CAR described herein can include a hinge region that can connect the antibody and the transmembrane domain.
  • the hinge region may comprise the amino acid sequence shown in SEQ ID No: 4 or a functional variant thereof.
  • the CARs described herein may include a signal peptide, which may, for example, be N-terminal to the extracellular domain that specifically binds CD5.
  • the signal peptide may comprise the amino acid sequence shown in SEQ ID No: 2 or a functional variant thereof.
  • the signal peptide can be the CD8a signal peptide, the sequence of which is shown in SEQ ID No:2.
  • the CAR can also be linked to a cleavage peptide.
  • the cleavage peptide may comprise the amino acid sequence from the T2A peptide.
  • the cleavage peptide may comprise the amino acid sequence shown in SEQ ID No: 12 or a functional variant thereof.
  • the cleavage peptide can be T2A, the sequence of which is shown in SEQ ID No: 12.
  • the CAR can also be linked to an EGFRt fragment, and the EGFRt fragment can be used for signal detection, or used as a molecular switch for CAR-T cells.
  • the CAR may comprise the amino acid sequence shown in SEQ ID No: 22-24, 98, 100 or 102 or a functional variant thereof.
  • the CAR can be selected from CAR001, the sequence of which is shown in SEQ ID No:22.
  • the CAR can be selected from CAR002, whose sequence is shown in SEQ ID No: 23; the CAR can be selected from CAR003, whose sequence is shown in SEQ ID No: 24; the CAR can be selected from CAR004, whose sequence is shown in SEQ ID No: 24.
  • the sequence is shown in SEQ ID No: 98;
  • the CAR can be selected from CAR005, whose sequence is shown in SEQ ID No: 100; the CAR can be selected from CAR006, whose sequence is shown in SEQ ID No: 102.
  • the CAR described herein may include, in order from the N-terminus, a CD5 binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.
  • the CAR can include a CD5 binding domain, and the CD5 binding domain sequence is shown in SEQ ID No:28.
  • the CD5 binding domain can include HCDR1-3, the sequence of which is shown in SEQ ID No: 43-45 in turn; and the CD5 binding domain can include LCDR1-3, whose sequence is shown in SEQ ID No: 46- 48 shown.
  • the CAR can include CAR001 or a CAR described herein that has the same LCDR1-3 and HCDR1-3.
  • the CD5 binding domain may include a heavy chain variable region, the sequence of which is shown in SEQ ID No: 30; and the CD5 binding domain may also include a light chain variable region, whose sequence is shown in SEQ ID No: 29 shown.
  • the CAR may comprise CAR001 or a CAR described herein having the same light chain variable region and heavy chain variable region.
  • a linking peptide may also be included between the light chain variable region and the heavy chain variable region, the sequence of which is shown in SEQ ID No: 18.
  • the CAR may comprise CAR001 or a CAR described herein with the same linker peptide.
  • the transmembrane domain may comprise the transmembrane domain from CD8a, the sequence of which may be as shown in SEQ ID No:6.
  • the CAR may comprise CAR001 or a CAR described herein having the same transmembrane domain.
  • the costimulatory domain may comprise a costimulatory structure from 4-1BB, the sequence of which may be set forth in SEQ ID No:8.
  • the CAR can comprise CAR001 or a CAR described herein that has the same costimulatory domain.
  • the intracellular signaling domain may comprise a signaling domain from CD3 ⁇ , the sequence of which is set forth in SEQ ID No:10.
  • the CAR may comprise CAR001 or a CAR described herein having the same intracellular signaling domain.
  • the CAR may further comprise a hinge region, which may be C-terminal to the CD5 binding domain and N-terminal to the transmembrane domain, the sequence of which may be, for example, as shown in SEQ ID No:4.
  • the CAR may comprise CAR001 or a CAR described herein that has the same hinge region.
  • the CAR can also be linked to a signal peptide, which can be located at the N-terminus of the CAR, and its sequence can be as shown in SEQ ID No:2.
  • the CAR can also be linked to a cleavage peptide such as T2A.
  • the cleavage peptide can be located at the C-terminus of the intracellular signal transduction domain, and its sequence can be shown in SEQ ID No: 12.
  • the CAR can also be linked to a CSF2RA signal peptide, which can be located before EGFRt, and its sequence can be, for example, as shown in SEQ ID No: 14.
  • the CAR can also be linked to a labeled detection signal, which can be located C-terminal to the CAR (alternatively, the cleavage peptide).
  • the marker detection signal may be selected from the group consisting of GFP, RFP, YFP or EGFRt, and the sequence of EGFRt may be, for example, as shown in SEQ ID No: 16.
  • the CAR described in this application can be CAR001, and the amino acid sequences of its LCDR1-3 are shown in SEQ ID No: 46, SEQ ID No: 47 and SEQ ID No: 48 respectively; the amino acid sequence of VL is shown in SEQ ID No: 48: 29; the amino acid sequences of HCDR1-3 are respectively shown in SEQ ID No: 43, SEQ ID No: 44 and SEQ ID No: 45; the amino acid sequence of VH is shown in SEQ ID No: 30; between VH and VL
  • the sequence of the connecting peptide is shown in SEQ ID No: 18; its hinge region is shown in SEQ ID No: 4; its transmembrane domain is shown in SEQ ID No: 6; its costimulatory domain is 4-1BB co-stimulatory domain.
  • Stimulatory domain as shown in SEQ ID No: 8; its CD3 ⁇ intracellular signaling domain as shown in SEQ ID No: 10; the CAR001 further comprises a cleavage peptide as shown in SEQ ID No: 12, as shown in SEQ ID No: 12 CSF2RA signal peptide shown in ID No: 14, and EGFRt shown in SEQ ID No: 16; CD8a signal peptide shown in SEQ ID No: 2.
  • the CAR described in this application can be CAR002, and the amino acid sequences of its LCDR1-3 are shown in SEQ ID No: 52, SEQ ID No: 53 and SEQ ID No: 54 respectively; the amino acid sequence of VL is shown in SEQ ID No: 54: shown in 35; the amino acid sequences of HCDR1-3 are shown in SEQ ID No: 49, SEQ ID No: 50 and SEQ ID No: 51 respectively; the amino acid sequence of VH is shown in SEQ ID No: 36; between VH and VL
  • the sequence of the connecting peptide is shown in SEQ ID No: 18; its hinge region is shown in SEQ ID No: 4; its transmembrane domain is shown in SEQ ID No: 6; its costimulatory domain is 4-1BB co-stimulatory domain.
  • Stimulatory domain as shown in SEQ ID No: 8; its CD3 ⁇ intracellular signaling domain as shown in SEQ ID No: 10; the CAR002 further comprises a cleavage peptide as shown in SEQ ID No: 12, as shown in SEQ ID No: 12 CSF2RA signal peptide shown in ID No: 14, and EGFRt shown in SEQ ID No: 16; CD8a signal peptide shown in SEQ ID No: 2.
  • the CAR described in this application can be CAR003, and the amino acid sequences of its LCDR1-3 are shown in SEQ ID No: 58, SEQ ID No: 59 and SEQ ID No: 60 respectively; the amino acid sequence of VL is shown in SEQ ID No: 60: 41; the amino acid sequences of HCDR1-3 are shown in SEQ ID No: 55, SEQ ID No: 56 and SEQ ID No: 57 respectively; the amino acid sequence of VH is shown in SEQ ID No: 42; between VH and VL
  • the sequence of the connecting peptide is shown in SEQ ID No: 18; its hinge region is shown in SEQ ID No: 4; its transmembrane domain is shown in SEQ ID No: 6; its costimulatory domain is 4-1BB co-stimulatory domain.
  • Stimulatory domain as shown in SEQ ID No: 8; its CD3 ⁇ intracellular signaling domain as shown in SEQ ID No: 10; the CAR002 further comprises a cleavage peptide as shown in SEQ ID No: 12, as shown in SEQ ID No: 12 CSF2RA signal peptide shown in ID No: 14, and EGFRt shown in SEQ ID No: 16; CD8a signal peptide shown in SEQ ID No: 2.
  • the CAR described in this application can be CAR004, and the amino acid sequences of its LCDR1-3 are shown in SEQ ID No: 64, SEQ ID No: 65 and SEQ ID No: 66, respectively; the amino acid sequence of VL is shown in SEQ ID No: 66: 89; the amino acid sequences of HCDR1-3 are shown in SEQ ID No: 61, SEQ ID No: 62 and SEQ ID No: 63 respectively; the amino acid sequence of VH is shown in SEQ ID No: 90; between VH and VL
  • the sequence of the connecting peptide is shown in SEQ ID No: 18; its hinge region is shown in SEQ ID No: 4; its transmembrane domain is shown in SEQ ID No: 6; its costimulatory domain is 4-1BB co-stimulatory domain.
  • Stimulatory domain as shown in SEQ ID No: 8; its CD3 ⁇ intracellular signaling domain as shown in SEQ ID No: 10; the CAR002 further comprises a cleavage peptide as shown in SEQ ID No: 12, as shown in SEQ ID No: 12 CSF2RA signal peptide shown in ID No: 14, and EGFRt shown in SEQ ID No: 16; CD8a signal peptide shown in SEQ ID No: 2.
  • the CAR described in this application can be CAR005, and the amino acid sequences of its LCDR1-3 are shown in SEQ ID No: 70, SEQ ID No: 71 and SEQ ID No: 72 respectively; the amino acid sequence of VL is shown in SEQ ID No: 72: 92; the amino acid sequences of HCDR1-3 are shown in SEQ ID No: 67, SEQ ID No: 68 and SEQ ID No: 69 respectively; the amino acid sequence of VH is shown in SEQ ID No: 93; between VH and VL
  • the sequence of the connecting peptide is shown in SEQ ID No: 18; its hinge region is shown in SEQ ID No: 4; its transmembrane domain is shown in SEQ ID No: 6; its costimulatory domain is 4-1BB co-stimulatory domain.
  • Stimulatory domain as shown in SEQ ID No: 8; its CD3 ⁇ intracellular signaling domain as shown in SEQ ID No: 10; the CAR002 further comprises a cleavage peptide as shown in SEQ ID No: 12, as shown in SEQ ID No: 12 CSF2RA signal peptide shown in ID No: 14, and EGFRt shown in SEQ ID No: 16; CD8a signal peptide shown in SEQ ID No: 2.
  • the CAR described in this application can be CAR006, and the amino acid sequences of its LCDR1-3 are shown in SEQ ID No: 76, SEQ ID No: 77 and SEQ ID No: 78 respectively; the amino acid sequence of VL is shown in SEQ ID No: 78: 95; the amino acid sequences of HCDR1-3 are respectively shown in SEQ ID No: 73, SEQ ID No: 74 and SEQ ID No: 75; the amino acid sequence of VH is shown in SEQ ID No: 96; between VH and VL
  • the sequence of the connecting peptide is shown in SEQ ID No: 18; its hinge region is shown in SEQ ID No: 4; its transmembrane domain is shown in SEQ ID No: 6; its costimulatory domain is 4-1BB co-stimulatory domain.
  • Stimulatory domain as shown in SEQ ID No: 8; its CD3 ⁇ intracellular signaling domain as shown in SEQ ID No: 10; the CAR002 further comprises a cleavage peptide as shown in SEQ ID No: 12, as shown in SEQ ID No: 12 CSF2RA signal peptide shown in ID No: 14, and EGFRt shown in SEQ ID No: 16; CD8a signal peptide shown in SEQ ID No: 2.
  • the application provides an isolated nucleic acid molecule encoding the CAR described herein.
  • the isolated nucleic acid molecule encoding a CAR described herein may comprise a nucleic acid molecule selected from the group consisting of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19-21, 25-27, 31-33 , 37-39, 79-87, 97, 99 or 101 or a functional variant thereof.
  • the nucleic acid molecules described herein can be isolated.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the present application provides a vector, which can contain the nucleic acid molecule.
  • the vector may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors.
  • the lentiviral vectors described herein may comprise a CAR.
  • the lentiviral vector described in the present application may comprise selected from SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19-21, 25-27, 31-33, 37-39 , 79-87, 97, 99 or 101 nucleic acid sequence or a functional variant thereof.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary depending on species or cell type function, but typically comprises 5' untranslated and 5' and 3' untranslated sequences involved in transcription and translation initiation, respectively, such as the TATA box, plus Cap sequences, CAAT sequences, etc.
  • a 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid.
  • One or more nucleic acid molecules described herein can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector is an expression vector, including a vector scFv plasmid and/or a CAR plasmid.
  • the application provides an immune effector cell, which can comprise the CAR described in the application, the nucleic acid molecule, or the vector.
  • the immune effector cells may be mammalian cells.
  • immune effector cells may be selected from T lymphocytes and natural killer (NK) cells.
  • the present application provides a method for preparing immune effector cells, which comprises knocking out the CD5 gene of the immune effector cells, and introducing the vector described in the present application into the immune effector cells.
  • the vectors described herein can be introduced into the immune effector cells, such as T lymphocytes or natural killer (NK) cells.
  • each or each cell may comprise one or one of the vectors described herein.
  • each or each cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the vectors described herein.
  • the vectors can be introduced into immune effector cells.
  • the vectors described herein can be introduced into the cells by methods known in the art.
  • immune effector cells can be transfected with retroviral vectors, and the viral genome with CAR molecules can be integrated into the host genome to ensure long-term and stable expression of the target gene.
  • a transposon a plasmid carrying a CAR (transposon) and a plasmid carrying a transposase are introduced into the target cell.
  • CAR molecules can be added to the genome by means of gene editing (eg, CRISPR/Cas9).
  • the vectors with CAR molecules described in the present application can be introduced into the cells by methods known in the art, such as electroporation, lipofectamine 2000, Invitrogen and the like.
  • the present application provides a pharmaceutical composition, which may comprise the immune effector cells and a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or non-ionic surfaces active agent, etc.
  • the pharmaceutical composition can be formulated for oral administration, intravenous administration (eg, intravenous injection, I.V.), intramuscular administration (eg, intramuscular injection, I.M.), original injection at the tumor site topical, inhalation, rectal, vaginal, transdermal, or via a subcutaneous depot.
  • the present application provides the use of the antibody, the CAR, the nucleic acid molecule, the vector or the immune effector cell for preparing a drug, wherein the drug is used for the treatment of patients with A disease or disorder associated with the expression of CD5.
  • the disease or disorder associated with the expression of CD5 may be cancer or malignancy.
  • the cancer or malignancy may be selected from a malignant T cell tumor or a malignant B cell tumor.
  • the malignant T-cell tumor can be selected from T-cell acute lymphoblastic leukemia (T-ALL), T-cell lymphoma (TCL) (such as peripheral T-cell lymphoma, cutaneous T-cell lymphoma (CTCL), T-cell lymphoma Hodgkin's lymphoma (T-NHL)); the malignant B-cell tumor may be selected from chronic lymphocytic leukemia (B-CLL) (eg, hairy cell leukemia cells (HCL)), mantle cell lymphoma (B-MCL) , Diffuse large B lymphoma (DLBCL).
  • T-ALL T-cell acute lymphoblastic leukemia
  • TCL T-cell lymphoma
  • CCL peripheral T-cell lymphoma
  • CTCL cutaneous T-cell lymphoma
  • T-NHL T-cell lymphoma Hodgkin's lymphoma
  • B-CLL chronic lymphocytic leukemia
  • HCL hair
  • the present application provides the antibody, the CAR, the nucleic acid molecule, the vector or the immune effector cell for treating a disease or condition associated with the expression of CD5.
  • the application provides a method of treating a disease or condition associated with the expression of CD5, comprising administering to a patient the antibody, the CAR, the nucleic acid molecule, the vector, or the described immune effector cells.
  • Example 1 Enrichment of specific antibody clones targeting the CD5 protein from a phage antibody library by affinity panning
  • the phage antibody library we constructed includes natural library, semi-synthetic library and single domain library.
  • Semi-synthetic phage antibody library used together with the natural library, to solve the problem that the natural library may lack CD5 high-affinity antibody clones.
  • the single-domain phage antibody library is an antibody library composed only of amino acids in the variable region of heavy chain antibodies, and its molecular weight is only 12-15kDa, but it has similar or higher specificity and affinity to traditional antibodies.
  • single-domain antibodies have attracted much attention due to their stable physicochemical properties, high affinity, easy recombinant expression and preparation, and easy combination with other target or epitope antibodies.
  • steps 1) to 6 usually 3 rounds of panning are required, until a significant increase in the recovery rate of phage (number of eluted phage/number of input phage) is observed.
  • the enriched phage pool can be used for subsequent monoclonal selection and ELISA/FACS screening.
  • Fully human phage antibody library including natural library, semi-synthetic library and single domain library;
  • Phage pools enriched by the affinity panning step contain phage antibodies of various properties: specific clones, non-specific clones, and negative clones.
  • specific clones we need to isolate monoclones from them, package them into monoclonal phages, and perform primary screening on a large number of monoclonal clones by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS), and select the specific binding at the same time.
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry
  • Monoclonal CD5 protein and CD5 positive cell line Jurkat The specific monoclonal was further determined by DNA sequencing to contain the unique antibody sequence.
  • the combination of Streptavidin and Biotin makes the biotinylated target protein (CD5-Fc-Bio) closer to the native antigen conformation in the reaction solution. Clones that bound only CD5-Fc-Bio but not the control antigen CD19-Fc-Bio were identified as specific clones.
  • the primary FACS screening was performed using the CD5-positive cell line Jurkat and the CD5-negative cell line Raji, and those that only bound to Jurkat cells but not Raji cells were identified as specific clones. Through ELISA and FACS two primary screenings, we can obtain candidate antibodies that can not only bind to the recombinantly expressed CD5 protein, but also recognize the native CD5 molecule on the cell surface for subsequent further screening.
  • step 7 Add 100 ⁇ L of the cultured phage supernatant in step 1) to the wells coated with the target antigen, and bind at room temperature for 2 hours;
  • mice anti M13 primary antibody diluted 1:2000, 100 ⁇ L/well, and incubate at room temperature for 45min;
  • mice anti M13 primary antibody diluted 1:2000, 100 ⁇ L/well, after pipetting and mixing, incubate at room temperature for 45min;
  • the monoclonal phages were randomly selected from the enriched phage antibody pool and packaged into phages.
  • the binding of monoclonal phages to CD5-Fc-Bio protein and the control protein CD19-Fc-Bio was detected by phage ELISA, and CD5-specific phage antibody clones were found. .
  • ELISA results of some clones are shown in FIG. 9 . It can be seen from the figure that clones H1, H2, H3, H4, H5, H6 and H7 bind strongly to the target antigen CD5 (CD5-Fc-Bio), but do not bind to the control antigen CD19-Fc-Bio, with good specificity .
  • Negative phage control is a negative control phage antibody clone, which does not bind to target antigen and control antigen.
  • Anti-M13 phage mouse Ab/anti-mouse HRP Ab is a negative antibody control with only primary antibody and secondary antibody added
  • anti-mouse HRP Ab is a negative antibody control with only secondary antibody added, which does not bind to the target antigen and control antigen.
  • Mouse anti human CD5 Ab/anti-mouse HRP Ab is a positive antibody control for the target antigen (CD5-Fc-Bio), Binds to the target antigen and does not bind to the control antigen.
  • Figure 10 shows the results of preliminary FACS screening of antibody clones corresponding to ELISA.
  • the H3, H4 and H7 clones bind to Jurkat, but not to Raji cells, and are specific clones; other clones are negative clones (the two cells do not bind).
  • Antibodies used in therapy must have very good target specificity, binding only to the target antigen and not to any unrelated antigen; on the other hand, the amino acid sequence of the same antigen will vary between cell lines mutants) or binding ligands, it is also necessary to investigate whether our antibodies can bind to cells positive for various target proteins. In order to further analyze the specificity and generality of these monoclones and find the best candidate clones, we further evaluated the specificity of the primary screened clones by flow cytometry.
  • CCRF-CEM cell line CD5 positive cell line
  • NALM6 cell line CD5 negative cell line
  • the rest of the reagents are the same as the FACS primary screening.
  • Antibodies used in therapy must have very good target specificity.
  • clone binds to two CD5-positive cell lines, Jurkat and CCRF-CEM, with strong or weak median fluorescence intensity (MFI), but does not bind to two CD5-negative cell lines, Raji and NALM6, with low MFI and good specificity; Clones #3, #13, #18, #23, #24, #28, #29, #40, #41, #50, #57 and #63 bind weakly or not to the positive cell line Jurkat, and bind to the positive cell line Jurkat.
  • MFI median fluorescence intensity
  • the positive cell line CCRF-CEM does not bind, so it is a negative clone; clones 52 and 53 only bind to the positive cell line Jurkat, but not CCRF-CEM, indicating that they cannot recognize the CD5 antigens of different conformations or isomers expressed by different cell lines. Does not meet the experimental needs; clone 56 can bind to both positive cell lines, but has a weaker binding to the negative cell line Raji, indicating that its binding may be non-specific binding, which does not meet the experimental needs.
  • Antibodies used for therapy must have very good target specificity, binding only to the target antigen and not to any unrelated antigen; on the other hand, the amino acid sequence of the same antigen produced by different companies will vary (isomers or mutants), it is also necessary to investigate whether our antibodies can bind to various target proteins.
  • ELISA enzyme-linked immunoassay
  • the rest of the reagents are the same as the ELISA primary screening.
  • Antibodies used in therapy must have very good target specificity.
  • ELISA enzyme-linked immunosorbent assay
  • Negative control is a negative control phage antibody clone, which does not bind to target antigen and control antigen, and anti-M13 phage mouse Ab/anti-mouse HRP Ab is only added with primary antibody and secondary antibody.
  • the negative antibody control of anti-mouse HRP Ab is the negative antibody control with only the second antibody added, they do not bind to the target antigen and the control antigen
  • Mouse anti human CD5 Ab/anti-mouse HRP Ab is the target antigen (CD5-Fc -Bio) positive antibody control that binds to the target antigen and does not bind to the control antigen.
  • Anti-human IgG-HRP Ab/anti-his-HRP Ab is a positive antibody control for the detection of antigen tags. It binds to antigens containing Fc tags or his tags, indicating that the coated antigens have been bound to the ELISA plate. Clones #1-64 bind to all three CD5 antigens, but none of the three unrelated antigens, indicating that they can bind to CD5 antigens from different companies with good specificity.
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • ScFv CDR sequences of some selected anti-CD5 antibodies
  • Mature T cells express CD5 antigen on the surface, and the CD5 CAR-T developed by Mamonkin M and other researchers without CD5 knockout has been reported to be suicidal to a certain extent1, and the duration in patients is limited, which greatly limits patients. Disease remission time and application of this CAR-T product.
  • this application uses CRISPR/Cas9 technology to knock out the CD5 antigen on the surface of T cells to minimize the self-activation and suicide of CD5 CAR-T and ensure its sustainability and effectiveness.
  • sgRNAs In order to screen sgRNAs with high knockout efficiency, we first used different sgRNAs to knock out the CD5 gene in jurkat cells that naturally highly express CD5, so as to determine the high-efficiency sgRNAs for CD5 antigen knockout of T cells, and obtain high knockout efficiency. After jurkat-CD5ko pooling, CD5-negative monoclonal jurkat-CD5ko cells were obtained by monoclonal screening. In this way, the difference between the jurkat/jurkat-CD5ko pair of cells mainly lies in the knocked out gene, which will have important applications in the identification of monoclonal antibody binding specificity and the functional verification of CAR-T.
  • sgRNA1 to sgRNA4 1) Select multiple sgRNA sequences (sgRNA1 to sgRNA4), and perform primer synthesis;
  • FACS detects the gene knockout efficiency of different sgRNAs, selects the jurkat-CD5ko pool with higher knockout efficiency, and isolates single clones by limiting dilution method
  • sgRNA1 aagcgtcaaaagtctgccag (SEQ ID NO: 103, the sequence is provided in the form of its target sequence, the same below)
  • sgRNA2 ccgttccaactcgaagtgcc (SEQ ID NO: 104)
  • sgRNA3 gctgtagaactccaccacgc (SEQ ID NO: 105)
  • sgRNA4 aatcatctgctacggacaac (SEQ ID NO: 106)
  • the cells corresponding to sgRNA3 had the highest CD5 knockout efficiency, with 95.46% of CD5 knocked out. Therefore, we selected the jurkat-CD5ko pool electroporated with sgRNA3 for single clone screening. Monoclones were isolated from the cell pool by limiting dilution method, and after monoclonal expansion, CD5 expression of these monoclonal was detected by FACS. The results are shown in Figure 2, in which the CD5 expression of the jurkat-CD5ko-1 and 2 clones is basically undetectable, which can be considered as a single clone with a successful knockout. The two clones were cultured and cryopreserved. We used clone 1 in subsequent studies.
  • lentiviral transfection is used to make knockout T cells express CAR.
  • CAR-T For the production process of CAR-T, please refer to the patent (Zhou J, Liu J, Hu G, et al. Chimeric antigen receptor (car) binding to bcma, and uses thereof: U.S. Patent Application 16/650,580[P].2020-8-6.).
  • the lentiviral vector is based on the genome of the lentivirus, and many sequence structures related to viral activity are removed to make it biologically safe, and then the sequence of the target gene required for the experiment is introduced into the genome backbone. and the vector prepared from the expression construct.
  • lentiviral vectors Compared with other retroviruses, lentiviral vectors have a wider range of hosts and have the ability to infect both dividing and non-dividing cells. For some cells that are difficult to transfect, such as primary cells, stem cells, undifferentiated cells, etc. It can greatly improve the transduction efficiency of the target gene (see Chen Chen and Wan Haisu, "Lentiviral Vectors and Their Research Progress, Chinese Journal of Lung Cancer 17.12(2014):870–876.PMC). Transfection by lentiviral vectors , CAR molecules can be integrated into host cells.
  • T cells were CD5 knocked out and transfected with CAR lentivirus with the structure shown in Figure 3A (the specific CAR molecular structure is shown in Table 2), and CD5, EGFRt expression and CD5 antigen binding were detected 5 to 7 days later (Figure 3B).
  • the CAR structure contains CD8 ⁇ signal peptide, scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB costimulatory molecule and CD3 ⁇ and a truncated EFGR molecule (EGFRt) linked with T2A can be used as a safety switch in clinical translation, and Since EGFRt is co-expressed with CAR molecules, it can be used as an indirect detection indicator of the distribution of CAR molecules on the surface of T cells without affecting the structure and function of CAR.
  • EGFRt truncated EFGR molecule
  • PE anti-human EGFR Antibody Clone AY13, BioLegend, Cat.No.352904;
  • the CD5 knockout efficiency on T cells can reach 86.6%, and all cloned CAR molecules can be expressed on the surface of T cells.
  • the CAR-T studied in this application targets CD5 targets.
  • - T cells function well, and after knockout and transfection of lentivirus, CAR-T cells can kill CAR-T/T cells that still express CD5 antigen.
  • Clone 8 CAR-T other cloned CAR-T cells can clear the cells without CD5 knockout.
  • CD5 protein-targeting specific antibody clones enriched, screened and identified from the phage antibody library by affinity panning were constructed on the second-generation CAR. Because the scFv that binds to the CD5 antigen is constructed into the CAR structure, it may not have Good activation function, its function on CAR-T cells needs to be further confirmed and screened for the most active CAR molecules. To this end, we prepared lentiviral vectors of these CAR molecular clones, and transduced T cells into CAR-T cells.
  • CD107a degranulation assay CD107a degranulation assay
  • in vitro cell killing assay in vitro cytotoxicity assay
  • CD107a is a marker for intracellular microvesicles, and CD107a on the cell membrane increases when granzyme-loaded microvesicles fuse with the cell membrane, and when its recovery is blocked with monesin (purchased from BioLegend) , which can quantitatively reflect the strength of microvesicle release.
  • monesin purchased from BioLegend
  • Target cells jurkat, jurkat CD5ko, CCRF-CEM, MOLT4, K562-CD5, K562, RAJI;
  • Alexa Fluor 488 anti-human EGFR BioLegend, Cat. No. 352908.
  • CAR-T cells were obtained by lentiviral transduction, and the CAR-T cells were cultured for 9-12 days in vitro for CD107a degranulation experiments.
  • the CAR-T cells to be tested and target cells, monensin and CD107a antibody were incubated together for 4h, and the cell density of both CAR-T cells and target cells was 5 ⁇ 10 5 cells/mL. Then, after labeling the samples with CD8 antibody and EGFR antibody, flow cytometry was performed.
  • the in vitro cell killing experiment used CCRF-CEM and MOLT4 as CD5 positive target cells, and K562 and RAJI cells as CD5 negative target cells to evaluate the antigen-specific killing ability of CD5 CAR-T cells.
  • the above cells were respectively transduced by lentivirus to obtain target cells stably expressing firefly luciferase, so the luciferase activity in the sample can reflect the number of target cells.
  • CAR-T cells and target cells were co-incubated. When target cells are killed by CAR-T cells, luciferase is released and quickly inactivated (firefly luciferase has a half-life of about 0.5h).
  • the target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as the target cells expand and continue to express luciferase. Therefore, the killing of target cells by CAR-T can be detected by the activity of luciferase.
  • the CAR-T cell samples and a fixed number of target cells (1 ⁇ 10 4 ) were mixed according to different effect-target ratios (E:T), incubated together for 24 h, and then the luciferase activity (RLU) in the samples was detected.
  • E:T effect-target ratios
  • RLU luciferase activity
  • CD5ko T cells are control samples containing only target cells. Since luciferase activity can reflect the number of target cells in the sample, the killing/inhibitory ability of CAR-T cells to target cells can be obtained through the change of luciferase activity in the sample. The lower the luciferase activity reading (RLU), the more target cells are killed.
  • Mitomycin-treated target cells were mixed with different groups of CD5 CAR-T cells for 4 times of stimulation, and then CAR-T cells and target cells were co-incubated to determine different scFv CARs. - The ability of T to proliferate after being continuously stimulated by target cells for multiple times.
  • the affinity between CD5 scFvs and antigens may also have an important impact on the killing effect and survival time of CAR-T in patients.
  • this application uses ForteBio's Octet molecular interaction technology to analyze it. Determination.
  • the biofilm interference technology used by the Octet system is a label-free technology that provides high-throughput biomolecular interaction information in real time.
  • the instrument emits white light to the sensor surface and collects the reflected light.
  • the reflected spectrum of different frequencies is affected by the thickness of the optical film layer of the biosensor. Some frequencies of reflected light form constructive interference (blue), while others are destructive interference (red).
  • interferences are detected by the spectrometer and form an interference spectrum, which is displayed as the phase-shift intensity (nm) of the interference spectrum. Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer will detect the displacement of the interference spectrum in real time, and this displacement directly reflects the thickness of the biofilm on the sensor surface, from which the interaction of biomolecules can be obtained.
  • High-quality data for the determination of biomolecular interaction kinetic parameters (Kon, Kdis and KD) provide important information for the R&D process.
  • Anti-CD5 IgG was diluted to 20 ⁇ g/mL with loading buffer (1 ⁇ PBS, pH 7.4, 0.01% BSA and 0.02% Tween 20) and loaded on the biosensor at approximately 0.8 nM.
  • Binding constants were analyzed by using a 1:1 binding site model (Biacore X-100 evaluation software).
  • Affinity refers to the strength with which a single molecule binds to its ligand, and is usually measured and reported by the equilibrium dissociation constant (KD), which can be used to assess and rank the strength of the interaction between two molecules.
  • KD equilibrium dissociation constant
  • the binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentration of the reactant. The smaller the KD value, the greater the affinity of the antibody for its target.
  • H65, Clone 10, Clone 32 and Clone 35 can all bind to the CD5 antigen, and Clone 10 showed a comparable affinity level to the control antibody H65, which was slightly higher than that of Clone 32 and Clone 35.
  • Ly-1 a membrane glycoprotein of mouse T lymphocytes and a subset of B cells:molecular homology to its human counterpart Leu-1/T1(CD5).Proceedings of the National Academy of Sciences of the United States of America, 1987.84(1):p.204-208.
  • CD8a signal nucleic acid sequence (SEQ ID NO:1)
  • CD8a signal protein sequence (SEQ ID NO:2)
  • CD8a hinge nucleic acid sequence (SEQ ID NO:3)
  • CD8a hinge protein sequence (SEQ ID NO:4)
  • CD8aTM nucleic acid sequence (SEQ ID NO:5)
  • CD8aTM protein sequence (SEQ ID NO:6)
  • CD3 ⁇ intracellular signaling domain nucleic acid sequence SEQ ID NO:9
  • CD3 ⁇ intracellular signaling domain protein sequence SEQ ID NO:10
  • VH-VL linker peptide nucleic acid sequence (SEQ ID NO: 17)
  • VH-VL linker peptide protein sequence (SEQ ID NO: 18)
  • VH (ie VH of Scfv10 in CAR001) protein sequence: (SEQ ID NO:30)
  • 35VL (ie the VL of Scfv35 in CAR003) nucleic acid sequence: 333bp (SEQ ID NO:38)
  • 35VH (ie the VH of Scfv35 in CAR003) nucleic acid sequence: 366bp (SEQ ID NO:39)
  • 35VL (ie VL of Scfv35 in CAR003) protein sequence: 111aa (SEQ ID NO:41)
  • 35VH (ie VH of Scfv35 in CAR003) protein sequence: 122aa (SEQ ID NO:42)
  • amino acid sequence of HCDR1 is GFTFNNYT (SEQ ID NO:43)
  • amino acid sequence of HCDR2 is ISSSSSYI (SEQ ID NO:44)
  • the amino acid sequence of HCDR3 is ARYFSGSAGDY (SEQ ID NO:45)
  • amino acid sequence of LCDR1 is SSNIGAGYD (SEQ ID NO:46)
  • amino acid sequence of LCDR2 is GNI (SEQ ID NO:47)
  • amino acid sequence of LCDR3 is GTWDNSLSAHYV (SEQ ID NO:48)
  • HCDR1 The amino acid sequence of HCDR1 is GFTFSSYS (SEQ ID NO:49)
  • amino acid sequence of HCDR2 is ISSSSSYI (SEQ ID NO:50)
  • the amino acid sequence of HCDR3 is ARYFSGSAGDY (SEQ ID NO: 51)
  • LCDR1 The amino acid sequence of LCDR1 is NSNIGNNY (SEQ ID NO:52)
  • the amino acid sequence of LCDR2 is DNN (SEQ ID NO:53)
  • the amino acid sequence of LCDR3 is GIWDSSLDAYV (SEQ ID NO:54)
  • HCDR1 The amino acid sequence of HCDR1 is GFTFSSYA (SEQ ID NO:55)
  • HCDR2 The amino acid sequence of HCDR2 is ITDSGDIT (SEQ ID NO: 56)
  • amino acid sequence of HCDR3 is ARMSSHWYFSADY (SEQ ID NO:57)
  • LCDR1 The amino acid sequence of LCDR1 is SSNIGNNY (SEQ ID NO:58)
  • the amino acid sequence of LCDR2 is DNN (SEQ ID NO:59)
  • amino acid sequence of LCDR3 is GTWDSSLSAVV (SEQ ID NO:60)
  • HCDR1 The amino acid sequence of HCDR1 is GGTFSSNA (SEQ ID NO:61)
  • HCDR2 The amino acid sequence of HCDR2 is IIPMFGTT (SEQ ID NO:62)
  • amino acid sequence of HCDR3 is ARDGGGRHPYYYYGMDV (SEQ ID NO:63)
  • LCDR1 The amino acid sequence of LCDR1 is SGSIARNY (SEQ ID NO:64)
  • the amino acid sequence of LCDR2 is EDN (SEQ ID NO:65)
  • amino acid sequence of LCDR3 is QSYDDNTSWV (SEQ ID NO:66)
  • HCDR1 The amino acid sequence of HCDR1 is GYSFTSYW (SEQ ID NO:67)
  • the amino acid sequence of HCDR2 is IYPDDSDT (SEQ ID NO:68)
  • amino acid sequence of HCDR3 is ARMSLTSYLATDG (SEQ ID NO:69)
  • LCDR1 The amino acid sequence of LCDR1 is SSNIGNND (SEQ ID NO:70)
  • the amino acid sequence of LCDR2 is DND (SEQ ID NO:71)
  • LCDR3 The amino acid sequence of LCDR3 is AVWDSSLSAAV (SEQ ID NO:72)
  • amino acid sequence of HCDR1 is TYTFTNYY (SEQ ID NO: 73)
  • HCDR2 The amino acid sequence of HCDR2 is INPSGDTT (SEQ ID NO:74)
  • amino acid sequence of HCDR3 is ARLSWYWGGSFDD (SEQ ID NO:75)
  • amino acid sequence of LCDR1 is SDDIGNYKR (SEQ ID NO:76)
  • the amino acid sequence of LCDR2 is EVT (SEQ ID NO:77)
  • LCDR3 The amino acid sequence of LCDR3 is SSYASGDTYV (SEQ ID NO:78)
  • VH i.e. VH of Scfv9 in CAR006 amino acid sequence (SEQ ID NO:96)

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Abstract

提供了CD5抗体。还提供了可以特异性结合CD5蛋白的嵌合抗原受体(CAR)，其包含CD5结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域。还提供了包含所述嵌合抗原受体的经工程化的免疫效应细胞(诸如T细胞)。还提供了所述CAR及工程化的免疫效应细胞在用于治疗与CD5的表达相关的疾病或病症中的应用。

Description

靶向CD5的全人源抗体、全人源嵌合抗原受体(CAR)及其应用
相关申请
本申请要求于2020年11月1日提交至中国专利局、申请号为202011200628.8的中国专利申请的优先权，在此通过引用将其全文并入本文。
序列表
本申请包括以电子方式提交的.txt格式的序列表。该.txt文件包含于2020年11月1日创建的名称为“CD5 CAR_ST25.txt”的序列表，其大小为约126千字节。所述.txt文件中所含的序列表是说明书的一部分，以全文引用的方式将其并入本文。
技术领域
本申请涉及生物医药领域，具体涉及能与CD5蛋白特异性结合的全人源抗体、全人源嵌合抗原受体及其应用。
背景技术
近年来，嵌合抗原受体T细胞(CAR-T)技术取得了突破性进展，尤其在针对B细胞恶性肿瘤的靶点取得了巨大的成功，以anti-CD19CAR-T为代表，迄今为止，已有Kymriah，Yescarta及Tecartus三款产品先后经美国FDA批准上市 [1]。临床试验已证实，特异性靶向CD19分子的CAR-T细胞能有效地靶向治疗B细胞性恶性肿瘤，包括复发/难治性的B-ALL、CLL和B细胞淋巴瘤 [2-9]。文献报道，CD19CAR-T对复发/难治性ALL的治疗有效率可高达90％，对CLL和部分B细胞淋巴瘤的有效率大于50％。尽管CAR-T疗法在B细胞恶性肿瘤治疗领域取得了巨大的成功，但其在T细胞恶性肿瘤的研究及应用十分有限。
T细胞恶性肿瘤包括急性T淋巴细胞白血病(T-ALL)及T细胞淋巴瘤(TCL)。T-ALL是T淋巴细胞异常增生造成的血液疾病，具有侵袭性并且进展迅速。T细胞淋巴瘤是T细胞的一种恶性肿瘤，可在淋巴组织(如淋巴结和脾脏)中或在淋巴组织之外(如胃肠道，肝脏，鼻腔，皮肤等)发展，约占非霍奇金淋巴瘤10％～15％，在我国比例更高。CD5在淋巴细胞前体，成熟T细胞和一部分成熟B细胞(Bl细胞)上组成性表达 [10][11]。CD5在约85％的T-ALL和约75％的外周T细胞淋巴瘤中高表达。此外，CD5在套细胞淋巴瘤，慢性淋巴细胞白血病(B-CLL)和毛细胞白血病细胞(HCL)中也常有表达。目前T细胞 恶性肿瘤放化疗后复发率高、预后差，是临床较难治愈的血液系统恶性肿瘤，急需开发针对该疾病的细胞治疗药物。在正常细胞中，CD5的表达仅限于成熟的T细胞和B细胞的部分亚型，CD5抗原的生物学特性允许CD5 CAR T细胞在体外和体内产生针对T-ALL和T淋巴瘤细胞的有效抗肿瘤活性 [12]。所以，CD5可以作为T细胞肿瘤的安全且可靠的靶点。
Mamonkin M等研究者开发了一个二代CD5 CAR，采用鼠源单克隆抗体H65的scFv，临床实验研究结果表明该CD5 CAR-T细胞在既往多线治疗的r/r CD5+T-ALL和T细胞非霍奇金淋巴瘤(T-NHL)患者中是安全的，而且能产生临床疗效，并且不会导致完全的T细胞消除。更重要的是，通过CD5 CAR-T细胞清除恶性T细胞或许能使既往不适合移植的患者接受HSCT(造血干细胞移植)。但根据现有临床随访数据得知该鼠源scFv CAR-T在输注后可出现CD5+恶性疾病的复发，CAR-T在患者体内存续时间有限可能与抗鼠抗体的产生有关，而全人源scFv可能可以解决CD5 CAR-T在患者体内存续时间短的问题。开发全人源的CD5抗体，对于研发下一代体内存续时间更长、长期疗效更好的CAR-T产品，有非常重要的意义。
发明内容
本申请提供了可以特异性结合CD5的全人源抗体、全人源嵌合抗原受体及其应用。
在一方面，本申请提供了靶向CD5的全人源抗体或其单链抗体或片段，其中所述全人源抗体包括重链可变区(HCVR)和/或轻链可变区(LCVR)，重链可变区包括HCDR1、HCDR2和HCDR3，轻链可变区包括LCDR1、LCDR2和LCDR3，所述HCDR1、HCDR2、HCDR3和/或LCDR1、LCDR2和LCDR3选自如下组合之一：
(1)LCDR1的氨基酸序列为SGSIARNY(SEQ ID NO：64)；
LCDR2的氨基酸序列为EDN(SEQ ID NO：65)；
LCDR3的氨基酸序列为QSYDDNTSWV(SEQ ID NO：66)；
HCDR1的氨基酸序列为GGTFSSNA(SEQ ID NO：61)；
HCDR2的氨基酸序列为IIPMFGTT(SEQ ID NO：62)；
HCDR3的氨基酸序列为ARDGGGRHPYYYYGMDV(SEQ ID NO：63)；
(2)LCDR1的氨基酸序列为SSNIGNND(SEQ ID NO：71)；
LCDR2的氨基酸序列为DND(SEQ ID NO：72)；
LCDR3的氨基酸序列为AVWDSSLSAAV(SEQ ID NO：73)；
HCDR1的氨基酸序列为GYSFTSYW(SEQ ID NO：67)；
HCDR2的氨基酸序列为IYPDDSDT(SEQ ID NO：68)；
HCDR3的氨基酸序列为ARMSLTSYLATDG(SEQ ID NO：69)；
(3)LCDR1的氨基酸序列为SDDIGNYKR(SEQ ID NO：76)；
LCDR2的氨基酸序列为EVT(SEQ ID NO：77)；
LCDR3的氨基酸序列为SSYASGDTYV(SEQ ID NO：78)；
HCDR1的氨基酸序列为TYTFTNYY(SEQ ID NO：73)；
HCDR2的氨基酸序列为INPSGDTT(SEQ ID NO：74)；
HCDR3的氨基酸序列为ARLSWYWGGSFDD(SEQ ID NO：75)；
(4)LCDR1的氨基酸序列为SSNIGAGYD(SEQ ID NO：46)；
LCDR2的氨基酸序列为GNI(SEQ ID NO：47)；
LCDR3的氨基酸序列为GTWDNSLSAHYV(SEQ ID NO：48)；
HCDR1的氨基酸序列为GFTFNNYT(SEQ ID NO：43)；
HCDR2的氨基酸序列为ISSSSSYI(SEQ ID NO：44)；
HCDR3的氨基酸序列为ARYFSGSAGDY(SEQ ID NO：45)；
(5)LCDR1的氨基酸序列为NSNIGNNY(SEQ ID NO：52)；
LCDR2的氨基酸序列为DNN(SEQ ID NO：53)；
LCDR3的氨基酸序列为GIWDSSLDAYV(SEQ ID NO：54)；
HCDR1的氨基酸序列为GFTFSSYS(SEQ ID NO：49)；
HCDR2的氨基酸序列为ISSSSSYI(SEQ ID NO：50)；
HCDR3的氨基酸序列为ARGNPWYGVDY(SEQ ID NO：51)；
(6)LCDR1的氨基酸序列为SSNIGNNY(SEQ ID NO：58)；
LCDR2的氨基酸序列为DNN(SEQ ID NO：59)；
LCDR3的氨基酸序列为GTWDSSLSAVV(SEQ ID NO：60)；
HCDR1的氨基酸序列为GFTFSSYA(SEQ ID NO：55)；
HCDR2的氨基酸序列为ITDSGDIT(SEQ ID NO：56)；
HCDR3的氨基酸序列为ARMSSHWYFSADY(SEQ ID NO：57)；或
所述全人源抗体包括(1)-(6)任一项中的CDR序列组合的变体，其中所述变体(1)-(6)任一项中的CDR序列相比，具有至少90％的序列一致性，或在CDR序列上共包含至少1个且不超过10，或不超过5、4、3、2或1个氨基酸改变。
在某些实施方案中，所述重链可变区和/或轻链可变区的氨基酸序列选自如下任一组合：
(1)SEQ ID NO：30所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：29所示序列或与其有至少90％序列一致性的轻链可变区序列；
(2)SEQ ID NO：36所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：35所示序列或与其有至少90％序列一致性的轻链可变区序列；
(3)SEQ ID NO：42所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：41所示序列或与其有至少90％序列一致性的轻链可变区序列；
(4)SEQ ID NO：90所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：89所示序列或与其有至少90％序列一致性的轻链可变区序列；
(5)SEQ ID NO：93所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：92所示序列或与其有至少90％序列一致性的轻链可变区序列；
(6)SEQ ID NO：96所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：95所示序列或与其有至少90％序列一致性的轻链可变区序列。
在某些实施方案中，所述重链可变区和/或轻链可变区的氨基酸序列选自如下任一组合：
(1)SEQ ID NO：30所示的重链可变区序列，和SEQ ID NO：29所示的轻链可变区序列；
(2)SEQ ID NO：36所示的重链可变区序列，和SEQ ID NO：35所示的轻链可变区序列；
(3)SEQ ID NO：42所示的重链可变区序列，和SEQ ID NO：41所示的轻链可变区序列；
(4)SEQ ID NO：90所示的重链可变区序列，和SEQ ID NO：89所示的轻链可变区序列；
(5)SEQ ID NO：93所示的重链可变区序列，和SEQ ID NO：92所示的轻链可变区序列；
(6)SEQ ID NO：96所示的重链可变区序列，和SEQ ID NO：95所示的轻链可变区序列。
在某些实施方案中，所述全人源抗体包括SEQ ID NO：28、34、40、88、91或94所示的氨基酸序列。
另一方面，本申请提供了编码上述全人源抗体或其单链抗体或片段的核酸分子。
在某些实施方案中，所述核酸分子包括SEQ ID NO:25-27、31-33、37-39、和79－87中任一项的核苷酸序列。
另一方面，本申请提供了一种表达载体，其包括上述核酸分子。
另一方面，本申请提供了一种宿主细胞，其包括上述表达载体。
另一方面，本申请提供了一种药物组合物，其包括上述全人源抗体或其单链抗体或片段，以及药学上可接受的载体或稀释剂。
另一方面，本申请提供了一种治疗疾病或病症的方法，其包括通过向有需要的患者施用治疗有效量的上述全人源抗体或其单链抗体或片段，宿主细胞，或药物组合物，来消除、抑制或降低CD5活性，从而预防、减轻、改善或抑制疾病或病症。
在某些实施方案中，所述疾病或病症为癌症。
在某些实施方案中，所述癌症为T细胞恶性肿瘤。
另一方面，本申请提供了上述全人源抗体或其单链抗体或片段竞争相同表位的抗体或片段。
另一方面，本申请提供了一种试剂盒，用于检测样品中CD5蛋白，其中，所述试剂盒包括上述全人源抗体或其单链抗体或片段。
另一方面，本申请提供了上述全人源抗体或其单链抗体或片段或宿主细胞在制备用于消除、抑制或降低CD5活性，从而预防、减轻、改善或抑制疾病或病症的药物中的用途。
在某些实施方案中，所述疾病或病症为癌症。
在某些实施方案中，所述癌症为恶性T细胞肿瘤或恶性B细胞肿瘤。
在某些实施方案中，所述恶性T细胞肿瘤选自T细胞急性淋巴细胞白血病(T-ALL)和T细胞淋巴瘤(TCL)，所述恶性B细胞肿瘤选自慢性淋巴细胞白血病(B-CLL)和套 细胞淋巴瘤(B-MCL)。
另一方面，本申请包括一种嵌合抗原受体(CAR)，其中所述CAR包含CD5结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，所述HCDR1、HCDR2、HCDR3的氨基酸序列选自以下组合：(1)如SEQ ID NO:43的氨基酸序列的HCDR1，如SEQ ID NO:44的氨基酸序列的HCDR2，和如SEQ ID NO:45的氨基酸序列的HCDR3；(2)如SEQ ID NO:49的氨基酸序列的HCDR1，如SEQ ID NO:50的氨基酸序列的HCDR2，和如SEQ ID NO:51的氨基酸序列的HCDR3；(3)如SEQ ID NO:55的氨基酸序列的HCDR1，如SEQ ID NO:56的氨基酸序列的HCDR2，和如SEQ ID NO:57的氨基酸序列的HCDR3；(4)如SEQ ID NO:61的氨基酸序列的HCDR1，如SEQ ID NO:62的氨基酸序列的HCDR2，和如SEQ ID NO:63的氨基酸序列的HCDR3；(5)如SEQ ID NO:67的氨基酸序列的HCDR1，如SEQ ID NO:68的氨基酸序列的HCDR2，和如SEQ ID NO:69的氨基酸序列的HCDR3；或(6)如SEQ ID NO:73的氨基酸序列的HCDR1，如SEQ ID NO:74的氨基酸序列的HCDR2，和如SEQ ID NO:75的氨基酸序列的HCDR3。
在某些实施方式中，所述抗体还包含轻链互补决定区1(LCDR1)，轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，所述LCDR1、LCDR2、LCDR3的氨基酸序列选自以下组合：(1)如SEQ ID NO:46的氨基酸序列的LCDR1，如SEQ ID NO:47的氨基酸序列的LCDR2，和如SEQ ID NO:48的氨基酸序列的LCDR3；(2)如SEQ ID NO:52的氨基酸序列的LCDR1，如SEQ ID NO:53的氨基酸序列的LCDR2，和如SEQ ID NO:54的氨基酸序列的LCDR3；(3)如SEQ ID NO:58的氨基酸序列的LCDR1，如SEQ ID NO:59的氨基酸序列的LCDR2，和如SEQ ID NO:60的氨基酸序列的LCDR3；(4)如SEQ ID NO:64的氨基酸序列的LCDR1，如SEQ ID NO:65的氨基酸序列的LCDR2，和如SEQ ID NO:66的氨基酸序列的LCDR3；(5)如SEQ ID NO:70的氨基酸序列的LCDR1，如SEQ ID NO:71的氨基酸序列的LCDR2，和如SEQ ID NO:72的氨基酸序列的LCDR3；或(6)如SEQ ID NO:76的氨基酸序列的LCDR1，如SEQ ID NO:77的氨基酸序列的LCDR2，和如SEQ ID NO:78的氨基酸序列的LCDR3。
在某些实施方式中，所述抗体包含的HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3选自以下组合的任一种：(1)如SEQ ID NO:43的氨基酸序列的HCDR1，如SEQ ID NO:44的氨基酸序列的HCDR2，和如SEQ ID NO:45的氨基酸序列的HCDR3，如SEQ ID NO:46的氨基酸序列的LCDR1，如SEQ ID NO:47的氨基酸序列的LCDR2，和如SEQ ID NO:48的氨基酸序列的LCDR3；(2)如SEQ ID NO:49的氨基酸序列的HCDR1，如SEQ ID NO:50的氨基酸序列的HCDR2，和如SEQ ID NO:51的氨基酸序列的HCDR3，如SEQ ID NO:52的氨基酸序列的LCDR1，如SEQ ID NO:53的氨基酸序列的LCDR2，和如SEQ ID NO:54的氨基酸序列的LCDR3；(3)如SEQ ID NO:55的氨基酸序列的HCDR1，如SEQ ID NO:56的氨基酸序列的HCDR2，和如SEQ ID NO:57的氨基酸序列的HCDR3，如SEQ ID NO:58的氨基酸序列的LCDR1，如SEQ ID NO:59的氨基酸序列的LCDR2，和如SEQ ID  NO:60的氨基酸序列的LCDR3；(4)如SEQ ID NO:61的氨基酸序列的HCDR1，如SEQ ID NO:62的氨基酸序列的HCDR2，和如SEQ ID NO:63的氨基酸序列的HCDR3，如SEQ ID NO:64的氨基酸序列的LCDR1，如SEQ ID NO:65的氨基酸序列的LCDR2，和如SEQ ID NO:66的氨基酸序列的LCDR3；(5)如SEQ ID NO:67的氨基酸序列的HCDR1，如SEQ ID NO:68的氨基酸序列的HCDR2，和如SEQ ID NO:69的氨基酸序列的HCDR3，如SEQ ID NO:70的氨基酸序列的LCDR1，如SEQ ID NO:71的氨基酸序列的LCDR2，和如SEQ ID NO:72的氨基酸序列的LCDR3；或(6)如SEQ ID NO:73的氨基酸序列的HCDR1，如SEQ ID NO:74的氨基酸序列的HCDR2，和如SEQ ID NO:75的氨基酸序列的HCDR3，如SEQ ID NO:76的氨基酸序列的LCDR1，如SEQ ID NO:77的氨基酸序列的LCDR2，和如SEQ ID NO:78的氨基酸序列的LCDR3。
在某些实施方式中，所述抗体包含重链可变区，所述重链可变区的氨基酸序列如SEQ ID NO：30、36、42、90、93或96所示。在某些实施方式中，所述抗体包含轻链可变区，所述轻链可变区的氨基酸序列如SEQ ID NO：29、35、41、89、92或95所示。在某些实施方式中，所述抗体为单链抗体。在某些实施方式中，所述抗体包含SEQ ID No:28、34、40、88、91或94所示的氨基酸序列或其功能性变体。
在某些实施方式中，所述CAR的跨膜结构域包含来自选自下述蛋白的多肽：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8a、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。在某些实施方式中，所述跨膜结构域包含SEQ ID No:6所示的氨基酸序列或其功能性变体。
在某些实施方式中，所述CAR的共刺激结构域包含选自下述蛋白的多肽：CD28、4-1BB、OX-40和ICOS。在某些实施方式中，所述共刺激结构域包含SEQ ID No:8所示的氨基酸序列或其功能性变体。
在某些实施方式中，所述CAR的胞内信号传导结构域包含来自CD3ζ的信号传导结构域。在某些实施方式中，所述胞内信号传导结构域包含SEQ ID No:10所示的氨基酸序列或其功能性变体。
在某些实施方式中，所述CAR还包含铰链区，所述铰链区连接所述抗体和所述跨膜结构域。在某些实施方式中，所述铰链区包含SEQ ID No:4所示的氨基酸序列或其功能性变体。
在某些实施方式中，所述CAR还连CD8α接信号肽。在某些实施方式中，所述信号肽包含SEQ ID No:2所示的氨基酸序列或其功能性变体。
在某些实施方式中，所述CAR还连接剪切肽。在某些实施方式中，所述剪切肽包含来自T2A肽的氨基酸序列。在某些实施方式中，所述剪切肽包含SEQ ID No:12所示的氨基酸序列或其功能性变体。在某些实施方式中，所述CAR通过接剪切肽和CSF2RA信号肽还连接一段截短形式的EGFR分子(tEGFR)。在某些实施方式中，所述截短形式的EGFR分子包含SEQ ID No:16所示的氨基酸序列或其功能性变体。所述CSF2RA信号肽包含SEQ  ID No:14所示的氨基酸序列或其功能性变体。
在某些实施方式中，所述的CAR包含选自SEQ ID No:22-24、98、100或102的氨基酸序列或其功能性变体。
另一方面，本申请还包括分离的核酸分子，其编码本申请所述的CAR。
另一方面，本申请还包括编码CAR的分离的核酸分子，其包含选自SEQ ID No:1、3、5、7、9、11、13、15、17、19-21、25-27、31-33、37-39、79－87、97、99或101的核酸序列或其功能性变体。
另一方面，本申请还包括一种载体，其包含本申请所述的核酸分子。在某些实施方式中，所述载体选自质粒、逆转录病毒载体和慢病毒载体。
另一方面，本申请还包括一种免疫效应细胞，其包含本申请所述的CAR，本申请所述的核酸分子，或本申请所述的载体。在某些实施方式中，所述免疫效应细胞选自T淋巴细胞和自然杀伤(NK)细胞。在某些实施方式中，所述免疫效应细胞上的CD5不表达。
另一方面，本申请还包括一种制备免疫效应细胞的方法，其包括敲除所述免疫效应细胞的CD5基因，并包括向免疫效应细胞中引入本申请所述的载体。在某些实施方案中，所述敲除采用SEQ ID NO：103-106任一项的sgRNA进行。
另一方面，本申请还包括一种药物组合物，其包含本申请所述的免疫效应细胞以及药学上可接受的佐剂。
另一方面，本申请还包括所述的CAR，所述的核酸分子，所述的载体，或所述的免疫效应细胞在制备药物中的用途，其中所述药物用于治疗与CD5的表达相关的疾病或病症。在某些实施方式中，所述与CD5的表达相关的疾病或病症为癌症或恶性肿瘤。
另一方面，本申请还涉及所述的CAR，所述的核酸分子，所述的载体，或所述的免疫效应细胞或所述的药物组合物用于治疗与CD5的表达相关的疾病或病症。在某些实施方式中，所述与CD5的表达相关的疾病或病症为癌症或恶性肿瘤。另一方面，本申请还包括一种治疗与CD5的表达相关的疾病或病症的方法，其包括向有需要的患者施用治疗有效量的本申请所述的免疫效应细，或本申请所述的药物组合物。在某些实施方式中，还可以进一步向有需要的患者施用EGFR抗体如西妥昔单抗(Cetuximab)来抑制所述免疫效应细胞或药物组合物的效果，其中，所述有需要的患者包括那些对本申请所述的免疫效应细胞或药物组合物产生严重不良反应的患者。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
图1用jurkat细胞系进行CD5ko sgRNA的筛选，显示利用不同sgRNA敲除jurkat细胞所获得的细胞克隆的流式细胞分析结果。
图2显示了用流式细胞术进行单克隆jurkat-CD5ko细胞系CD5表达鉴定结果。
图3显示了本实验中采用的CAR结构(图3A)及转染后CAR-T细胞的CD5和EGFRt表达检测(图3B)。
图4显示了不同靶细胞对CAR-T细胞的CD107a脱粒作用结果。其中在每个CAR分子的结果中，从左至右分别为与Jurkat、Jurkat CD5ko、CCRF-CEM、MOLT4、K562-CD5、K562、RAJI共孵育以及CAR-T细胞单独孵育后的结果。
图5显示了CAR-T细胞对多种靶细胞的杀伤结果。
图6显示了用丝裂霉素处理后的CCRF-CEM细胞反复刺激CAR-T细胞增殖的实验结果。
图7显示了H65、Clone 10、Clone 32和Clone 35与CD5抗原的亲和力测定的动态结合曲线及KD、kon、kdis参数。
图8显示了本发明从噬菌体抗体库筛选靶向CD5的特异抗体的大体流程。
图9显示了所淘选的部分噬菌体单克隆与靶抗原和对照抗原的酶联免疫吸附测定(ELISA)结果。
图10显示了部分噬菌体单克隆与Raji和Jurkat细胞结合的流式细胞分析结果。
图11A-F显示了所筛选的噬菌体单克隆#1～64与多种不同的CD5阳性和阴性细胞系的结合的流式细胞分析结果(峰形图和MFI值)。Negative Control为阴性对照噬菌体抗体克隆。
图12A-F显示了所筛选的噬菌体单克隆#1～64与多种不同公司的CD5抗原蛋白和非相关抗原的酶联免疫吸附测定分析结果。Negative control为阴性对照噬菌体抗体克隆，anti-M13 phage mouse Ab/anti-mouse HRP Ab为只加第一抗体和第二抗体的阴性抗体对照，anti-mouse HRP Ab为只加第二抗体的阴性抗体对照，Mouse anti human CD5 Ab/anti-mouse HRP Ab为靶抗原(CD5-Fc-Bio)的阳性抗体对照，anti-human IgG-HRP Ab/anti-his-HRP Ab为检测抗原标签的阳性抗体对照。其中，图12A-F中，每个测试抗体和对照组对应的柱形图从左至右依次表示与蛋白Kactus-CD5-Fc-Bio、SB-CD5-his-Bio、Acro-CD5-his、Kactus-BAFFR-his-Bio、Kactus-CD19-FC-Bio、SA的测试结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。本申请所述的CAR可特异性结合CD5，利用所述CAR制得的CAR-T细胞可以稳定表达所述CAR，利用所述CAR制得的CAR-T细胞有较高的CAR阳性率。此外，所述CAR可促进细胞因子的释放，并可用于治疗与CD5的表达相关的疾病或病症。
除非明确指明相反，否则本申请的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。这些方法的描述可以参见，例如，Sambrook等人，Molecular Cloning:A Laboratory Manual(第3版，2001)；Sambrook等人，Molecular Cloning:A Laboratory Manual(第2版，1989)；Maniatis等人，Molecular Cloning:A Laboratory Manual(1982)；Ausubel等人，Current Protocols in Molecular Biology(John Wiley和Sons，2008年7月更新)；Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology，Greene Pub.Associates和Wiley-Interscience；Glover，DNA Cloning:A Practical Approach，vol.I&II(IRL Press，Oxford，1985)；Anand，Techniques for the Analysis of Complex Genomes，(Academic Press，New York，1992)；Transcription and Translation(B.Hames&S.Higgins，Eds.，1984)；Perbal，A Practical Guide to Molecular Cloning(1984)；Harlow和Lane，Antibodies，(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1998)Current Protocols in Immunology Q.E.Coligan，A.M.Kruisbeek，D.H.Margulies，E.M.Shevach和W.Strober，eds.，1991)；Annual Review of Immunology；以及期刊专著如Advances in Immunology。
除非另有定义，否则本申请中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本申请的目的，下文定义了以下术语。
在本申请中，术语“嵌合抗原受体”(Chimeric Antigen Receptor，CAR)是单链抗体的可变区和T细胞信号分子的融合蛋白。它使T细胞可以通过非MHC限制性的方式识别特异性抗原，发挥杀伤作用。CAR是嵌合抗原受体T细胞(CAR-T)的核心部件，其可以包括肿瘤相关抗原(tumor-associated antigen，TAA)结合区、跨膜结构域、共刺激结构域和胞内信号结构域，在肿瘤相关抗原结合区和跨膜结构域之间还可以进一步包含铰链区。在本申请中，所述CAR可以是一种能够将免疫效应细胞的细胞毒性重定向至B细胞的基因工程嵌合蛋白，其将基于抗体的抗原(例如CD5)特异性与T细胞受体活化胞内结构域组合在一起。经遗传修饰表达CAR的T细胞可以特异地识别和消除表达靶抗原的恶性细胞。关于CAR和CAR T细胞的描述，可以参见例如Sadelain M,Brentjens R,Rivi`ere I.The basicprinciples of chimeric antigen receptor design.Cancer Discov.2013；3(4):388-398；Turtle CJ,Hudecek M,Jensen MC,Riddell SR.Engineered T cells for anti-cancer therapy.Curr Opin Immunol.2012；24(5):633-639；Dotti G,Gottschalk S,Savoldo B,Brenner MK.Design and development of therapies using chimeric antigen receptor-expressing T cells.Immunol Rev.2014；257(1):107-126；以及WO2013154760、WO2016014789。
在本申请中，术语“CD5”是I型跨膜糖基化蛋白，在T细胞受体信号传导的负调控中起着重要作用，并促进正常和恶性淋巴细胞的存活。CD5是恶性T细胞肿瘤的特征性表面标志物之一，80％的T细胞急性淋巴细胞白血病(T-ALL)和外周T细胞淋巴瘤都表达CD5。在本申请中，所述CD5可以为人CD5，其GenBank登录号为NM_014207.4。CD5 蛋白也可包括CD5的片段，诸如胞外结构域及其片段。
在本申请中，术语“CD5结合结构域”通常是指CD5 CAR的胞外结构域，该结构域可以与抗原特异性结合。例如，所述CD5胞外结合结构域可包含能特异性结合人体细胞上表达的CD5多肽的嵌合抗原受体，抗CD5抗体或其抗原结合片段。在本申请中使用的术语“结合结构域”、“胞外结构域”、“胞外结合结构域”、“抗原特异性结合结构域”和“胞外抗原特异性结合结构域”可互换使用，并且提供了具有特异性结合目标靶抗原(例如CD5)的能力的CAR。CD5结合结构域可以为天然来源、合成来源、半合成来源或重组来源。
在本申请中，术语“抗体”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。例如，抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且包括任何包含其抗原结合部分的分子。术语“抗体”包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于人抗体、人源化抗体、嵌合抗体、单域抗体(例如，dAb)，单链抗体(例如，scFv)。本申请中，抗体的“片段”可指抗体的抗原结合片段，例如，Fab、Fab’和(Fab’) 2片段等。术语“抗体”还包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(例如，效应细胞)和经典补体系统的第一成分(Clq)。
在本申请中，术语“单链抗体”也可以被称为scFv，是指由重链可变区和轻链可变区通过连接肽连接而成的由一条链组成的抗体。
“鼠源抗体”是由鼠类针对特异抗原产生的抗体，通常指小鼠B淋巴细胞产生的抗体。在大多情况下，该鼠源抗体为杂交瘤细胞产生的单克隆抗体。本申请的全人源抗体是从人源噬菌体抗体库筛选获得，其相对于鼠源抗体降低了免疫原性，更利于人体的治疗用途。
本申请所述的“全人源抗体或其单链抗体或片段”通常是指能够与靶抗原结合的任何形式的抗原结合分子，例如该抗原结合分子可为蛋白质或多肽，包括例如抗体及其抗原结合片段、单链scFv抗体、单域抗体、基于scFv构建的各种融合物和缀合物，例如scFv-Fc抗体、免疫缀合物、抗体药物偶联物(ADC)、多/双特异性抗体、嵌合抗原受体(CAR)。
“表位”是指受抗原结合蛋白(例如，抗体)约束的分子的部分。表位可以包含该分子的非相邻的部分(例如，多肽中，在该多肽的主要序列上不相邻、但在该多肽的三价和四价结构中彼此足够靠近以被抗原结合蛋白约束的氨基酸残基)。
在本申请中，术语“跨膜结构域”(Transmembrane Domain)通常是指CAR中穿过 细胞膜的结构域，其与细胞内信号转导结构域相连接，起着传递信号的作用。本发明中，跨膜结构域可以是CD28跨膜结构域。
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激分子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。所述共刺激结构域可包括CD28的共刺激结构域，还可包括TNF受体家族的共刺激结构域，例如OX40和4-1BB的共刺激结构域。
在本申请中，术语“铰链区”通常是指抗原结合区和免疫细胞Fc受体(FcR)结合区之间的连接区。本发明中，铰链区可以是CD28铰链区。
在本申请中，术语“胞内信号传导结构域”通常是指CAR位于细胞内信号传导的组分，其包含信号传导结构域和特异性结合所述受体组分的结构域，例如：其可选自CD3ζ胞内域，CD28胞内域，CD28胞内域，4-1BB胞内域和OX40胞内域。
在本申请中，术语“CD8α信号肽”(Signal peptide)通常是指引导新合成的蛋白质向分泌通路转移的短(长度5-30个氨基酸)肽链。
在本申请中，术语“剪切肽”是指自剪切2A肽，其可经核糖体跳跃而非蛋白酶水解来实现剪切蛋白的功能，其可包括T2A，F2A和P2A等。
在本申请中，术语“标记检测信号”通常是指已知功能或序列的能够起到特异性标记作用，发出可以被检测到的信号的基因、蛋白质或其他分子。所述标记检测信号可以为荧光蛋白，如：GFP、RFP和YFP等。所述标记检测信号可以为EGFRt。术语“EGFRt”或“tEGFR”在本发明中可以互换使用，是指编码截短的人表皮生长因子受体多肽的基因，其缺乏远端膜EGF结合域和细胞质信号传导尾，但保留了由抗EGFR抗体识别的细胞外表位。EGFRt可用作具有遗传修饰细胞功能的非免疫原性选择工具以及追踪标记。在本申请中，其可作为CAR-T细胞的标记分子，用于必要时清除体内的CAR-T细胞EGFR抗体(例如，西妥昔单抗)介导的ADCC途径(cetuximab mediated ADCC pathway)(参见US8802374B2)，即在临床转化时作为安全开关使用，。
本发明中，术语“CSF2RA信号肽”，即集落刺激因子2受体α亚基(colony stimulating factor 2 receptor subunit alpha)信号肽，是可引导新合成的蛋白质在CAR-T细胞表面表达的肽链。
在本申请中，“EGFR抗体”是指能引发抗体依赖型细胞媒介毒性作用(antibody dependent cell-mediated cytotoxicity)，使免疫细胞攻击具有截短型表皮生长因子受体(EGFRt)的CAR-T细胞，协助清除CAR-T细胞的抗体。所述EGFR抗体可在患者输注CAR-T后发生严重不良反应或其他需要清除CAR-T细胞的情况时使用，其可以协助清除CAR-T细胞，减轻CAR-T治疗相关症状。所述EGFR抗体包括但不限于西妥昔单抗，帕尼单抗，耐昔妥珠单抗和尼妥珠单抗。
在本申请中，“序列同一性”或“序列一致性”通常是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将 两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
在一些实施方案中，本申请提供的全人源抗体还包括与SEQ ID NO：28、34、40、88、91或94任一项所示序列有至少90％序列一致性(例如，至少95％、至少98％、至少99％或甚至100％序列一致性)的氨基酸序列。
本领域技术人员可以理解的是，在本文提供的具体序列基础上，可以通过对少数氨基酸进行替换、删除、添加并验证或筛选所得产物与相应抗原CD5的结合能力或生物学活性，从而获得本文提供的靶向CD5抗体的相应变体，这些变体也应包括在本发明的范围内。例如，本申请的全人源抗体或其单链抗体或片段在全长或CDR序列上，可以有至少1个且不超过10，或不超过5、4、3、2或1个氨基酸的改变。
本领域技术人员还可以理解的是，在本文提供的具体重链可变区序列基础上，可以通过以CD5作为抗原筛选抗体轻链库(如人源噬菌体轻链库)，从而获得与所述重链可变区匹配并维持CD5结合能力的轻链可变区。以此方式可获得的抗CD5抗体分子也包括在本发明的范围内。
在一些实施方案中，本申请的抗原结合分子可以进一步包含转译后修饰。转译后蛋白修饰的示例包括：磷酸化、乙酰化、甲基化、ADP-核糖化、泛素化、糖基化、羰基化、类泛素化、生物素化或加入多肽侧链或疏水基团。因此，经修饰的可溶性多肽可以包含非氨基酸成分，例如类脂、多聚糖或单糖、以及磷酸盐。糖基化的一种优选形式是唾液酸化修饰，将一个或多个唾液酸基团与多肽结合。唾液酸基团改善蛋白质的溶解性和血清半衰期，同时也降低蛋白质可能的免疫遗传性。参见Raju et al.Biochemistry.2001 31；40(30):8868-76。
术语蛋白质或多肽序列的“功能性变体”在本申请中指，通过1个或更多个，例如1-30，或1-20或1-10个，例如1或2或3或4或5个氨基酸取代、缺失和/或插入而与亲本相比具有氨基酸改变的序列。功能性变体基本上保持改变之前的蛋白质或多肽序列的生物学特性。在一方面，本申请涵盖在本申请中所述的任何蛋白质或多肽序列的变体。在某些实施方式中，蛋白质或多肽序列的功能性变体保持改变前亲本的至少60％，70％，80％，90％，或100％的生物学活性。本发明所述的功能性变体可以是信号肽、抗体、铰链区、跨膜结构域、共刺激结构域胞内信号传导域、剪切肽、CSF2RA信号肽、EGFRt的功能性变体。当提及抗体的功能性变体时，还包括抗体可变区(如VH或VL)、抗体恒定区(如CH或CL)、重链CDR区(HCDR1、HCDR2或HCDR3)、轻链CDR区(LCDR1、LCDR2或 LCDR3)等的功能性变体。氨基酸的取代、缺失和/或插入可以发生在重链CDR区或轻链CDR区，或者重链FR区或轻链FR区，或者重链恒定区或轻链恒定区，其中变体基本上保持改变之前的抗体分子的生物学特性。对于抗体而言，其生物学活性例如包括抗原结合能力。在某些实施方式中，抗体的功能性变体包含的氨基酸改变不会导致抗体变体丧失对抗原的结合，但任选地可以赋予诸如提高的抗原亲和力和不同的效应子功能等性质。可以理解的，抗体的重链可变区或轻链可变区、或各CDR区可以单独改变或组合改变。在某些实施方案中，在一个或多个或全部三个重链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。在某些实施方式中，所述氨基酸改变可以为氨基酸取代，例如可以为保守取代。在某些实施方式中，功能性变体与亲本具有至少80％、85％、90％或95％或99％或更高的氨基酸同一性。类似地，核酸分子的“功能性变体”在本申请中指，能与亲本核酸分子编码相同氨基酸序列的核酸分子。
在本申请中，术语“分离的”通常是指抗体是已经与它的天然环境中的组分分离的抗体。在某些实施方式中，将抗体纯化至大于95％或99％纯度，所述纯度通过例如电泳(例如，SDS-PAGE、等电聚焦(IEF)、毛细管电泳)或色谱(例如，离子交换或反相HPLC)确定。关于评价抗体纯度的方法的综述可参见Flatman，S.等，J.Chrom.B 848(2007)79-87。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体或其抗原结合片段的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning,A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor,N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology,Greene Publishing and Wiley-Interscience,New York N.Y.,1993。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，用以将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。在本申请中，所述载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所 熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。本申请所述载体可选自质粒、逆转录病毒载体和慢病毒载体。本申请所述质粒、逆转录病毒载体和慢病毒载体可包含CAR。
在本申请中，术语“质粒”通常是指细菌、酵母菌等生物中染色体或拟核以外的DNA分子，存在于细胞质中，具有自主复制能力，使其能够在子代细胞中保持恒定的拷贝数，并表达所携带的遗传信息。质粒在遗传工程研究中被用作基因的载体。
在本申请中，术语“逆转录病毒载体”通常是指RNA病毒的一种，其遗传信息存储在核糖核酸上，此类病毒多具有反转录酶。反转录病毒至少含有三种基因：gag，包含组成病毒中心和结构的蛋白质的基因；pol，包含反转录酶的基因和env，包含组成病毒外壳的基因。通过逆转录病毒转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，术语“慢病毒载体”通常是指属于逆转录病毒的一种二倍体RNA病毒载体。慢病毒载体是以慢病毒的基因组为基础，将其中多个和病毒活性相关的序列结构去除，使其具有生物学的安全性，然后再在这个基因组骨架中引入实验所需要的目标基因的序列和表达结构制备成的载体。与其他逆转录病毒相比，慢病毒载体有着更广泛的宿主，对于分裂和非分裂细胞均具有感染能力，对于一些较难转染的细胞，如原代细胞、干细胞、不分化的细胞等，能大大提高目的基因的转导效率(参见陈琛和万海粟，“慢病毒载体及其研究进展，Chinese Journal of Lung Cancer 17.12(2014):870–876.PMC)。通过慢病毒载体转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，术语“转座子”通常是指含有转座酶基因的离散DNA片段，侧翼是含有转座酶结合位点的末端反向重复序列(TIR)。转座酶可与TIR结合并使转座子转移到新的位点。本申请所述转座子是由一个携带CAR(转座子)的质粒和另一个携带转座酶的质粒组成的双组分系统。所述转座子可以通过电转导等方式导入靶细胞。例如，首先，两种组分被电穿孔到外周血单核细胞(Peripheral Blood Mononuclear Cell，PBMC)中，表达的转座酶作用于CAR两侧的末端反向重复序列(terminal inverted repeat，TIR)，切割CAR(转座子)并随后整合到靶细胞(例如T细胞)基因组中的TA二核苷酸序列上。转座和稳定的基因组整合完成后，靶细胞表面就能表达出CAR蛋白(参见Cheng Zhang,Jun Liu,Jiang F Zhong,et al.Engineering CAR-T cells.Biomarker Research.2017，5:22)。
在本申请中，术语“基因编辑”通常是指对基因组进行定点修饰的技术，可包括基于锌指核酸酶(zinc finger nucleases，ZFNs)、转录激活子样效应因子核酸酶(transcription  activator like effector nucleases，TALENs)、规律性重复短回文序列簇(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(Cas9)，CRISPR/Cas9)等的技术。其可以对基因组进行高效靶向修饰，通过在基因组的特定位置添加、去除或改变遗传物质来进行修饰。本申请所述基因编辑可包括通过基因编辑的技术(例如CRISPR-Cas9)，将CAR分子导入受体细胞的基因组中。
在本申请中，术语“免疫效应细胞”通常是指在免疫应答中参与清除异物抗原和行使效应功能的免疫细胞。例如浆细胞、细胞毒性T细胞、NK细胞、APSC多能细胞、肥大细胞等。
在本申请中，术语“药学上可接受的佐剂”通常是指药学可接受的制剂载体、溶液或加强制剂特性的添加剂。此类添加剂是所属领域技术人员熟知的。
在本申请中，术语“癌症”通常是指或描述哺乳动物的生理状况，其典型特征在于细胞增殖或存活失调。在本申请中，被称为癌症的过度增殖性疾病包括但不限于实体瘤，例如发生在乳腺、呼吸道、脑、生殖器官、消化道、尿道、眼、肝脏、皮肤、头颈、甲状腺、甲状旁腺的癌症，以及它们的远端转移。这类疾病还包括淋巴瘤、肉瘤和白血病。乳腺癌的实例包括但不限于浸润性导管癌、浸润性小叶癌、乳腺导管原位癌和乳腺小叶原位癌。呼吸道癌症的实例包括但不限于小细胞肺癌和非小细胞肺癌，以及支气管腺瘤和胸膜肺母细胞瘤。脑癌的实例包括但不限于脑干和下丘脑角质瘤、小脑和大脑星状细胞瘤、髓母细胞瘤、室管膜瘤，以及神经外胚层和松果体肿瘤。男性生殖器肿瘤包括但不限于前列腺和睾丸癌。女性生殖器肿瘤包括但不限于子宫内膜癌、宫颈癌、卵巢癌、阴道癌和外阴癌，以及子宫瘤。消化道肿瘤包括但不限于肛门、结肠、结肠直肠、食管、胆囊、胃、胰腺、直肠、小肠和唾液腺癌。尿道肿瘤包括但不限于膀胱、阴茎、肾、肾盂、输尿管和尿道癌。眼癌包括但不限于眼球内黑素瘤和视网膜母细胞瘤。肝癌的实例包括但不限于肝细胞癌(有或没有纤维板层变异的肝细胞瘤)、胆管癌(肝内胆管癌)和混合型肝细胞胆管细胞癌。皮肤癌包括但不限于鳞状细胞癌、卡波西(Kaposi’s)肉瘤、恶性黑素瘤、Merkel细胞皮肤癌和非黑素瘤型皮肤癌。头颈癌包括但不限于喉/下咽/鼻咽/口咽癌，和嘴唇和口腔癌。淋巴癌包括但不限于AIDS相关的淋巴癌、非霍奇金淋巴癌、皮肤T细胞淋巴癌、霍奇金氏病，以及中枢神经系统淋巴癌。肉瘤包括但不限于软组织肉瘤、骨肉瘤、恶性纤维组织细胞瘤、淋巴肉瘤和横纹肌肉瘤。白血病包括但不限于急性髓样白血病、急性淋巴细胞白血病、慢性淋巴细胞白血病、慢性粒细胞白血病和毛细胞白血病。
提及药物组合物，所使用的“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。
“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物或细胞的量。“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，对于抗体而言，典型的日剂量范围可以为每kg体重0.01mg至 100mg活性成分。本申请的抗体的施用方式包括但不限于注射，例如通过静脉内、肌内、动脉内、皮下、腹膜内等。
术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
如本申请中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本申请中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
CD5抗体
本发明使用全人源噬菌体进行抗体筛选，直接获得全人源的单克隆抗体。与传统杂交瘤技术相比，省却了困难的鼠源抗体人源化步骤，而且全人源抗体比人源化的鼠源抗体具有更低的免疫原性，在抗体药物(包括单抗，双抗，ADC等)、细胞治疗药物(包括CAR-T，CAR-NK等)、检测试剂等应用上有更好的潜力。
本发明使用了抗原蛋白淘选的方法，能高效地富集的同时结合重组CD5和细胞膜上天然结构CD5的抗体，大大降低了后期抗体筛选的难度，提高了效率。
我们应用大容量噬菌体抗体库筛选全人源的CD5特异抗体，并通过ELISA和FACS实验来评估这些抗体在phage水平的特异性。最终，我们获得了若干特异性良好的全人源抗体克隆。
我们使用不同的抗体库，经过重组CD5蛋白淘选，总共挑选了184个单克隆进行酶联免疫吸附测定(ELISA)和流式细胞术(FACS)检测初筛，其中93个克隆特异结合CD5-Fc-Bio蛋白和CD5表达阳性细胞Jurkat，而不结合对照蛋白CD19-Fc-Bio和CD5表达阴性细胞Raji。测序后得到了64种不同的单克隆序列。随后，我们将这64种抗体与多种CD5阳性(Jurkat，CCRF-CEM)和阴性细胞系(Raji，NALM6)进行流式细胞分析(FACS)鉴定，与不同公司的CD5蛋白(Kactus-CD5-Fc-Bio,Acro-CD5-his,SB-CD5-his-Bio)，非相关蛋白(Kactus-BAFFR-his-Bio，Kactus-CD19-FC-Bio，SA)进行酶联免疫吸附(ELISA)鉴定，其中49个克隆在多个细胞系和多种蛋白抗原上都表现出良好的结合力和特异性。这些克隆的获得为后续开发全人源的CD5 CAR-T产品或者抗体药奠定了基础。总体项目流程如图8所示。
嵌合抗原受体
在本申请中，所述CAR可以包含特异性结合CD5的胞外结构域、跨膜结构域、胞内共刺激信号传导结构域和胞内信号传导结构域。在本申请中，所述CAR的胞外结构域可 以包含本发明的单链scFv抗体。例如，所述单链抗体可以通过铰链区，例如CD8铰链，与跨膜结构域连接。在本申请中，所述CAR可以用于转导免疫效应细胞(例如T细胞)并在细胞表面表达。由此本申请也可提供表达所述嵌合抗原受体的T细胞，以及该T细胞和/或所述CAR用于制备治疗CD5相关疾病的药物中的用途。
在本申请中，所述嵌合抗原受体(CAR)可包含CD5结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域。在本申请中，所述CD5结合结构域可包含特异性结合CD5的抗体或其片段，所述抗体可包含重链互补决定区1(HCDR1)，重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，所述HCDR1-3的氨基酸序列如SEQ ID NO：43-45、49-51、55-57、61-63、67-69或73-75所示，所述抗体还可包含轻链互补决定区1(LCDR1)，轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，所述LCDR1-3的氨基酸序列如SEQ ID No:46-48、52-54、58-60、64-66、70-72或76-78所示。在本申请中，所述抗体可包含重链可变区，所述重链可变区的氨基酸序列如SEQ ID No:30、36、42、90、93或96所示。在本申请中，所述抗体可包含轻链可变区，所述轻链可变区的氨基酸序列如SEQ ID No:29、35、41、89、92或95所示。
在本申请中，所述抗体可为单链抗体。在某些实施方案中，所述抗体可包含SEQ ID No:28、3440、88、91或94所示的氨基酸序列或其功能性变体。例如，所述单链抗体可包括scFv10，其序列如SEQ ID No:28所示；所述单链抗体可包括scFv32，其序列如SEQ ID No:34所示；所述单链抗体可包括scFv35，其序列如SEQ ID No:40所示；所述单链抗体可包括scFv6，其序列如SEQ ID No:88所示；所述单链抗体可包括scFv7，其序列如SEQ ID No:91所示；所述单链抗体可包括scFv9，其序列如SEQ ID No:94所示；。
例如，本申请所述单链抗体可以为scFv10(原始编号为clone 10)，其序列如SEQ ID No:28所示。单链抗体scFv10的LCDR1-3的氨基酸序列分别如SEQ ID No:46、SEQ ID No:47和SEQ ID No:48所示；VL的氨基酸序列如SEQ ID No:29所示；HCDR1-3的氨基酸序列分别如SEQ ID No:43、SEQ ID No:44和SEQ ID No:45所示；VH的氨基酸序列如SEQ ID No:30所示。本申请所述单链抗体可以为scFv32(原始编号为clone 32)，其序列如SEQ ID No:34所示。单链抗体scFv32的LCDR1-3的氨基酸序列分别如SEQ ID No:52、SEQ ID No:53和SEQ ID No:54所示；VL的氨基酸序列如SEQ ID No:35所示；HCDR1-3的氨基酸序列分别如SEQ ID No:49、SEQ ID No:50和SEQ ID No:51所示；VH的氨基酸序列如SEQ ID No:36所示。本申请所述单链抗体可以为scFv35(原始编号为clone 35)，其序列如SEQ ID No:40所示。单链抗体scFv35的LCDR1-3的氨基酸序列分别如SEQ ID No:58、SEQ ID No:59和SEQ ID No:60所示；VL的氨基酸序列如SEQ ID No:41所示；HCDR1-3的氨基酸序列分别如SEQ ID No:55、SEQ ID No:56和SEQ ID No:57所示；VH的氨基酸序列如SEQ ID No:42所示。本申请所述单链抗体可以为scFv6(原始编号为clone 6)，其序列如SEQ ID No:88所示。单链抗体scFv6的LCDR1-3的氨基酸序列分别如SEQ ID No:64、SEQ ID No:65和SEQ ID No:66所示；VL的氨基酸序列如SEQ ID No:89所示； HCDR1-3的氨基酸序列分别如SEQ ID No:61、SEQ ID No:62和SEQ ID No:63所示；VH的氨基酸序列如SEQ ID No:90所示。本申请所述单链抗体可以为scFv7(原始编号为clone7)，其序列如SEQ ID No:91所示。单链抗体scFv7的LCDR1-3的氨基酸序列分别如SEQ ID No:70、SEQ ID No:71和SEQ ID No:72所示；VL的氨基酸序列如SEQ ID No:92所示；HCDR1-3的氨基酸序列分别如SEQ ID No:67、SEQ ID No:68和SEQ ID No:69所示；VH的氨基酸序列如SEQ ID No:93所示。本申请所述单链抗体可以为scFv9(原始编号为clone9)，其序列如SEQ ID No:94所示。单链抗体scFv9的LCDR1-3的氨基酸序列分别如SEQ ID No:76、SEQ ID No:77和SEQ ID No:78所示；VL的氨基酸序列如SEQ ID No:95所示；HCDR1-3的氨基酸序列分别如SEQ ID No:73、SEQ ID No:74和SEQ ID No:75所示；VH的氨基酸序列如SEQ ID No:96所示。
本申请所述CAR可包括跨膜结构域，所述跨膜结构域可包含来自选自下述蛋白的多肽：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8a、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。在本申请中，所述跨膜结构域可包含SEQ ID No:6所示的氨基酸序列或其功能性变体。例如，本申请的跨膜结构域可包括CD8a，其序列如SEQ ID No:6所示。
在本申请中，所述共刺激结构域可包含来自选自下述蛋白的多肽：CD28、4-1BB、OX40和ICOS。在本申请中，所述共刺激结构域可包含SEQ ID No:8所示的氨基酸序列或其功能性变体。
本申请所述CAR可包括胞内信号传导结构域，所述胞内信号传导结构域可包含来自CD3ζ的信号传导结构域。在本申请中，所述胞内信号传导结构域可包含SEQ ID No:10所示的氨基酸序列或其功能性变体。
本申请所述CAR可包括铰链区，所述铰链区可连接所述抗体和所述跨膜结构域。在本申请中，所述铰链区可包含SEQ ID No:4所示的氨基酸序列或其功能性变体。
本申请所述CAR可包括信号肽，所述信号肽例如可以位于特异性结合CD5的胞外结构域的N端。所述信号肽可包含SEQ ID No:2所示的氨基酸序列或其功能性变体。例如，所述信号肽可为CD8a信号肽，其序列如SEQ ID No:2所示。
在本申请中，所述CAR还可连接剪切肽。在本申请中，所述剪切肽可包含来自T2A肽的氨基酸序列。在本申请中，所述剪切肽可包含SEQ ID No:12所示的氨基酸序列或其功能性变体。例如，所述剪切肽可为T2A，其序列如SEQ ID No:12所示。
在本申请中，所述CAR还可连接EGFRt片段，所述EGFRt片段可用于信号检测，或作为CAR-T细胞的分子开关使用。
在本申请中，所述CAR可包含SEQ ID No:22-24、98、100或102所示的氨基酸序列或其功能性变体。例如，所述CAR可选自CAR001，其序列如SEQ ID No:22所示。又例如，所述CAR可选自CAR002，其序列如SEQ ID No:23所示；所述CAR可选自CAR003，其序列如SEQ ID No:24所示；所述CAR可选自CAR004，其序列如SEQ ID No:98所示； 所述CAR可选自CAR005，其序列如SEQ ID No:100所示；所述CAR可选自CAR006，其序列如SEQ ID No:102所示。
在某些实施方式中，本申请所述CAR可自N端依次包括CD5结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域。其中，所述CAR可包括CD5结合结构域，所述CD5结合结构域序列如SEQ ID No:28所示。其中，所述CD5结合结构域可包括HCDR1-3，其序列依次如SEQ ID No:43-45所示；并且，CD5结合结构域可包括LCDR1-3，其序列依次如SEQ ID No:46-48所示。例如，所述CAR可包括CAR001或与其具有相同的LCDR1-3及HCDR1-3的本申请所述的CAR。所述CD5结合结构域可包括重链可变区，其序列如SEQ ID No:30所示；并且，所述CD5结合结构域还可包括轻链可变区，其序列如SEQ ID No:29所示。例如，所述CAR可包括CAR001或与其具有相同的轻链可变区及重链可变区的本申请所述的CAR。所述轻链可变区和所述重链可变区之间还可包括连接肽，其序列如SEQ ID No:18所示。例如，所述CAR可包括CAR001或与其具有相同的连接肽的本申请所述的CAR。所述跨膜结构域的可包含来自CD8a的跨膜结构域，其序列可以如SEQ ID No:6所示。例如，所述CAR可包括CAR001或与其具有相同的跨膜结构域的本申请所述的CAR。所述共刺激结构域可包含来自4-1BB的共刺激结构，其序列可以如SEQ ID No:8所示。例如，所述CAR可包括CAR001或与其具有相同的共刺激结构域的本申请所述的CAR。所述胞内信号传导结构域可包含来自CD3ζ的信号传导结构域，其序列如SEQ ID No:10所示。例如，所述CAR可包括CAR001或与其具有相同的胞内信号转导结构域的本申请所述CAR。所述CAR还可包含铰链区，所述铰链区可位于所述CD5结合结构域的C端且位于所述跨膜结构域的N端，其序列例如可以如SEQ ID No:4所示。例如，所述CAR可包括CAR001或与其具有相同的铰链区的本申请所述CAR。所述CAR还可连接信号肽，其可位于所述CAR的N端，其序列可以如SEQ ID No:2所示。
所述CAR还可连接剪切肽，例如：T2A。所述剪切肽可以位于所述胞内信号转导域的C端，其序列可以如SEQ ID No:12所示。所述CAR还可以连接CSF2RA信号肽，其可以位于EGFRt之前，其序列例如可以如SEQ ID No:14所示。所述CAR还可连接标记检测信号，其可位于所述CAR(或者，所述剪切肽)的C端。所述标记检测信号可选自以下组：GFP、RFP、YFP或EGFRt，EGFRt的序列例如可以如SEQ ID No:16所示。
例如，本申请所述的CAR可以为CAR001，其LCDR1-3的氨基酸序列分别如SEQ ID No:46、SEQ ID No:47和SEQ ID No:48所示；VL的氨基酸序列如SEQ ID No:29所示；HCDR1-3的氨基酸序列分别如SEQ ID No:43、SEQ ID No:44和SEQ ID No:45所示；VH的氨基酸序列如SEQ ID No:30所示；VH与VL之间的连接肽的序列如SEQ ID No:18所示；其铰链区如SEQ ID No:4所示；其跨膜结构域如SEQ ID No:6所示；其共刺激结构域为4-1BB共刺激结构域，如SEQ ID No:8所示；其CD3ζ胞内信号传导结构域如SEQ ID No：10所示；所述CAR001还包含如SEQ ID No:12所示的剪切肽，如SEQ ID No:14所示的CSF2RA信号肽，以及如SEQ ID No:16所示的EGFRt；如SEQ ID No:2所示的CD8a信 号肽。
例如，本申请所述的CAR可以为CAR002，其LCDR1-3的氨基酸序列分别如SEQ ID No:52、SEQ ID No:53和SEQ ID No:54所示；VL的氨基酸序列如SEQ ID No:35所示；HCDR1-3的氨基酸序列分别如SEQ ID No:49、SEQ ID No:50和SEQ ID No:51所示；VH的氨基酸序列如SEQ ID No:36所示；VH与VL之间的连接肽的序列如SEQ ID No:18所示；其铰链区如SEQ ID No:4所示；其跨膜结构域如SEQ ID No:6所示；其共刺激结构域为4-1BB共刺激结构域，如SEQ ID No:8所示；其CD3ζ胞内信号传导结构域如SEQ ID No:10所示；所述CAR002还包含如SEQ ID No:12所示的剪切肽，如SEQ ID No:14所示的CSF2RA信号肽，以及如SEQ ID No:16所示的EGFRt；如SEQ ID No:2所示的CD8a信号肽。
例如，本申请所述的CAR可以为CAR003，其LCDR1-3的氨基酸序列分别如SEQ ID No:58、SEQ ID No:59和SEQ ID No:60所示；VL的氨基酸序列如SEQ ID No:41所示；HCDR1-3的氨基酸序列分别如SEQ ID No:55、SEQ ID No:56和SEQ ID No:57所示；VH的氨基酸序列如SEQ ID No:42所示；VH与VL之间的连接肽的序列如SEQ ID No:18所示；其铰链区如SEQ ID No:4所示；其跨膜结构域如SEQ ID No:6所示；其共刺激结构域为4-1BB共刺激结构域，如SEQ ID No:8所示；其CD3ζ胞内信号传导结构域如SEQ ID No:10所示；所述CAR002还包含如SEQ ID No:12所示的剪切肽，如SEQ ID No:14所示的CSF2RA信号肽，以及如SEQ ID No:16所示的EGFRt；如SEQ ID No:2所示的CD8a信号肽。
例如，本申请所述的CAR可以为CAR004，其LCDR1-3的氨基酸序列分别如SEQ ID No:64、SEQ ID No:65和SEQ ID No:66所示；VL的氨基酸序列如SEQ ID No:89所示；HCDR1-3的氨基酸序列分别如SEQ ID No:61、SEQ ID No:62和SEQ ID No:63所示；VH的氨基酸序列如SEQ ID No:90所示；VH与VL之间的连接肽的序列如SEQ ID No:18所示；其铰链区如SEQ ID No:4所示；其跨膜结构域如SEQ ID No:6所示；其共刺激结构域为4-1BB共刺激结构域，如SEQ ID No:8所示；其CD3ζ胞内信号传导结构域如SEQ ID No:10所示；所述CAR002还包含如SEQ ID No:12所示的剪切肽，如SEQ ID No:14所示的CSF2RA信号肽，以及如SEQ ID No:16所示的EGFRt；如SEQ ID No:2所示的CD8a信号肽。
例如，本申请所述的CAR可以为CAR005，其LCDR1-3的氨基酸序列分别如SEQ ID No:70、SEQ ID No:71和SEQ ID No:72所示；VL的氨基酸序列如SEQ ID No:92所示；HCDR1-3的氨基酸序列分别如SEQ ID No:67、SEQ ID No:68和SEQ ID No:69所示；VH的氨基酸序列如SEQ ID No:93所示；VH与VL之间的连接肽的序列如SEQ ID No:18所示；其铰链区如SEQ ID No:4所示；其跨膜结构域如SEQ ID No:6所示；其共刺激结构域为4-1BB共刺激结构域，如SEQ ID No:8所示；其CD3ζ胞内信号传导结构域如SEQ ID No:10所示；所述CAR002还包含如SEQ ID No:12所示的剪切肽，如SEQ ID No:14所示 的CSF2RA信号肽，以及如SEQ ID No:16所示的EGFRt；如SEQ ID No:2所示的CD8a信号肽。
例如，本申请所述的CAR可以为CAR006，其LCDR1-3的氨基酸序列分别如SEQ ID No:76、SEQ ID No:77和SEQ ID No:78所示；VL的氨基酸序列如SEQ ID No:95所示；HCDR1-3的氨基酸序列分别如SEQ ID No:73、SEQ ID No:74和SEQ ID No:75所示；VH的氨基酸序列如SEQ ID No:96所示；VH与VL之间的连接肽的序列如SEQ ID No:18所示；其铰链区如SEQ ID No:4所示；其跨膜结构域如SEQ ID No:6所示；其共刺激结构域为4-1BB共刺激结构域，如SEQ ID No:8所示；其CD3ζ胞内信号传导结构域如SEQ ID No:10所示；所述CAR002还包含如SEQ ID No:12所示的剪切肽，如SEQ ID No:14所示的CSF2RA信号肽，以及如SEQ ID No:16所示的EGFRt；如SEQ ID No:2所示的CD8a信号肽。
核酸、载体、细胞、制备方法和组合物
另一方面，本申请提供了一种分离的核酸分子，其可编码本申请所述的CAR。本申请所述编码CAR的分离的核酸分子，其可包含选自SEQ ID No:1、3、5、7、9、11、13、15、17、19-21、25-27、31-33、37-39、79－87、97、99或101的核酸序列或其功能性变体。本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
另一方面，本申请提供了一种载体，其可包含所述的核酸分子。在本申请中，所述载体可选自质粒、逆转录病毒载体和慢病毒载体中的一种或多种。本申请所述慢病毒载体可包含CAR。例如，本申请所述慢病毒载体可包含选自SEQ ID No:1、3、5、7、9、11、13、15、17、19-21、25-27、31-33、37-39、79－87、97、99或101的核酸序列或其功能性变体。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体，包括载体scFv质粒和/或CAR质粒。
另一方面，本申请提供给了一种免疫效应细胞，其可包含本申请所述的CAR，所述的 核酸分子，或所述的载体。在本申请中，所述免疫效应细胞可为哺乳动物细胞。在本申请中，免疫效应细胞可选自T淋巴细胞和自然杀伤(NK)细胞。
另一方面，本申请提供了一种制备免疫效应细胞的方法，其包括敲除所述免疫效应细胞的CD5基因，并向免疫效应细胞中引入本申请所述的载体。例如，可将本申请所述的载体引入所述免疫效应细胞中，例如T淋巴细胞或自然杀伤(NK)细胞。在某些实施方式中，每种或每个细胞可包含一个或一种本申请所述的载体。在某些实施方式中，每种或每个细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的载体。在本申请中，可将所述载体引入免疫效应细胞中可通过本领域已知的方法将本申请所述的载体引入所述细胞中。例如，可以通过逆转录病毒载体进行转染免疫效应细胞，将带有CAR分子的病毒基因组能整合到宿主基因组，保证目的基因长期、稳定地表达。又例如，利用转座子，通过携带CAR(转座子)的质粒和携带转座酶的质粒导入到靶细胞中。又例如，可以通过基因编辑的方式(例如CRISPR/Cas9)将CAR分子添加进基因组中。在本申请中，可通过本领域已知的方法将本申请所述的带有CAR分子的载体引入所述细胞中，例如电穿孔、脂质体法转染(lipofectamine 2000，Invitrogen)等。
另一方面，本申请提供了一种药物组合物，其可包含所述的免疫效应细胞和药学上可接受的佐剂。所述药学上可接受的佐剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。在本申请中，所述药物组合物可被配制用于口服给药，静脉内给药(例如，静脉注射，I.V.)，肌肉内给药(例如，肌肉注射，I.M.)，在肿瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。
制药用途
另一方面，本申请提供了所述的抗体、所述的CAR、所述的核酸分子、所述的载体或所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗与CD5的表达相关的疾病或病症。在本申请中，所述与CD5的表达相关的疾病或病症可为癌症或恶性肿瘤。在某些实施方式中，所述癌症或恶性肿瘤可选自恶性T细胞肿瘤或恶性B细胞肿瘤。其中，所述恶性T细胞肿瘤可选自T细胞急性淋巴细胞白血病(T-ALL)、T细胞淋巴瘤(TCL)(例如外周T细胞淋巴瘤、皮肤T细胞淋巴瘤(CTCL)、T细胞非霍奇金淋巴瘤(T-NHL))；所述恶性B细胞肿瘤可选自慢性淋巴细胞白血病(B-CLL)(例如毛细胞白血病细胞(HCL))、套细胞淋巴瘤(B-MCL)、弥漫大B淋巴瘤(DLBCL)。
另一方面，本申请提供了所述的抗体、所述的CAR、所述的核酸分子、所述的载体或所述的免疫效应细胞，其治疗与CD5的表达相关的疾病或病症。
另一方面，本申请提供了一种治疗与CD5的表达相关的疾病或病症的方法，包括向患者施用所述的抗体、所述的CAR，所述的核酸分子，所述的载体，或所述的免疫效应细胞。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的嵌合抗原受体、载体、 细胞、组合物的工作方式，而不用于限制本申请发明的范围。
研究概述
研究者使用全人源噬菌体进行抗体筛选，直接获得全人源的单克隆抗体。与传统杂交瘤技术相比，省却了困难的鼠源抗体人源化步骤，而且全人源抗体比人源化的鼠源抗体具有更低的免疫原性，在CAR-T开发上有更好的潜力。在获得了特异性结合细胞表面CD5抗原及CD5重组蛋白的抗体克隆后，本申请进行了深入研究，将这些克隆及对照克隆H65构建至二代CAR结构上，然后进行慢病毒包装，并转染T细胞，在CAR-T细胞水平，从靶细胞激活和杀伤、靶细胞刺激增殖等角度筛选出了功能强于对照克隆H65的全人源CD5抗体克隆及候选CAR-T分子。
此外，文献报道在大多数构建的肿瘤动物模型中，重新出现的肿瘤细胞保留了CD5表达，这表明肿瘤复发并非源于抗原的丧失，未能根除所有异种移植物是由于CD5 CAR-T细胞在小鼠中的存续性差 [13]。本申请采用CRISPR技术对jurkat细胞系进行了CD5基因的敲除，成功挑选出了敲除效率高的sgRNA。实验结果表明，CD5敲除的CAR-T细胞能正常扩增，并保持对CD5阳性靶细胞的杀伤功能，同时最大限度的减少CD5 CAR-T的自激活及自杀现象，保证其在临床验证中的持续性和有效性。
实施例
实施例1.通过亲和淘选从噬菌体抗体库富集靶向CD5蛋白的特异抗体克隆
采用合适的负淘选和正淘选策略从噬菌体抗体库中富集我们所需的特异性抗体克隆。
噬菌体抗体库的构建
我们构建的噬菌体抗体库包括天然库、半合成库和单域库。半合成噬菌体抗体库，与天然库一起使用，解决天然库可能缺乏CD5高亲和力抗体克隆的问题。单域噬菌体抗体库是只由重链抗体的可变区氨基酸组成的抗体库，其分子量仅有12-15kDa，但却具备与传统抗体相似或更高的特异性和亲和力。此外，单域抗体理化性质稳定、亲和力高、易于重组表达制备、易于与其它靶点或表位抗体组合等特点使其备受关注。
CD5蛋白淘选
以CD5-Fc-Bio作为正淘选蛋白，以CD19-Fc-Bio作为负淘选蛋白进行多轮淘选，获得富集目的抗体克隆的噬菌体池(pool)。实验步骤简述如下：
1)将SA磁珠先用封闭液封闭2h，然后再将靶抗原(CD5-Fc-Bio)与封闭好的SA磁珠结合；
2)加入噬菌体文库(含5x10 12个噬菌体颗粒)和和对照抗原一起孵育，以便扣除非特异结合Fc标签的噬菌体抗体克隆；
3)孵育后将上清转移到结合了靶抗原的SA磁珠中，继续孵育，使噬菌体和靶抗原结合；
4)用洗涤液洗涤磁珠，将未结合的噬菌体洗去；
5)用洗脱液将阳性噬菌体从靶抗原上洗脱下来，加入中和液中和；
6)以洗脱后的噬菌体重新感染宿主菌XL1-blue，扩增回收的噬菌体。留少量样品梯度稀释，感染宿主菌，涂Amp抗性平板，计算回收噬菌体数量；
7)重复步骤1)至6)，通常需要进行3轮淘选，直到观察到噬菌体的回收率(洗脱噬菌体数/投入噬菌体数)有明显上升。
富集好的噬菌体池可用于进行随后的单克隆挑选以及ELISA/FACS筛选。
主要材料和试剂：
全人源噬菌体抗体库，包含天然库，半合成库和单域库；
辅助噬菌体KO7，Thermo/Invitrogen,18311019；
Recombinant biotinylated Human CD5-Fc Protein,Kactus,CD5-HM401；
Recombinant biotinylated Human CD19 Protein-Fc,Kactus,CD2-HE121
BeaverBeads TM Streptavidin,海狸生物,22307-10；
High binding ELSIA plate,Costar，#3590
封闭液：PBS+3％BSA
漂洗液：PBS+0.1％Tween20
洗脱液：0.2M Glycine，pH2.2
中和液：1M Tris，pH9.1
实验结果：
使用不同的抗体库，通过3轮蛋白淘选，每个淘选都观察到了回收率的显著上升(表1)，证明抗体克隆得到了有效富集。
表1蛋白淘选实验结果
Figure PCTCN2021127995-appb-000001
可以看到经过3轮淘选，不同的抗体库都得到了富集(第3轮回收率比上一轮显著提高)。
实施例2.采用酶联免疫吸附测定(ELISA)和流式细胞术(FACS)从经富集的噬菌体池筛选 特异性克隆
目的和原理：
通过亲和淘选步骤富集的噬菌体池中包含各种性质的噬菌体抗体：特异克隆、非特异克隆、以及阴性克隆。为了获得特异克隆，我们需要从中分离单克隆，包装成单克隆的噬菌体，并通过酶联免疫检测(ELISA)和流式细胞术(FACS)对大量单克隆进行初筛，从中挑选到同时特异结合CD5蛋白和CD5阳性细胞系Jurkat的单克隆。特异的单克隆再进一步通过DNA测序确定其中包含的唯一的抗体序列。
在ELISA初筛中，通过链霉亲和素Streptavidin与生物素Biotin的结合，使得生物素化的靶蛋白(CD5-Fc-Bio)在反应液中更接近于天然状态的抗原构象。只结合CD5-Fc-Bio而不结合对照抗原CD19-Fc-Bio的被认定为特异克隆。FACS初筛使用CD5高表达的阳性细胞系Jurkat和CD5阴性的细胞系Raji来进行，只结合细胞Jurkat且不结合Raji细胞的被认定为特异克隆。通过ELISA和FACS两种初筛，我们可以获得既能结合重组表达的CD5蛋白，又能识别细胞表面天然状态CD5分子的候选抗体，供随后进一步筛选。
ELISA初筛实验简要步骤：
1)用深孔96孔板培养和包装单克隆噬菌体；
2)将Strepavidin用PBS稀释到2μg/mL，以100μL/孔加入到高结合酶标板中，室温结合2h；
3)弃掉包被液，每孔加入250μL封闭液，4℃封闭过夜；
4)250μL漂洗液洗板2次；
5)将带生物素标签的靶蛋白和对照蛋白用PBS稀释至2μg/mL，以100μg/孔加入预包被Strepavidin的酶标板中，室温结合1h；
6)250μL漂洗液洗板2次；
7)加入100μL步骤1)培养好的噬菌体上清到包被好靶抗原的孔，室温结合2h；
8)250μL漂洗液洗板4次；
9)加入1:2000稀释的mouse anti M13第一抗体，100μL/孔，室温孵育45min；
10)250μL漂洗液洗板4次；
11)加入1:2000稀释的HRP Donkey anti-mouse IgG，100μL/孔，室温孵育45min；
12)250μL漂洗液洗板6次；
13)加入100μL TMB显色底物，显色5至10min；
14)加入100μL 2M H 2SO 4终止反应，在酶标仪上读取结果。
FACS初筛实验简要步骤：
1)用深孔96孔板培养和包装单克隆噬菌体；
2)Raji和Jurkat细胞用PBS洗2次，用PBS重悬成1x10 7/mL浓度，以50μL分装到96孔深孔板中；
3)每孔加入50μL包装好的单克隆噬菌体，混匀后，4℃结合2h；
4)200μL PBS洗涤2次；
5)加入1:2000稀释的mouse anti M13第一抗体，100μL/孔，吹打混匀后，室温孵育45min；
6)200μL PBS洗涤2次；
7)加入1:300稀释的FITC horse anti mouse-IgG(H+L)，100μL/孔，吹打混匀后，室温孵育45min；
8)200μL PBS洗涤2次；最后用200μL PBS重悬细胞；
9)在流式细胞仪上检测样品FITC通道的荧光强度，分析结果。
主要材料和试剂：
辅助噬菌体KO7，Thermo/Invitrogen,18311019
Streptavidin,Pierce,21125
Recombinant biotinylated Human CD5 Protein,Kactus,CD5-HM401；
Recombinant biotinylated Human CD19 Protein,Kactus,CD2-HE121
High binding ELSIA plate,Costar,#3590
Corning 96 Well Clear Round Bottom TC-Treated Microplate,Costar,#3799
封闭液：PBS+3％BSA
漂洗液：PBS+0.1％Tween20
可溶型单组分TMB底物溶液,Tiangen,PA-107-02
Anti-M13 Bacteriophage Coat Protein g8p antibody,abcam,ab9225
HRP Goat anti-mouse IgG(minimal x-reactivity)Antibody,Biolegend,405306
FITC horse anti mouse-IgG(H+L),Vector,FI2000
实验结果：
从富集后的噬菌体抗体池随机挑选单克隆，包装成噬菌体后，通过噬菌体ELISA检测单克隆噬菌体与CD5-Fc-Bio蛋白、对照蛋白CD19-Fc-Bio的结合，找到CD5特异的噬菌体抗体克隆。部分克隆的ELISA结果如图9所示。从图中可知，H1，H2，H3，H4，H5，H6和H7号克隆与靶抗原CD5(CD5-Fc-Bio)结合较强，且不与对照抗原CD19-Fc-Bio结合，特异性良好。Negative phage control为阴性对照噬菌体抗体克隆，与靶抗原和对照抗原都不结合，anti-M13 phage mouse Ab/anti-mouse HRP Ab为只加第一抗体和第二抗体的阴性抗体对照，anti-mouse HRP Ab为只加第二抗体的阴性抗体对照，它们与靶抗原和对照抗原都不结合，Mouse anti human CD5 Ab/anti-mouse HRP Ab为靶抗原(CD5-Fc-Bio)的阳性抗体对照，与靶抗原结合，与对照抗原不结合。
与ELISA相对应的抗体克隆的FACS初筛结果如图10所示。其中H3，H4和H7克隆结合Jurkat，不结合Raji细胞，是特异克隆；其它克隆是阴性克隆(2种细胞都不结合)。
通过ELISA检测和FACS初筛，我们总共获得93个ELISA和FACS双阳性且特异性良好的克隆，随后我们将获得的93个双阳性且特异性良好的克隆测序，测序后得到了64种不同的单克隆序列，然后将这64个不同序列的单克隆进一步通过多细胞系的FACS鉴定和多种抗原的ELISA鉴定检测候选克隆的结合特异性。
实施例3.采用多个细胞系通过FACS鉴定单克隆特异性
实验目的和原理：
用于治疗的抗体必须具有非常好的靶点特异性，只结合靶抗原，而不结合任何无关的抗原；另一方面，不同的细胞系上同一抗原的氨基酸序列会有差异(异构体或突变体)或结合的配体不一样，也需要考察我们的抗体能否与各种靶蛋白阳性的细胞都结合。为了进一步分析这些单克隆的特异性和普适性，寻找最佳的候选克隆，我们通过流式细胞术进一步评估初筛克隆的特异性。在这个实验中，我们采用多种CD5阳性的细胞系和多种CD5阴性的细胞系与这些单克隆噬菌体抗体进行反应，分析这些克隆是否可以结合不同的细胞系上的CD5抗原，以及是否与其它不表达CD5的细胞系有任何非特异的结合。通过这个实验，我们获得了若干具有优良特异性的克隆。
实验方法：与FACS初筛相同；
主要样品和试剂：
Jurkat细胞系，CD5阳性细胞系；
Raji细胞系，CD5阴性细胞系；
CCRF-CEM细胞系,CD5阳性细胞系；
NALM6细胞系,CD5阴性细胞系；
其余试剂与FACS初筛相同。
实验结果：
用于治疗的抗体必须具有非常好的靶点特异性。为了进一步分析这些单克隆抗体的特异性，我们将实施例2获得的唯一特异性克隆在更多的抗原和细胞系上应用酶联免疫吸附和流式细胞术进行了鉴定。结果显示在图11A-F中，Negative Control为阴性对照噬菌体抗体克隆。#1～2，#4～12，#14～17，#19～22，#25～27，#30～39，#42～49，#51，#54～55，#58～62，#64号克隆与2种CD5阳性细胞系Jurkat和CCRF-CEM都结合，中位荧光强度(MFI)有强有弱，与2种CD5阴性细胞系Raji和NALM6都不结合，MFI低，特异性良好；#3，#13，#18，#23，#24，#28，#29，#40，#41，#50，#57和#63号克隆与阳性细胞系Jurkat结合很弱或者不结合，与阳性细胞系CCRF-CEM不结合，为阴性克隆；52和53号克隆只结合阳性细胞系Jurkat，不结合CCRF-CEM，说明其不能识别不同细胞系表达的不同构象或异构体的CD5抗原，不符合实验需要；56号克隆与两种阳性细胞系都能结合，但是与阴性细 胞系Raji有较弱的结合，说明其结合可能为非特异性结合，不符合实验需要。
实施例4.采用不同公司的抗原通过ELISA鉴定单克隆特异性
实验目的和原理：
用于治疗的抗体必须具有非常好的靶点特异性，只结合靶抗原，而不结合任何无关的抗原；另一方面，不同的公司生产的同一抗原的氨基酸序列会有差异(异构体或突变体)，也需要考察我们的抗体能否与各种靶蛋白都结合。为了进一步分析这些单克隆的特异性和普适性，寻找最佳的候选克隆，我们通过酶联免疫检测(ELISA)进一步评估初筛克隆的特异性。在这个实验中，我们采用购买自不同公司生产的CD5抗原和多种CD5不相关抗原与这些单克隆噬菌体抗体进行反应，分析这些克隆是否可以结合不同的CD5抗原，以及是否与其它CD5不相关抗原有任何非特异的结合。通过这个实验，我们获得了若干具有优良特异性的克隆。
实验方法：与ELISA初筛相同；
主要样品和试剂：
Figure PCTCN2021127995-appb-000002
其余试剂与ELISA初筛相同。
实验结果：
用于治疗的抗体必须具有非常好的靶点特异性。为了进一步分析这些单克隆抗体的特异性，我们将实施例2获得的多个克隆在多种抗原应用酶联免疫吸附(ELISA)进行了鉴定。结果显示在图12A-F中，Negative control为阴性对照噬菌体抗体克隆，与靶抗原和对照抗原都不结合，anti-M13 phage mouse Ab/anti-mouse HRP Ab为只加第一抗体和第二抗体 的阴性抗体对照，anti-mouse HRP Ab为只加第二抗体的阴性抗体对照，它们与靶抗原和对照抗原都不结合，Mouse anti human CD5 Ab/anti-mouse HRP Ab为靶抗原(CD5-Fc-Bio)的阳性抗体对照，与靶抗原结合，与对照抗原不结合。anti-human IgG-HRP Ab/anti-his-HRP Ab为检测抗原标签的阳性抗体对照，与含有Fc标签或者his标签的抗原结合，说明包被的抗原已经结合到酶标板上。#1～64号克隆与3种CD5抗原都结合，与3种非相关抗原都不结合，说明能够结合不同公司的CD5抗原且特异性良好。
下表列出了部分所选的抗CD5抗体的重链可变区(HCVR)、轻链可变区(LCVR)、ScFv和CDR序列对应的氨基酸及核酸序列的编号：
Figure PCTCN2021127995-appb-000003
实施例5.利用CRISPR/Cas9技术敲除T细胞表面的CD5抗原
(1)实验目的和原理：
成熟的T细胞表面均表达CD5抗原，而Mamonkin M等研究者开发的未进行CD5敲除的CD5 CAR-T被报道存在一定程度的自杀情况 1，在患者体内存续时间有限，极大地限制了患者疾病缓解时间和该CAR-T产品的应用。为了解决这一问题，本申请采用CRISPR/Cas9技术将T细胞表面的CD5抗原敲除，最大限度地减少CD5 CAR-T的自激活及自杀现象，保证其持续性和有效性。
为了筛选敲除效率高的sgRNA，我们首先采用不同的sgRNA对天然高表达CD5的jurkat细胞进行CD5基因敲除，从而确定效率高的sgRNA进行T细胞的CD5抗原敲除，获得敲除效率高的jurkat-CD5ko pool后，通过单克隆筛选，获得了CD5阴性的单克隆化的jurkat-CD5ko细胞。这样，jurkat/jurkat-CD5ko这一对细胞的差别主要在于这一个被敲除的基因，这对细胞将在单克隆抗体结合特异性鉴定以及CAR-T功能验证方面都有很重要的应用。
(2)简要实验步骤如下：
1)选择多种sgRNA序列(sgRNA1至sgRNA4)，并进行引物合成；
2)化学合成法合成sgRNA。
3)sgRNA/Cas9 RNP电转jurkat细胞；
4)FACS检测不同sgRNA的基因敲除效率，挑选敲除效率较高的jurkat-CD5ko pool，用有限稀释法分离单克隆；
5)单克隆的FACS鉴定；
6)jurkat-CD5ko单克隆细胞库构建及保存。
(3)主要材料和试剂：
化学合成EasyEdit sgRNA，南京金斯瑞生物科技有限公司
sgRNA1:aagcgtcaaaagtctgccag(SEQ ID NO：103，该序列以其靶序列形式提供，以下同)
sgRNA2:ccgttccaactcgaagtgcc(SEQ ID NO：104)
sgRNA3:gctgtagaactccaccacgc(SEQ ID NO：105)
sgRNA4:aatcatctgctacggacaac(SEQ ID NO：106)
TrueCut TM Cas9 Protein v2,thermo,A36498
APC Mouse Anti-Human CD5 antibody,Clone UCHT2,BD Pharmingen,555355
(4)实验结果：
如图1所示，sgRNA3对应的细胞CD5敲除效率最高，有95.46％的CD5被敲除。因此我们选择sgRNA3电转的jurkat-CD5ko pool进行单克隆的筛选。采用有限稀释法从该细胞池分离单克隆，等单克隆扩增起来后，通过FACS检测这些单克隆的CD5表达。结果如图2所示，其中jurkat-CD5ko-1,2号克隆的CD5表达基本检测不到，可以认为是敲除成功的单克隆。将这2个克隆进行培养冻存。我们在随后的研究中使用克隆1。
实施例6.CAR-T细胞的制备与检测
本申请采用慢病毒转染的方式使敲除后的T细胞表达CAR，CAR-T制备流程参见专利(Zhou J,Liu J,Hu G,et al.Chimeric antigen receptor(car)binding to bcma,and uses thereof:U.S.Patent Application 16/650,580[P].2020-8-6.)。慢病毒载体是以慢病毒的基因组为基础，将其中多个和病毒活性相关的序列结构去除，使其具有生物学的安全性，然后再在这个基因组骨架中引入实验所需要的目标基因的序列和表达结构制备成的载体。与其他逆转录病毒相比，慢病毒载体有着更广泛的宿主，对于分裂和非分裂细胞均具有感染能力，对于一些较难转染的细胞，如原代细胞、干细胞、不分化的细胞等，能大大提高目的基因的转导效率(参见陈琛和万海粟，“慢病毒载体及其研究进展，Chinese Journal of Lung Cancer 17.12(2014):870–876.PMC)。通过慢病毒载体转染，可将CAR分子整合进宿主细胞中。
T细胞进行CD5敲除并转染如图3A所示结构的CAR慢病毒(具体CAR分子结构如表2所示)，5～7天后检测CD5、EGFRt表达和CD5抗原结合情况(图3B)。CAR结构中 包含CD8α信号肽，scFv，CD8α铰链区，CD8α跨膜区，4-1BB共刺激分子和CD3ζ并用T2A连接一个截短的EFGR分子(EGFRt)可在临床转化时作为安全开关使用，且由于EGFRt与CAR分子共表达，所以可以作为CAR分子在T细胞表面分布的间接检测指标且不影响CAR的结构及功能。
表2.示例性CAR分子及其抗原识别部分
Figure PCTCN2021127995-appb-000004
Figure PCTCN2021127995-appb-000005
CAR-T/T细胞CD5抗原表达检测及EGFRt表达检测简要实验步骤如下：
1)取1×10 6CAR-T/T细胞每孔，加入PBS洗一遍，300g离心5分钟，弃上清。
2)用100μL PBS重悬细胞沉淀，分别加入5μL APC-CD5抗体及5μL PE-EGFR抗体，4℃避光孵育15分钟。
3)用PBS洗两遍，300g离心5分钟。
4)用200μL PBS重悬，流式上机检测。
主要材料和试剂：
APC Mouse Anti-Human CD5 antibody,Clone UCHT2,BD Pharmingen,Cat.No.555355；
PE anti-human EGFR Antibody,Clone AY13,BioLegend,Cat.No.352904；
胎牛血清(FBS),Gibco，Cat.No.10099141；
实验结果：
如图3B所示，第七天检测，在T细胞上CD5敲除效率可达86.6％，全部克隆CAR分子均可表达在T细胞表面，本申请研究的CAR-T针对CD5靶点，若CAR-T细胞功能良好，敲除并转染慢病毒后CAR-T细胞可杀伤仍然表达CD5抗原的CAR-T/T细胞。本次实验中，除Clone 8 CAR-T外，其他克隆的CAR-T细胞均可将未敲除CD5的细胞清除干净。
实施例7.CAR-T细胞的体外功能验证
实验目的和原理：
本申请将通过亲和淘选从噬菌体抗体库中富集、筛选和鉴定的多种靶向CD5蛋白的特异抗体克隆构建至二代CAR上，由于结合CD5抗原的scFv构建至CAR结构后未必具有 良好的激活功能，其在CAR-T细胞上的功能需要进一步确认并筛选出活性最好的CAR分子。为此，我们制备了这些CAR分子克隆的慢病毒载体，并转导T细胞制备成CAR-T细胞。然后，通过CD107a脱粒实验(CD107a degranulation assay)和体外细胞杀伤实验(in vitro cytotoxicity assay)进行CAR-T细胞的体外生物学效力评估。通过这些CAR-T水平的功能验证，最终筛选有效性和安全性都理想的CAR分子进行下游CAR-T产品开发。
CD107a脱粒实验
CD107a是细胞内微囊泡的标志物，当负载有颗粒酶的微囊泡与细胞膜融合后，细胞膜上的CD107a会增加，当用莫能酶素(monesin，购自BioLegend)阻断其回收时，可以定量反映微囊泡释放的强度。当CAR-T受到靶细胞上靶抗原刺激后，会造成颗粒酶释放，并可通过流式检测CD107a的增加来判断T细胞的激活情况。
CD107a脱粒简要实验步骤：
1)将待测的CAR-T细胞和靶细胞分别在室温下以300g离心5min，弃上清后，用1640培养基+10％FBS重悬为1x10 6个细胞/mL；
2)在24孔板中，分别加入500μL待测的CAR-T细胞和500μL靶细胞，并混匀；
3)在每孔细胞中加入5μL PE/Cy7 mouse anti-human CD107a抗体和1μLmonensin，然后放入细胞培养箱中(37℃,5％CO 2)孵育3h；
4)孵育完成后，从24孔板中取出500μL细胞悬液，在4℃下300g离心5min后弃上清，用1mL PBS洗细胞2次；
5)用100μLPBS重悬细胞，并分别加入5μL APC mouse anti-human CD8和5μL Alexa Fluor 488 anti-human EGFR抗体，混匀后在冰上避光孵育20min；
6)孵育完成后，用1mL PBS洗细胞3次；用400μLPBS重悬后，用流式细胞仪检测。主要样品和试剂：
靶细胞jurkat,jurkat CD5ko,CCRF-CEM,MOLT4,K562-CD5,K562,RAJI；
胎牛血清(FBS),Gibco，Cat.No.10099141；
Monensin,BioLegend,Cat.No.420701；
PE/Cy7 mouse anti-human CD107a,BD,Cat.No.561348；
APC mouse anti-human CD8,BD,Cat.No.555369；
Alexa Fluor 488 anti-human EGFR,BioLegend,Cat.No.352908。
实验结果：
通过慢病毒转导的方式获得CAR-T细胞，将该CAR-T细胞在体外培养9-12天后进行CD107a脱粒实验。待检测的CAR-T细胞和靶细胞、莫能酶素和CD107a抗体共同孵育4h，CAR-T细胞与靶细胞的细胞密度均为5×10 5个细胞/mL。然后用CD8抗体、EGFR抗体标记样品后，进行流式检测。在Flowjo软件中分析，散点图中选取活细胞门(P1)，去除细胞 碎片；在P1门中的细胞，经过分析选取单个分散细胞门(P2)；然后，在P2门中进一步选取CD8阳性的细胞(P3)；最后，在P3门中，分析EGFR抗体染色呈阳性的细胞(即CAR阳性细胞)中CD107a阳性的比例。分析结果如图4所示，提示Clone 8，Clone 9，Clone 10和Clone 32的CAR-T细胞具有比对照CAR-T细胞(H65 CAR-T细胞)强的CD107a脱粒功能，而Clone 6，Clone 7和Clone 35的CAR-T细胞与CD5-的细胞系RAJI细胞共孵育时存在一定的非特异激活，且由于上文实验中Clone 8的CAR-T细胞无法完全清除CD5+T细胞，故Clone 9，Clone 10和Clone 32 CAR-T细胞可作为靶细胞特异性激活细胞。
体外细胞杀伤实验
实验目的和原理：体外细胞杀伤实验采用CCRF-CEM和MOLT4作为CD5阳性靶细胞，K562和RAJI细胞作为CD5阴性靶细胞，进行CD5 CAR-T细胞的抗原特异性杀伤能力评价。其中，以上细胞分别通过慢病毒转导方式，获得稳定表达萤火虫荧光素酶的靶细胞，因此样品中荧光素酶的活性可以反映靶细胞的数量。将CAR-T细胞和靶细胞共孵育培养。当靶细胞被CAR-T细胞杀伤时，荧光素酶会被释放并且很快失活(萤火虫荧光素酶半衰期约0.5h)。如果靶细胞没有被CAR-T细胞杀伤或者抑制，随着靶细胞的扩增和荧光素酶的持续表达，将会产生更多的荧光素酶。因此，可以通过荧光素酶的活性来检测CAR-T对靶细胞的杀伤情况。
体外细胞杀伤简要实验步骤：
1)将上述细胞分别在室温下以300g离心5min，弃上清后，用T细胞培养基重悬为2x10 5个细胞/mL；在96孔板的每孔中分别加入100μL靶细胞；
2)根据待测CAR-T样品的CAR阳性率和效靶比，分别在96孔板的每孔中加入100μLCAR-T细胞，并和靶细胞混匀；然后放入二氧化碳培养箱中孵育培养24h；
3)使用荧光素酶检测试剂盒分别检测每孔样品中的荧光素酶活性。
主要样品和试剂：
靶细胞CCRF-CEM,MOLT4,K562,RAJI；
Steady-Glo Luciferase Assay System,Promega,Cat.No.E2520。
实验结果：
将CAR-T细胞样品和固定数量的靶细胞(1x10 4个)按照不同效靶比(E:T)混合后，共同孵育24h，然后检测样品中的荧光素酶活性(RLU)。其中，CD5ko T cells为只含有靶细胞的对照样品。由于荧光素酶活性可以反映靶细胞在样品中的数量，通过样品中荧光素酶活性的变化，可以得到CAR-T细胞对靶细胞的杀伤/抑制能力。荧光素酶活性读数(RLU)越低，靶细胞被杀伤的越多。
如图5所示，除Clone 8 CAR-T细胞以外的其他克隆CAR-T细胞样品，对阳性靶细胞的杀伤均强于对照H65 CAR-T细胞。当T细胞和阳性靶细胞共孵育时无明显杀伤。所有CAR-T和T细胞样品与阴性靶细胞共同孵育时都无明显杀伤。因此，Clone 6，clone 7，clone  9，clone 10，clone 32和clone 35的CAR-T样品都可以特异性地杀伤CD5阳性靶细胞，并且对CD5阴性靶细胞没有非特异性杀伤。
反复刺激增殖实验
实验目的和原理：
采用丝裂霉素(Mitomycin)处理过的靶细胞(CCRF-CEM)与不同组别CD5 CAR-T细胞混合进行4次刺激后将CAR-T细胞和靶细胞共孵育培养，从而确定不同scFv CAR-T在被靶细胞持续多次刺激后的增殖能力。
反复刺激后杀伤实验简要实验步骤：
1)取CCRF-CEM细胞4×10 6细胞，300g，室温，离心5min；
2)完全培养基调整密度至0.2×10 6细胞/mL，加入4μL Mitomycin母液(5μg/μL)混匀后培养24h后待用。
3)取处理24h后的CCRF-CEM-Mitomycin细胞，300g，离心换液，用PBS洗涤6次，CTS培养基重悬CCRF-CEM-Mitomycin细胞，并计数且调整密度至2×10 6细胞/mL，待用。
4)依据CAR-T CAR阳性率，分别取1×10 5 CAR+细胞，转入24孔板中；每孔CAR-T加入CCRF-CEM-Mitomycin细胞50μL，调整效靶比(效应细胞以CAR+计)E:T＝1：1。用CTS完全培养基补液至培养终体积至500μL，混匀，37℃，5％CO 2培养72h并计数，再次用Mitomycin处理靶细胞(CCRF-CEM)及检测CAR-T CAR阳性率，重复以上步骤4次后绘制扩增曲线。
实验结果：
如图6所示，5组CAR-T/T细胞样品反复刺激后，增殖能力：Clone 32 CAR-T>Clone 10 CAR-T>H65 CAR-T>Clone 35 CAR-T>CD5ko T cells，靶细胞刺激4次后，Clone 10,Clone32及H65的CAR-T细胞依然能够有效扩增。CAR-T细胞被靶细胞刺激后的增殖能力与患者长期预后密切相关，因此，Clone 32 CAR-T及Clone 10CAR-T可被认为具有在体内长期增殖并清除肿瘤细胞的潜能。
实施例8.抗CD5 scFvs亲和力的测定
实验目的和原理：
CD5 scFvs与抗原间的亲和力大小也可能对CAR-T在患者体内发挥杀伤作用及存续时间有着重要影响，为了确定这一重要性质，本申请采用了ForteBio公司的Octet分子相互作用技术对其进行了测定。Octet系统所运用的生物膜干涉技术，是一种免标记技术，实时提供高通量的生物分子相互作用信息。该仪器发射白光到传感器表面并收集反射光，不同频率的反射光谱受到生物传感器的光膜层厚度的影响，一些频率的反射光形成了相长干涉(蓝色)，而另一些受到了相消干涉(红色)。这些干涉被光谱仪所检测到，并形成一幅干涉光谱，并以干涉光谱的相位位移强度(nm)显示。因此，结合到传感器表面的分子一旦有数量 上的增减，光谱仪便会实时地检测到干涉光谱的位移，而这种位移直接反应出传感器表面生物膜的厚度，从中可以获取生物分子相互作用的高质量的数据，从而进行生物分子间相互作用动力学参数测定(Kon,Kdis和KD)，为研发过程提供重要的信息。
简要实验步骤：
1)用上样缓冲液(1×PBS，pH 7.4，0.01％BSA和0.02％Tween 20)将抗CD5 IgG稀释至20μg/mL，并在生物传感器上上样，约0.8nM。
2)在60s平衡阶段后，在多种抗原浓度(400至12.5nM)下监测CD5抗原的结合动力学。在每个浓度下分别进行至160s结合和300s解离。
3)用10mMGlycine-HCl,pH1.5洗涤3次使芯片再生。
4)通过使用1：1结合位点模型(Biacore X-100评估软件)分析结合常数。
实验结果：
亲和力系指单个分子与其配体结合的强度，通常通过平衡解离常数(KD)进行测定和报告，平衡解离常数可用于评估两分子间相互作用的强度并对此进行排序。抗体与其抗原的结合是一个可逆的过程，结合反应的速率与反应物的浓度成正比。KD值越小，抗体对其靶标的亲和力越大。如表3及图7所示，H65、Clone 10、Clone 32和Clone 35均可与CD5抗原结合，且Clone 10显示了与对照抗体H65相当的亲和力水平，其稍高于Clone 32和Clone 35。
表3抗CD5 IgG亲和力测定
Analyte KD(M) kon(1/Ms) kdis(1/s)
H65 2.25E-09 3.61E+05 8.13E-04
Clone 10 4.49E-09 3.18E+05 1.43E-03
Clone 32 2.83E-08 7.65E+04 2.17E-03
Clone 35 1.36E-08 2.81E+04 3.82E-04
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。
参考文献：
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2.Gill,S.,et al.,CD19 CAR-T cells combined with ibrutinib to induce complete remission in CLL.Journal of Clinical Oncology,2017.35(15_suppl):p.7509-7509.
3.Neelapu,S.S.,et al.,Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma.New England Journal of Medicine,2017.377(26):p.2531-2544.
4.Maude,S.L.,et al.,CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia.Blood,2015.125(26):p.4017.
5.Jacobson,C.A.,CD19Chimeric Antigen Receptor Therapy for Refractory Aggressive B-Cell Lymphoma.Journal of Clinical Oncology,2018.37(4):p.328-335.
6.Kochenderfer,J.,et al.,Anti-CD19chimeric antigen receptor T cells preceded by low-dose chemotherapy to induce remissions of advanced lymphoma.Journal of Clinical Oncology,2016.34(18_suppl):p.LBA3010-LBA3010.
7.Grupp,S.A.,et al.,Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia.New England Journal of Medicine,2013.368(16):p.1509-1518.
8.Hirayama,A.V.,et al.,The response to lymphodepletion impacts PFS in aggressive non-Hodgkin lymphoma patients treated with CD19 CAR-T cells.Blood,2019:p.blood-2018-11-887067.
9.Davila,M.L.,et al.,Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.Sci Transl Med,2014.6(224):p.224ra25.
10.Jones,N.H.,et al.,Isolation of complementary DNA clones encoding the human lymphocyte glycoprotein T1/Leu-1.Nature,1986.323(6086):p.346-349.
11.Huang,H.J.,et al.,Molecular cloning of Ly-1,a membrane glycoprotein of mouse T lymphocytes and a subset of B cells:molecular homology to its human counterpart Leu-1/T1(CD5).Proceedings of the National Academy of Sciences of the United States of America,1987.84(1):p.204-208.
12.Fleischer,L.C.,H.T.Spencer,and S.S.Raikar,Targeting T cell malignancies using CAR-based immunotherapy:challenges and potential solutions.Journal of Hematology&Oncology,2019.12(1):p.141.
13.Mamonkin,M.,et al.,A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies.Blood,2015.126(8):p.983-992.
本申请实施例中的具体序列如下：
CD8a signal核酸序列(SEQ ID NO:1)
Figure PCTCN2021127995-appb-000006
CD8a signal蛋白序列(SEQ ID NO:2)
Figure PCTCN2021127995-appb-000007
CD8a hinge核酸序列(SEQ ID NO:3)
Figure PCTCN2021127995-appb-000008
Figure PCTCN2021127995-appb-000009
CD8a hinge蛋白序列(SEQ ID NO:4)
Figure PCTCN2021127995-appb-000010
CD8a TM核酸序列(SEQ ID NO:5)
Figure PCTCN2021127995-appb-000011
CD8a TM蛋白序列(SEQ ID NO:6)
Figure PCTCN2021127995-appb-000012
4-1BB胞内结构域核酸序列(SEQ ID NO:7)
Figure PCTCN2021127995-appb-000013
4-1BB胞内结构域蛋白序列(SEQ ID NO:8)
Figure PCTCN2021127995-appb-000014
CD3ζ胞内信号结构域核酸序列(SEQ ID NO:9)
Figure PCTCN2021127995-appb-000015
CD3ζ胞内信号结构域蛋白序列(SEQ ID NO:10)
Figure PCTCN2021127995-appb-000016
剪切肽T2A核酸序列(SEQ ID NO:11)
Figure PCTCN2021127995-appb-000017
剪切肽T2A蛋白序列(SEQ ID NO:12)
Figure PCTCN2021127995-appb-000018
CSF2RA signal核酸序列(SEQ ID NO:13)
Figure PCTCN2021127995-appb-000019
CSF2RA signal蛋白序列(SEQ ID NO:14)
Figure PCTCN2021127995-appb-000020
tEGFR核酸序列(SEQ ID NO:15)
Figure PCTCN2021127995-appb-000021
tEGFR蛋白序列(SEQ ID NO:16)
Figure PCTCN2021127995-appb-000022
VH-VL连接肽核酸序列(SEQ ID NO:17)
Figure PCTCN2021127995-appb-000023
VH-VL连接肽蛋白序列(SEQ ID NO:18)
Figure PCTCN2021127995-appb-000024
CAR001核酸序列，2658bp，(SEQ ID NO:19)
Figure PCTCN2021127995-appb-000025
Figure PCTCN2021127995-appb-000026
CAR002核酸序列，2652bp，(SEQ ID NO:20)
Figure PCTCN2021127995-appb-000027
Figure PCTCN2021127995-appb-000028
Figure PCTCN2021127995-appb-000029
CAR003核酸序列，2664bp，(SEQ ID NO:21)
Figure PCTCN2021127995-appb-000030
Figure PCTCN2021127995-appb-000031
CAR001蛋白序列，886aa(SEQ ID NO:22)
Figure PCTCN2021127995-appb-000032
CAR002蛋白序列，884aa(SEQ ID NO:23)
Figure PCTCN2021127995-appb-000033
CAR003蛋白序列,888aa(SEQ ID NO:24)
Figure PCTCN2021127995-appb-000034
Scfv10核酸序列(SEQ ID NO:25)
Figure PCTCN2021127995-appb-000035
10VL(即CAR001中的Scfv10的VL)核酸序列：339bp(SEQ ID NO:26)
Figure PCTCN2021127995-appb-000036
10VH(即CAR001中的Scfv10的VH)核酸序列：354bp(SEQ ID NO:27)
Figure PCTCN2021127995-appb-000037
Figure PCTCN2021127995-appb-000038
Scfv10蛋白序列(SEQ ID NO:28)
Figure PCTCN2021127995-appb-000039
10VL(即CAR001中的Scfv10的VL)蛋白序列：(SEQ ID NO:29)
Figure PCTCN2021127995-appb-000040
10VH(即CAR001中的Scfv10的VH)蛋白序列：(SEQ ID NO:30)
Figure PCTCN2021127995-appb-000041
Scfv32核酸序列(SEQ ID NO:31)
Figure PCTCN2021127995-appb-000042
32VL(即CAR002中的Scfv32的VL)核酸序列：333bp(SEQ ID NO:32)
Figure PCTCN2021127995-appb-000043
32VH(即CAR002中的Scfv32的VH)核酸序列：354bp(SEQ ID NO:33)
Figure PCTCN2021127995-appb-000044
Scfv32蛋白序列(SEQ ID NO:34)
Figure PCTCN2021127995-appb-000045
32VL(即CAR002中的Scfv32的VL)蛋白序列：111aa(SEQ ID NO:35)
Figure PCTCN2021127995-appb-000046
32VH(即CAR002中的Scfv32的VH)蛋白序列：118aa(SEQ ID NO：36)
Figure PCTCN2021127995-appb-000047
Scfv35核酸序列(SEQ ID NO:37)
Figure PCTCN2021127995-appb-000048
35VL(即CAR003中的Scfv35的VL)核酸序列：333bp(SEQ ID NO:38)
Figure PCTCN2021127995-appb-000049
Figure PCTCN2021127995-appb-000050
35VH(即CAR003中的Scfv35的VH)核酸序列：366bp(SEQ ID NO:39)
Figure PCTCN2021127995-appb-000051
Scfv35蛋白序列(SEQ ID NO:40)
Figure PCTCN2021127995-appb-000052
35VL(即CAR003中的Scfv35的VL)蛋白序列：111aa(SEQ ID NO:41)
Figure PCTCN2021127995-appb-000053
35VH(即CAR003中的Scfv35的VH)蛋白序列：122aa(SEQ ID NO:42)
Figure PCTCN2021127995-appb-000054
CAR001中的Scfv10(clone 10)抗体的CDRs：
HCDR1的氨基酸序列为GFTFNNYT(SEQ ID NO:43)
HCDR2的氨基酸序列为ISSSSSYI(SEQ ID NO:44)
HCDR3的氨基酸序列为ARYFSGSAGDY(SEQ ID NO:45)
LCDR1的氨基酸序列为SSNIGAGYD(SEQ ID NO:46)
LCDR2的氨基酸序列为GNI(SEQ ID NO:47)
LCDR3的氨基酸序列为GTWDNSLSAHYV(SEQ ID NO:48)
CAR002中的Scfv32(clone 32)抗体的CDRs：
HCDR1的氨基酸序列为GFTFSSYS(SEQ ID NO:49)
HCDR2的氨基酸序列为ISSSSSYI(SEQ ID NO:50)
HCDR3的氨基酸序列为ARYFSGSAGDY(SEQ ID NO:51)
LCDR1的氨基酸序列为NSNIGNNY(SEQ ID NO:52)
LCDR2的氨基酸序列为DNN(SEQ ID NO:53)
LCDR3的氨基酸序列为GIWDSSLDAYV(SEQ ID NO:54)
CAR003中的Scfv35(clone 35)抗体的CDRs：
HCDR1的氨基酸序列为GFTFSSYA(SEQ ID NO:55)
HCDR2的氨基酸序列为ITDSGDIT(SEQ ID NO:56)
HCDR3的氨基酸序列为ARMSSHWYFSADY(SEQ ID NO:57)
LCDR1的氨基酸序列为SSNIGNNY(SEQ ID NO:58)
LCDR2的氨基酸序列为DNN(SEQ ID NO:59)
LCDR3的氨基酸序列为GTWDSSLSAVV(SEQ ID NO:60)
CAR004中的Scfv6(clone6)抗体的CDRs：
HCDR1的氨基酸序列为GGTFSSNA(SEQ ID NO:61)
HCDR2的氨基酸序列为IIPMFGTT(SEQ ID NO:62)
HCDR3的氨基酸序列为ARDGGGRHPYYYYGMDV(SEQ ID NO:63)
LCDR1的氨基酸序列为SGSIARNY(SEQ ID NO:64)
LCDR2的氨基酸序列为EDN(SEQ ID NO:65)
LCDR3的氨基酸序列为QSYDDNTSWV(SEQ ID NO:66)
CAR005中的Scfv7(clone 7)抗体的CDRs：
HCDR1的氨基酸序列为GYSFTSYW(SEQ ID NO:67)
HCDR2的氨基酸序列为IYPDDSDT(SEQ ID NO:68)
HCDR3的氨基酸序列为ARMSLTSYLATDG(SEQ ID NO:69)
LCDR1的氨基酸序列为SSNIGNND(SEQ ID NO:70)
LCDR2的氨基酸序列为DND(SEQ ID NO:71)
LCDR3的氨基酸序列为AVWDSSLSAAV(SEQ ID NO:72)
CAR006中的Scfv9(clone 9)抗体的CDRs：
HCDR1的氨基酸序列为TYTFTNYY(SEQ ID NO:73)
HCDR2的氨基酸序列为INPSGDTT(SEQ ID NO:74)
HCDR3的氨基酸序列为ARLSWYWGGSFDD(SEQ ID NO:75)
LCDR1的氨基酸序列为SDDIGNYKR(SEQ ID NO:76)
LCDR2的氨基酸序列为EVT(SEQ ID NO:77)
LCDR3的氨基酸序列为SSYASGDTYV(SEQ ID NO:78)
ScFv6 DNA序列：(SEQ ID NO:79)
Figure PCTCN2021127995-appb-000055
10VL(即CAR004中的Scfv6的VL)DNA序列：(SEQ ID NO:80)
Figure PCTCN2021127995-appb-000056
6VH(即CAR004中的Scfv6的VH)DNA序列(SEQ ID NO:81)
Figure PCTCN2021127995-appb-000057
ScFv7 DNA序列(SEQ ID NO:82)
Figure PCTCN2021127995-appb-000058
7VL(即CAR005中的Scfv7的VL)DNA序列(SEQ ID NO:83)
Figure PCTCN2021127995-appb-000059
7VH(即CAR005中的Scfv7的VH)DNA序列(SEQ ID NO:84)
Figure PCTCN2021127995-appb-000060
ScFv9 DNA序列(SEQ ID NO:85)
Figure PCTCN2021127995-appb-000061
9VL(即CAR006中的Scfv9的VL)DNA序列(SEQ ID NO:86)
Figure PCTCN2021127995-appb-000062
9VH(即CAR006中的Scfv9的VH)DNA序列(SEQ ID NO:87)
Figure PCTCN2021127995-appb-000063
Figure PCTCN2021127995-appb-000064
ScFv6氨基酸序列(SEQ ID NO:88)
Figure PCTCN2021127995-appb-000065
6VL(即CAR004中的Scfv6的VL)氨基酸序列(SEQ ID NO:89)
Figure PCTCN2021127995-appb-000066
6VH(即CAR004中的Scfv6的VH)氨基酸序列(SEQ ID NO:90)
Figure PCTCN2021127995-appb-000067
ScFv 7氨基酸序列(SEQ ID NO:91)
Figure PCTCN2021127995-appb-000068
7VL(即CAR005中的Scfv7的VL)氨基酸序列(SEQ ID NO:92)
Figure PCTCN2021127995-appb-000069
7VH(即CAR005中的Scfv7的VH)氨基酸序列(SEQ ID NO:93)
Figure PCTCN2021127995-appb-000070
ScFv9氨基酸序列(SEQ ID NO:94)
Figure PCTCN2021127995-appb-000071
9VL(即CAR006中的Scfv9的VL)氨基酸序列(SEQ ID NO:95)
Figure PCTCN2021127995-appb-000072
9VH(即CAR006中的Scfv9的VH)氨基酸序列(SEQ ID NO:96)
Figure PCTCN2021127995-appb-000073
CAR004核酸序列(SEQ ID NO:97)
Figure PCTCN2021127995-appb-000074
Figure PCTCN2021127995-appb-000075
CAR004氨基酸序列(SEQ ID NO:98)
Figure PCTCN2021127995-appb-000076
CAR005核酸序列(SEQ ID NO:99)
Figure PCTCN2021127995-appb-000077
Figure PCTCN2021127995-appb-000078
CAR005氨基酸序列(SEQ ID NO:100)
Figure PCTCN2021127995-appb-000079
Figure PCTCN2021127995-appb-000080
CAR006核酸序列(SEQ ID NO:101)
Figure PCTCN2021127995-appb-000081
Figure PCTCN2021127995-appb-000082
CAR006氨基酸序列(SEQ ID NO:102)
Figure PCTCN2021127995-appb-000083

Claims (54)

  1. 嵌合抗原受体(CAR)，其中所述CAR包含CD5结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，所述CD5结合结构域包含特异性结合CD5的抗体或其片段，其中所述抗体包含重链互补决定区1(HCDR1)，重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，所述HCDR1、HCDR2、HCDR3的氨基酸序列选自以下组合：
    (1)如SEQ ID NO:43的氨基酸序列的HCDR1，如SEQ ID NO:44的氨基酸序列的HCDR2，和如SEQ ID NO:45的氨基酸序列的HCDR3；
    (2)如SEQ ID NO:49的氨基酸序列的HCDR1，如SEQ ID NO:50的氨基酸序列的HCDR2，和如SEQ ID NO:51的氨基酸序列的HCDR3；
    (3)如SEQ ID NO:55的氨基酸序列的HCDR1，如SEQ ID NO:56的氨基酸序列的HCDR2，和如SEQ ID NO:57的氨基酸序列的HCDR3；
    (4)如SEQ ID NO:61的氨基酸序列的HCDR1，如SEQ ID NO:62的氨基酸序列的HCDR2，和如SEQ ID NO:63的氨基酸序列的HCDR3；
    (5)如SEQ ID NO:67的氨基酸序列的HCDR1，如SEQ ID NO:68的氨基酸序列的HCDR2，和如SEQ ID NO:69的氨基酸序列的HCDR3；或
    (6)如SEQ ID NO:73的氨基酸序列的HCDR1，如SEQ ID NO:74的氨基酸序列的HCDR2，和如SEQ ID NO:75的氨基酸序列的HCDR3。
  2. 根据权利要求1所述的CAR，其中所述抗体还包含轻链互补决定区1(LCDR1)，轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，所述LCDR1、LCDR2、LCDR3的氨基酸序列选自以下组合：
    (1)如SEQ ID NO:46的氨基酸序列的LCDR1，如SEQ ID NO:47的氨基酸序列的LCDR2，和如SEQ ID NO:48的氨基酸序列的LCDR3；
    (2)如SEQ ID NO:52的氨基酸序列的LCDR1，如SEQ ID NO:53的氨基酸序列的LCDR2，和如SEQ ID NO:54的氨基酸序列的LCDR3；
    (3)如SEQ ID NO:58的氨基酸序列的LCDR1，如SEQ ID NO:59的氨基酸序列的LCDR2，和如SEQ ID NO:60的氨基酸序列的LCDR3；
    (4)如SEQ ID NO:64的氨基酸序列的LCDR1，如SEQ ID NO:65的氨基酸序列的LCDR2，和如SEQ ID NO:66的氨基酸序列的LCDR3；
    (5)如SEQ ID NO:70的氨基酸序列的LCDR1，如SEQ ID NO:71的氨基酸序列的LCDR2，和如SEQ ID NO:72的氨基酸序列的LCDR3；或
    (6)如SEQ ID NO:76的氨基酸序列的LCDR1，如SEQ ID NO:77的氨基酸序列的LCDR2， 和如SEQ ID NO:78的氨基酸序列的LCDR3。
  3. 根据权利要求1-2中任一项所述的CAR，其中所述抗体包含的HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3选自以下组合的任一种：
    (1)如SEQ ID NO:43的氨基酸序列的HCDR1，如SEQ ID NO:44的氨基酸序列的HCDR2，和如SEQ ID NO:45的氨基酸序列的HCDR3，如SEQ ID NO:46的氨基酸序列的LCDR1，如SEQ ID NO:47的氨基酸序列的LCDR2，和如SEQ ID NO:48的氨基酸序列的LCDR3；
    (2)如SEQ ID NO:49的氨基酸序列的HCDR1，如SEQ ID NO:50的氨基酸序列的HCDR2，和如SEQ ID NO:51的氨基酸序列的HCDR3，如SEQ ID NO:52的氨基酸序列的LCDR1，如SEQ ID NO:53的氨基酸序列的LCDR2，和如SEQ ID NO:54的氨基酸序列的LCDR3；
    (3)如SEQ ID NO:55的氨基酸序列的HCDR1，如SEQ ID NO:56的氨基酸序列的HCDR2，和如SEQ ID NO:57的氨基酸序列的HCDR3，如SEQ ID NO:58的氨基酸序列的LCDR1，如SEQ ID NO:59的氨基酸序列的LCDR2，和如SEQ ID NO:60的氨基酸序列的LCDR3。
    (4)如SEQ ID NO:61的氨基酸序列的HCDR1，如SEQ ID NO:62的氨基酸序列的HCDR2，和如SEQ ID NO:63的氨基酸序列的HCDR3，如SEQ ID NO:64的氨基酸序列的LCDR1，如SEQ ID NO:65的氨基酸序列的LCDR2，和如SEQ ID NO:66的氨基酸序列的LCDR3；
    (5)如SEQ ID NO:67的氨基酸序列的HCDR1，如SEQ ID NO:68的氨基酸序列的HCDR2，和如SEQ ID NO:69的氨基酸序列的HCDR3，如SEQ ID NO:70的氨基酸序列的LCDR1，如SEQ ID NO:71的氨基酸序列的LCDR2，和如SEQ ID NO:72的氨基酸序列的LCDR3；或
    (6)如SEQ ID NO:73的氨基酸序列的HCDR1，如SEQ ID NO:74的氨基酸序列的HCDR2，和如SEQ ID NO:75的氨基酸序列的HCDR3，如SEQ ID NO:76的氨基酸序列的LCDR1，如SEQ ID NO:77的氨基酸序列的LCDR2，和如SEQ ID NO:78的氨基酸序列的LCDR3。
  4. 根据权利要求1-3中任一项所述的CAR，其中所述抗体包含重链可变区，所述重链可变区的氨基酸序列如SEQ ID NO：30、36、42、90、93或96所示。
  5. 根据权利要求1-4中任一项所述的CAR，其中所述抗体包含轻链可变区，所述轻链可变区的氨基酸序列如SEQ ID NO：29、35、41、89、92或95所示。
  6. 根据权利要求1-5中任一项所述的CAR，其中所述抗体为单链抗体。
  7. 根据权利要求1-6中任一项所述的CAR，其中所述抗体包含SEQ ID NO:28、34、40、88、91或94所示的氨基酸序列或其功能性变体。
  8. 根据权利要求1-7中任一项所述的CAR，其中所述跨膜结构域包含来自选自下述蛋白的多肽：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8a、CD9、CD16、 CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  9. 根据权利要求1-8中任一项所述的CAR，其中所述跨膜结构域包含SEQ ID NO:6所示的氨基酸序列或其功能性变体。
  10. 根据权利要求1-9中任一项所述的CAR，其中所述共刺激结构域包含选自下述蛋白的多肽：CD28、4-1BB、OX-40和ICOS。
  11. 根据权利要求1-10中任一项所述的CAR，其中所述共刺激结构域包含SEQ ID NO:8所示的氨基酸序列或其功能性变体。
  12. 根据权利要求1-11中任一项所述的CAR，其中所述胞内信号传导结构域包含来自CD3ζ的信号传导结构域。
  13. 根据权利要求1-12中任一项所述的CAR，其中所述胞内信号传导结构域包含SEQ ID NO:10所示的氨基酸序列或其功能性变体。
  14. 根据权利要求1-13中任一项所述的CAR，其中所述CAR还包含铰链区，所述铰链区连接所述抗体和所述跨膜结构域。
  15. 根据权利要求14所述的CAR，其中所述铰链区包含SEQ ID NO:4所示的氨基酸序列或其功能性变体。
  16. 根据权利要求1-15中任一项所述的CAR，其中所述CAR还连接CD8α信号肽。
  17. 根据权利要求16所述的CAR，其中所述信号肽包含SEQ ID NO:2所示的氨基酸序列或其功能性变体。
  18. 根据权利要求1-17中任一项所述的CAR，其中所述CAR还连接剪切肽。
  19. 根据权利要求18所述的CAR，其中所述剪切肽包含来自T2A肽的氨基酸序列。
  20. 根据权利要求18-19中任一项所述的CAR，其中所述剪切肽包含SEQ ID NO:12所示的氨基酸序列或其功能性变体。
  21. 根据权利要求1-20中任一项所述的CAR，其中所述CAR通过接剪切肽和CSF2RA信号肽还连接一段截短形式的EGFR分子(tEGFR)。
  22. 根据权利要求21所述的CAR，其中所述CSF2RA信号肽包含SEQ ID NO:14所示的氨基酸序列或其功能性变体，所述截短形式的EGFR分子包含SEQ ID NO:16所示的氨基酸序列或其功能性变体。
  23. 根据权利要求1-22中任一项所述的CAR，其包含选自SEQ ID NO:22-24、98、100或102的氨基酸序列或其功能性变体。
  24. 分离的核酸分子，其编码权利要求1-23中任一项所述的CAR。
  25. 根据权利要求24所述的编码CAR的分离的核酸分子，其包含选自SEQ ID NO:1、3、5、7、9、11、13、15、17、19-21、25-27、31-33、37-39、79－87、97、99或101的核酸序列或其功能性变体。
  26. 一种载体，其包含权利要求24-25中任一项所述的核酸分子。
  27. 根据权利要求26所述的载体，其中所述载体选自质粒、逆转录病毒载体和慢病毒载体。
  28. 一种免疫效应细胞，其包含权利要求1-23中任一项所述的CAR，权利要求24-25中任一项所述的核酸分子，或权利要求26-27中任一项所述的载体。
  29. 根据权利要求28所述的细胞，其中所述免疫效应细胞上的CD5不表达。
  30. 根据权利要求28-29中任一项所述的细胞，其中所述免疫效应细胞选自T淋巴细胞和自然杀伤(NK)细胞。
  31. 一种制备免疫效应细胞的方法，其包括敲除所述免疫效应细胞的CD5基因，并向免疫效应细胞中引入权利要求26或27所述的载体；优选地，所述敲除采用SEQ ID NO：103-106任一项的sgRNA进行。
  32. 药物组合物，其包含权利要求28-30中任一项所述的免疫效应细胞以及药学上可接受的佐剂。
  33. 权利要求1-23中任一项所述的CAR，权利要求24-25中任一项所述的核酸分子，权利要求26-27所述的载体，或权利要求28-30中任一项所述的免疫效应细胞在制备药物中的用途，其中所述药物用于治疗与CD5的表达相关的疾病或病症。
  34. 一种治疗与CD5的表达相关的疾病或病症的方法，其包括向有需要的患者施用治疗有效量的权利要求28-30中任一项所述的免疫效应细胞，或权利要求32所述的药物组合物。
  35. 如权利要求34所述的方法，其还包括进一步向有需要的患者施用EGFR抗体来抑制所述免疫效应细胞或药物组合物的效果。
  36. 根据权利要求33所述的用途或权利要求34-35中任一项所述的方法，其中所述与CD5的表达相关的疾病或病症为癌症或恶性肿瘤。
  37. 靶向CD5的全人源抗体或其单链抗体或片段，其中所述全人源抗体包括重链可变区(HCVR)和/或轻链可变区(LCVR)，重链可变区包括HCDR1、HCDR2和HCDR3，轻链可变区包括LCDR1、LCDR2和LCDR3，所述HCDR1、HCDR2、HCDR3和/或LCDR1、LCDR2和LCDR3选自如下组合之一：
    (1)LCDR1的氨基酸序列为SGSIARNY(SEQ ID NO：64)；
    LCDR2的氨基酸序列为EDN(SEQ ID NO：65)；
    LCDR3的氨基酸序列为QSYDDNTSWV(SEQ ID NO：66)；
    HCDR1的氨基酸序列为GGTFSSNA(SEQ ID NO：61)；
    HCDR2的氨基酸序列为IIPMFGTT(SEQ ID NO：62)；
    HCDR3的氨基酸序列为ARDGGGRHPYYYYGMDV(SEQ ID NO：63)；
    (2)LCDR1的氨基酸序列为SSNIGNND(SEQ ID NO：71)；
    LCDR2的氨基酸序列为DND(SEQ ID NO：72)；
    LCDR3的氨基酸序列为AVWDSSLSAAV(SEQ ID NO：73)；
    HCDR1的氨基酸序列为GYSFTSYW(SEQ ID NO：67)；
    HCDR2的氨基酸序列为IYPDDSDT(SEQ ID NO：68)；
    HCDR3的氨基酸序列为ARMSLTSYLATDG(SEQ ID NO：69)；
    (3)LCDR1的氨基酸序列为SDDIGNYKR(SEQ ID NO：76)；
    LCDR2的氨基酸序列为EVT(SEQ ID NO：77)；
    LCDR3的氨基酸序列为SSYASGDTYV(SEQ ID NO：78)；
    HCDR1的氨基酸序列为TYTFTNYY(SEQ ID NO：73)；
    HCDR2的氨基酸序列为INPSGDTT(SEQ ID NO：74)；
    HCDR3的氨基酸序列为ARLSWYWGGSFDD(SEQ ID NO：75)；
    (4)LCDR1的氨基酸序列为SSNIGAGYD(SEQ ID NO：46)；
    LCDR2的氨基酸序列为GNI(SEQ ID NO：47)；
    LCDR3的氨基酸序列为GTWDNSLSAHYV(SEQ ID NO：48)；
    HCDR1的氨基酸序列为GFTFNNYT(SEQ ID NO：43)；
    HCDR2的氨基酸序列为ISSSSSYI(SEQ ID NO：44)；
    HCDR3的氨基酸序列为ARYFSGSAGDY(SEQ ID NO：45)；
    (5)LCDR1的氨基酸序列为NSNIGNNY(SEQ ID NO：52)；
    LCDR2的氨基酸序列为DNN(SEQ ID NO：53)；
    LCDR3的氨基酸序列为GIWDSSLDAYV(SEQ ID NO：54)；
    HCDR1的氨基酸序列为GFTFSSYS(SEQ ID NO：49)；
    HCDR2的氨基酸序列为ISSSSSYI(SEQ ID NO：50)；
    HCDR3的氨基酸序列为ARGNPWYGVDY(SEQ ID NO：51)；
    (6)LCDR1的氨基酸序列为SSNIGNNY(SEQ ID NO：58)；
    LCDR2的氨基酸序列为DNN(SEQ ID NO：59)；
    LCDR3的氨基酸序列为GTWDSSLSAVV(SEQ ID NO：60)；
    HCDR1的氨基酸序列为GFTFSSYA(SEQ ID NO：55)；
    HCDR2的氨基酸序列为ITDSGDIT(SEQ ID NO：56)；
    HCDR3的氨基酸序列为ARMSSHWYFSADY(SEQ ID NO：57)；或
    所述全人源抗体包括(1)-(6)任一项中的CDR序列组合的变体，其中所述变体(1) -(6)任一项中的CDR序列相比，具有至少90％的序列一致性，或在CDR序列上共包含至少1个且不超过10，或不超过5、4、3、2或1个氨基酸改变。
  38. 如权利要求37所述的全人源抗体或其单链抗体或片段，其中所述重链可变区和/或轻链可变区的氨基酸序列选自如下任一组合：
    (1)SEQ ID NO：30所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：29所示序列或与其有至少90％序列一致性的轻链可变区序列；
    (2)SEQ ID NO：36所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：35所示序列或与其有至少90％序列一致性的轻链可变区序列；
    (3)SEQ ID NO：42所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：41所示序列或与其有至少90％序列一致性的轻链可变区序列；
    (4)SEQ ID NO：90所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：89所示序列或与其有至少90％序列一致性的轻链可变区序列；
    (5)SEQ ID NO：93所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：92所示序列或与其有至少90％序列一致性的轻链可变区序列；
    (6)SEQ ID NO：96所示序列或与其有至少90％序列一致性的重链可变区序列，和SEQ ID NO：95所示序列或与其有至少90％序列一致性的轻链可变区序列。
  39. 如权利要求37或38所述的全人源抗体或其单链抗体或片段，其中所述重链可变区和/或轻链可变区的氨基酸序列选自如下任一组合：
    (1)SEQ ID NO：30所示的重链可变区序列，和SEQ ID NO：29所示的轻链可变区序列；
    (2)SEQ ID NO：36所示的重链可变区序列，和SEQ ID NO：35所示的轻链可变区序列；
    (3)SEQ ID NO：42所示的重链可变区序列，和SEQ ID NO：41所示的轻链可变区序列；
    (4)SEQ ID NO：90所示的重链可变区序列，和SEQ ID NO：89所示的轻链可变区序列；
    (5)SEQ ID NO：93所示的重链可变区序列，和SEQ ID NO：92所示的轻链可变区序列；
    (6)SEQ ID NO：96所示的重链可变区序列，和SEQ ID NO：95所示的轻链可变区序列。
  40. 如权利要求37-39任一项所述的全人源抗体或其单链抗体或片段，其中，所述全人源抗体包括SEQ ID NO：28、34、40、88、91或94所示的氨基酸序列。
  41. 编码权利要求37-40任一项的全人源抗体或其单链抗体或片段的核酸分子。
  42. 如权利要求41所述的核酸分子，其包括SEQ ID NO:25-27、31-33、37-39、和79－87中任一项的核苷酸序列。
  43. 一种表达载体，其包括权利要求41或42所述的核酸分子。
  44. 一种宿主细胞，其包括权利要求43所述的表达载体。
  45. 一种药物组合物，其包括权利要求37-40任一项的全人源抗体或其单链抗体或片段，以及药学上可接受的载体或稀释剂。
  46. 一种治疗疾病或病症的方法，所述方法包括通过向有需要的患者施用治疗有效量的权利要求37-40任一项所述的全人源抗体或其单链抗体或片段，权利要求44所述的宿主细胞， 或权利要求45所述的药物组合物，来消除、抑制或降低CD5活性，从而预防、减轻、改善或抑制疾病或病症。
  47. 如权利要求46所述的方法，其中所述疾病或病症为癌症。
  48. 如权利要求47所述的方法，所述癌症为T细胞恶性肿瘤。
  49. 与权利要求37-40任一项的全人源抗体或其单链抗体或片段竞争相同表位的抗体或片段。
  50. 一种试剂盒，用于检测样品中CD5蛋白，其中，所述试剂盒包括权利要求37-40任一项所述的全人源抗体或其单链抗体或片段。
  51. 权利要求37-40任一项所述的全人源抗体或其单链抗体或片段或权利要求44所述的宿主细胞在制备用于消除、抑制或降低CD5活性，从而预防、减轻、改善或抑制疾病或病症的药物中的用途。
  52. 如权利要求51所述的用途，其中所述疾病或病症为癌症。
  53. 如权利要求52所述的用途，其中所述癌症为恶性T细胞肿瘤或恶性B细胞肿瘤。
  54. 如权利要求53所述的方法，其中所述恶性T细胞肿瘤选自T细胞急性淋巴细胞白血病(T-ALL)和T细胞淋巴瘤(TCL)，所述恶性B细胞肿瘤选自慢性淋巴细胞白血病(B-CLL)和套细胞淋巴瘤(B-MCL)。
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