WO2018045811A1 - 融合蛋白及其应用 - Google Patents

融合蛋白及其应用 Download PDF

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WO2018045811A1
WO2018045811A1 PCT/CN2017/092108 CN2017092108W WO2018045811A1 WO 2018045811 A1 WO2018045811 A1 WO 2018045811A1 CN 2017092108 W CN2017092108 W CN 2017092108W WO 2018045811 A1 WO2018045811 A1 WO 2018045811A1
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WIPO (PCT)
Prior art keywords
immune effector
seq
endocytic
cells
amino acid
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PCT/CN2017/092108
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English (en)
French (fr)
Inventor
李宗海
吴秀奇
王华茂
蒋华
石必枝
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科济生物医药（上海）有限公司
上海市肿瘤研究所
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Application filed by 科济生物医药（上海）有限公司, 上海市肿瘤研究所 filed Critical 科济生物医药（上海）有限公司
Priority to US16/331,786 priority Critical patent/US20190359989A1/en
Priority to GB1904563.2A priority patent/GB2570063B/en
Publication of WO2018045811A1 publication Critical patent/WO2018045811A1/zh

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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to the field of immunotherapy. More specifically, the present invention relates to a fusion protein for controlling chimeric antigen receptor immune effector cells or TCR-T cells and uses thereof.
  • the safety switches currently used in cell therapy mainly include two forms: suicide genes and marker genes.
  • the suicide genes mainly include herpes simplex virus thymidine kinase (HSV-TK) and inducible cysteine-containing aspartate proteolytic enzyme 9 (iCasp9).
  • HSV-TK herpes simplex virus thymidine kinase
  • iCasp9 inducible cysteine-containing aspartate proteolytic enzyme 9
  • the HSV-TK suicide gene greatly enhances the sensitivity of T cells to ganciclovir by expressing HSV-TK on T cells.
  • iCasp9 induces apoptosis of T cells expressing the iCasp9 suicide gene by applying a small molecule drug (AP20187) in the patient. Only AP20187 has not been commercialized, which limits the popularity of iCasp9 suicide gene.
  • Marker genes enable T cells to be sorted, detected, and cleared by expressing a specific marker on the surface of T cells that can be recognized by antibodies.
  • Hum Gene Ther, 11(4): 611-20 reports the expression of the CD20 receptor on the surface of T cells, allowing T cells to be recognized and killed by anti-CD20 monoclonal antibodies; Blood, 118(5): 1255-1263 A truncated EGFR receptor capable of being recognized by an anti-EGFR monoclonal antibody was co-expressed on CAR-T cells.
  • marker genes broadens the range of applications for safety switches, but its killing effect is often complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity). , ADCC) mediated, its killing effect depends on the complement system and the activity of NK cells in vivo. When a patient's body complement system or NK cell activity is defective, its killing ability is often limited. These shortcomings limit the application of these marker genes.
  • the object of the present invention is to provide an immune effector cell expressing a chimeric antigen receptor, wherein the surface of the immune effector cell simultaneously expresses a fusion protein, by which the immune effect can be conjugated by a specific antibody.
  • the cells are highly effective in killing.
  • the present invention provides an immune effector cell which expresses a chimeric antigen receptor on the surface, the immune cell further expressing the fusion protein of Expression I,
  • Z is an optional signal peptide
  • A is an antibody binding region
  • L is an optional joint portion
  • B is the endocytic functional area.
  • the present invention also provides such an immune effector cell expressing a chimeric antigen receptor, the immune cell further expressing a fusion protein comprising an antibody binding region and an endocytic functional region.
  • the antibody binding region is a polypeptide that is absent in normal cells, or is in a concealed state in normal cells, or is low expressed in normal cells.
  • the antibody binding region is selected from the group consisting of EGFRvIII, EGFR, CD20, CD22, CD19, BCMA, proBDNF precursor protein, GPC3, CLD18.2, CLD6, mesothelin, PD-L1, PD-1, WT-1, IL13Ra2, Her-2, Her-1, Her-3;
  • the antibody binding region comprises any one of the following amino acid sequences or contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and the following amino acid sequence, Or 99% identity amino acid sequence: SEQ ID NO: 28, 29, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43;
  • the antibody binding region comprises an active fragment of any of the following amino acid sequences: SEQ ID NO: 28, 29, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43.
  • the antibody binding region specifically binds to an EGFR antibody.
  • the extracellular portion of the chimeric antigen receptor does not have binding ability to the fusion protein.
  • the endocytic domain is derived from a folate receptor, LDL, CD30, CD33, CD3, EGFR, TFR1; preferably derived from a folate receptor and CD30; more preferably, the endocytic domain Having the amino acid sequence set forth in SEQ ID NO: 32 or 44, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 32 or 44 Or an amino acid sequence of 99% identity, or an active fragment of the amino acid sequence set forth in SEQ ID NO: 32 or 44.
  • the signal peptide is a folate receptor signal peptide.
  • the fusion protein has the amino acid sequence set forth in SEQ ID NO: 10 or contains at least 90%, 91%, 92%, 93%, 94%, 95%, and SEQ ID NO: A 96%, 97%, 98%, or 99% identity amino acid sequence or an active fragment thereof.
  • the fusion protein and the chimeric antigen receptor are expressed separately or fused on the surface of the immune effector cell, preferably expressed separately.
  • the endocytic domain is capable of transporting a substance that binds to the antibody binding region or endocytic domain into the immune effector cell.
  • the substance is activated into the immune effector cells to initiate killing of the immune effector cells.
  • the substance is an antibody drug conjugate or an antibody drug conjugate (ADC).
  • ADC antibody drug conjugate
  • the present invention provides an immune effector cell expressing a chimeric antigen receptor, the cell further expressing an endocytic functional region, the endocytic functional region capable of binding a substance to the endocytic functional region Transfer into the immune effector cells as described.
  • the substance is activated into the immune effector cells to initiate killing of the immune effector cells.
  • the substance is an antibody drug conjugate or an antibody drug conjugate (ADC).
  • ADC antibody drug conjugate
  • the endocytic domain is derived from a folate receptor, LDL, CD30, CD33, CD3, EGFR, TFR1; preferably derived from a folate receptor and CD30; more preferably, the endocytic domain Having the amino acid sequence set forth in SEQ ID NO: 32 or 44, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 32 or 44 Or an amino acid sequence of 99% identity, or an active fragment of the amino acid sequence set forth in SEQ ID NO: 32 or 44.
  • the endocytic functional region and the chimeric antigen receptor are expressed separately or fused on the surface of the immune effector cell, preferably expressed separately.
  • the invention provides a fusion protein of Formula I,
  • Z is an optional signal peptide
  • A is an antibody binding region
  • L is an optional joint portion
  • B is the endocytic functional area.
  • the invention also provides a fusion protein comprising an antibody binding region and an endocytic functional region.
  • the antibody binding region is a polypeptide that is absent in normal cells, or is in a concealed state in normal cells, or is low expressed in normal cells.
  • the antibody binding region is selected from the group consisting of EGFRvIII, EGFR, CD20, CD22, CD19, BCMA, proBDNF precursor protein, GPC3, CLD18.2, CLD6, mesothelin, PD-L1, PD-1, WT-1, IL13Ra2, Her-2, Her-1, Her-3;
  • the antibody binding region contains any one of the following amino acid sequences or has the following amino acid sequence Amino acid sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity: SEQ ID NOs: 28, 29, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43;
  • the antibody binding region comprises an active fragment of any of the following amino acid sequences: SEQ ID NO: 28, 29, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43.
  • the antibody binding region specifically binds to an EGFR antibody.
  • the endocytic domain is derived from a folate receptor, LDL, CD30, CD33, CD3, EGFR, TFR1; preferably derived from a folate receptor and CD30; more preferably, the endocytic domain Having the amino acid sequence set forth in SEQ ID NO: 32 or 44, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 32 or 44 Or an amino acid sequence of 99% identity, or an active fragment of the amino acid sequence set forth in SEQ ID NO: 32 or 44.
  • the signal peptide is a folate receptor signal peptide.
  • the fusion protein has the amino acid sequence set forth in SEQ ID NO: 10 or contains at least 90%, 91%, 92%, 93%, 94%, 95%, and SEQ ID NO: A 96%, 97%, 98%, or 99% identity amino acid sequence or an active fragment thereof.
  • the present invention provides the nucleic acid encoding the fusion protein of the third aspect of the invention.
  • the invention provides an expression vector comprising the encoding nucleic acid of the third aspect of the invention.
  • the present invention provides a host cell comprising the expression vector of the fifth aspect of the present invention or the genome encoding the nucleic acid of the fourth aspect of the present invention.
  • the present invention provides an immunoconjugate comprising:
  • the cell killing functional moiety is a small molecule drug or a killing cytokine, including but not limited to MMAF, Auristatin, calicheamicin, maytansine, maytansin, doxorubicin, paclitaxel , 5-fluorouracil, methotrexate, DM1, DM4, MGBA, SN-38 (see: Sassoon I, Blanc V. Antibody-Drug Conjugate (ADC) Clinical Pipeline: A Review [M]//Antibody-Drug Conjugates. Humana Press, 2013: 1-27).
  • ADC Antibody-Drug Conjugate
  • the present invention provides the use of the immunoconjugate of the seventh aspect of the invention for specifically killing the immune effector cells of the first or second aspect of the invention.
  • the present invention provides a kit comprising the immune effector cell of the first or second aspect of the invention or the immunoconjugate of the seventh aspect of the invention.
  • the present invention provides a method of specifically eliminating the immune effector cells of the first or second aspect of the invention, the method comprising the step of administering the immunoconjugate of the seventh aspect of the invention.
  • the immunoconjugate is administered at a concentration of not less than 0.1 ⁇ g/ml; preferably from 0.1 ⁇ g/ml to 100 ⁇ g/ml; more preferably, from 1 ⁇ g/ml to 100 ⁇ g/ml; , is 10 ⁇ g / ml.
  • the substance is substantially non-killing against cells that do not express the fusion protein of the third aspect of the invention.
  • the present invention provides a method of sorting or enriching the immune effector cells of the first or second aspect of the invention, the method comprising the steps of:
  • a sorting reagent is added to the system comprising the immune effector cells, the sorting reagent comprising a substance or specific binding capable of specifically binding to an antibody binding region or an endocytic functional region in the immune effector cells of the first aspect of the invention a substance of an endocytic functional region in an immune effector cell of the second aspect of the invention;
  • a step of separating a substance that binds the immune effector cells from the system is separating a substance that binds the immune effector cells from the system.
  • the substance is an antibody or an active fragment thereof.
  • the substance capable of specifically binding to the antibody binding region or the endocytic domain of the immune effector cell of the first aspect of the invention or specifically binds to the inside of the immune effector cell of the second aspect of the invention The substance of the swallowing functional region is immobilized on the solid phase carrier, thereby enabling separation of the substance to which the immune effector cells are bound from the system.
  • the solid support is a magnetic bead or a resin.
  • the substance is an antibody or an active fragment thereof.
  • the concentration of the sorting reagent is not less than 0.01 ⁇ g/ml; preferably 0.01 ⁇ g/ml to 100 ⁇ g/ml; more preferably, 0.1 ⁇ g/ml to 10 ⁇ g/ml; more preferably, It is 10 ⁇ g/ml.
  • the sorting reagent has a sorting efficiency of greater than 80% for the immune effector cells.
  • the present invention provides a method of detecting an immune effector cell of the first or second aspect of the invention, the method comprising:
  • a detection reagent that specifically binds to an antibody binding region or an endocytic domain in an immune effector cell of the first aspect of the invention or a detection reagent that specifically binds to an endocytic domain in an immune effector cell of the second aspect of the invention is linked to the detectable label;
  • a complex formed by the detection reagent and the immune effector cells is detected.
  • the detection reagent is an antibody or an active fragment thereof.
  • Figure 1 shows a schematic representation of the construction of a fusion protein of the invention
  • Figure 2A shows a flow test chart of T cells and CH12 antibodies expressing FR806 fusion protein
  • Figure 2B shows a flow test chart of Keratinocyte expressing EGFR and HEK-293T cells and CH12 antibody
  • Figure 3 shows the affinity of CH12-biotin for FR806
  • Figure 4 shows the results of sorting FR806 positive cells using CH12-biotin
  • Figure 5 shows that FR806 fusion receptor mediates endocytosis of CH12 antibody
  • Figure 6A shows the binding ability of CH12-MMAF and CH12 to FR806-expressing T cells
  • Figure 6B shows the endocytosis of CH12-MMAF by FR806+T-cells
  • Figure 6C shows different concentrations of CH12-MMAF in different Time killing of T cells expressing FR806
  • Figure 6D shows the killing effect of CH12-MMAF on human Keratinocy cells
  • Figure 7A shows the killing effect of CH12-MMAF and free MMAF detected by CCK8 on FR806 positive and negative T cells
  • Fig. 7B shows the killing effect of CH12-MMAF and free MMAF on FR806 positive and negative 293T cells
  • Figure 8A shows the splicing pattern of FR806 and ⁇ CD19CAR and with eGFP;
  • Figure 8B shows the results of flow analysis of CAR-T cells surface-expressing CAR19 and FR806;
  • Figure 8C shows T cells using FR806-CAR19 with CH12-biotin Sorting
  • Figure 9A shows the splicing manner of FR806 and ⁇ CD19CAR
  • Figure 9B shows the results of flow cytometry expressing T cells according to CAR19 and FR806;
  • Figure 10A shows the killing results of tumor cells by CAR-T cells expressing FR806 and not expressing FR806
  • Figure 10B shows the results of cytokine release of CAR-T cells expressing FR806 and not expressing FR806;
  • Figure 11A shows the killing of CH12-MMAF against T cells co-expressing FR806 and CAR
  • Figure 11B is the killing effect of CH12-MMAF concentration on T cells co-expressing FR806 and CAR;
  • Figure 12A is a graph showing the eGFP positive rate of human CD3+ cell circle analysis
  • Figure 12B shows the in vivo killing effect of CH12-MMAF and physiological saline on FR806-CAR19-eGFP-expressing CAR-T cells
  • Fig. 12B shows the results of flow analysis of CAR-T cells with CAR19 and FR806 on the surface
  • Figure 13 shows the killing of CH12-MMAF against T cells co-expressing CD30806 and CAR.
  • the immunogenic effector cells expressing the chimeric antigen receptor express a fusion protein comprising an antibody binding region, an optional linker portion, and an endocytic functional region, and the resulting immunization is obtained.
  • Effector cells can be killed by specific antibodies to the antibody binding region.
  • the antibody binding region is preferably absent from normal cells, and when administered an antibody that specifically binds to the antibody binding region, does not bind to normal cells, and therefore does not kill normal cells; even if exposed on normal cells Because the amount of cells used to kill immune cells is small, it does not cause too much impact on normal cells.
  • the fusion protein is capable of mediating endocytosis, killing of the cells is completed inside the cell membrane.
  • the present invention also provides an immune effector cell expressing a chimeric antigen receptor expressing only an endocytic functional region, the endocytic functional region capable of binding a substance to the endocytic functional region or to the surface of the immune effector cell
  • the antigen-bound substance is transported into the immune effector cells. Since the killing effect of the substance on the immune effector cells after endocytosis is also completed in the cell membrane, the killing ability is remarkable.
  • the present invention has been completed on this basis.
  • the inventors expressed a fusion protein consisting of an antibody binding region, an optional linker portion, and an endocytic functional region on the surface of an immune effector cell expressing a chimeric antigen receptor, ie, a safety switch.
  • the fusion protein of the present invention has the same meaning as the "safety switch”.
  • the immune effector cells include, but are not limited to, T cells or NK cells.
  • the term "active fragment” refers to a portion of a protein or polypeptide having an activity, ie, the active fragment is not a full-length protein or polypeptide, but has the same or similar activity as the protein or polypeptide. .
  • the fusion protein of the invention is as shown in Formula I
  • Z is an optional signal peptide
  • A is an antibody binding region
  • L is an optional joint portion
  • B is the endocytic functional area.
  • fusion proteins of the present invention can be any suitable linker in the art, as long as the linker is capable of functioning as a fusion protein of the invention. Each part does not adversely affect the function of the final fusion protein.
  • the above optional linkers include a linker and no linker, and thus, in a specific embodiment, the fusion protein of the present invention may comprise only the antibody binding region and the endocytic function region.
  • the fusion protein of the present invention binds to a specific antibody through an antibody binding region, and then the endocytic domain allows the fusion protein and antibody to be endocytosed into the interior of the immune cell.
  • an "antibody binding region” as described herein based on the teachings of the present invention.
  • the antibody binding region in the fusion protein of the present invention is preferably a polypeptide which is not present in normal cells or which is concealed or underexpressed in normal cells.
  • the antibody binding region epitope is in an epitope-concealed state in normal cells, including but not limited to normal cells expressing EGFR.
  • the antibody may be, but is not limited to, an EGFR antibody, a GPC3 antibody, a mesothelin antibody, or the like, such as a CH12 antibody.
  • the antibody binding region is specifically selected from the group consisting of EGFRvIII, EGFR, CD20, CD22, CD19, BCMA, proBDNF precursor protein, GPC3, CLD18.2, CLD6, mesothelin, PD-L1, PD-1 WT-1, IL13Ra2, Her-2, Her-1, Her-3; preferably, the antibody binding region contains any one of the following amino acid sequences or contains at least 90%, 91%, 92% with the following amino acid sequence , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity amino acid sequence: SEQ ID NO: 28, 29, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43; more preferably, the antibody binding region comprises an active fragment of any of the following amino acid sequences: SEQ ID NO: 28, 29,
  • the functional part inside the cell may be derived from folate receptor, LDL, CD30, CD33, CD3, EGFR, TFR1; preferably derived from a folate receptor and CD30; more preferably, the endocytic domain has SEQ ID NO: 32 Or the amino acid sequence shown at 44, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32 or 44.
  • the amino acid sequence is either an active fragment of the amino acid sequence shown in SEQ ID NO: 32 or 44.
  • the signal peptide in the fusion protein of the present invention functions to help the fusion protein pull out of the cell membrane.
  • Specific signal peptides can be determined by those skilled in the art.
  • the signal peptide can be a folate receptor signal peptide, a CD30 receptor signal peptide, a CD33 signal peptide, a CD8 signal peptide, preferably a folate receptor signal peptide.
  • the signal peptide and endocytic functional regions in the fusion proteins of the invention may be derived from the same or different proteins.
  • the fusion protein of the invention may have the amino acid sequence set forth in SEQ ID NO: 10 or contain at least 90%, 91%, 92%, 93%, 94%, 95 with SEQ ID NO: 10. Amino acid sequence of %, 96%, 97%, 98%, or 99% identity or an active fragment thereof.
  • the fusion protein of the present invention can be expressed alone or in fusion with a chimeric antigen receptor on the surface of an immune effector cell.
  • the fusion protein of the invention and the chimeric antigen receptor are expressed separately on the surface of an immune effector cell.
  • single expression means that the fusion protein and the chimeric antigen receptor are expressed on the surface of the immune effector cell, respectively, and the two are not in a fused state; and "fusion expression” refers to fusion of the fusion protein and the chimeric antigen.
  • the form of the protein is expressed on the surface of immune effector cells.
  • the fusion protein of the invention is expressed in fusion with a chimeric antigen receptor on the surface of an immune effector cell.
  • chimeric antigen receptors for different tumor antigens, for example, CD19-CAR, GPC3-CAR, CD30-CAR, Mesothelin-CAR, and the like.
  • the nucleotide sequence encoding the chimeric antigen receptor is set forth in SEQ ID NO: 12.
  • Those skilled in the art can also use the technical means known in the art to promote fusion expression of the fusion protein of the present invention and the chimeric antigen receptor on the surface of immune effector cells, including but not limited to fusion protein and chimeric using self-cleaving sequences. Fusion expression of antigen receptors.
  • the self-shearing sequence is preferably F2A or P2A.
  • F2A is a core sequence derived from foot-and-mouth disease virus 2A (or "self-cleaving polypeptide 2A"), and has a "self-shearing" function of 2A, which can achieve co-expression of upstream and downstream genes.
  • 2A has an effective and feasible strategy for constructing gene therapy polycistronic vectors due to its high shear efficiency, high balance of upstream and downstream gene expression and short self-sequence.
  • the self-shearing sequence is Vkqtlnfdllklagdvesnpgp (SEQ ID NO: 30).
  • the fusion protein of the invention is set forth in SEQ ID NO:31.
  • the immune effector cells expressing the fusion protein of the present invention can achieve high-efficiency killing by using specific antibodies of the antibody binding region, particularly when the antibody binding region in the fusion protein is absent or concealed in normal cells, and the antibody is utilized.
  • the specific antibody of the binding region kills the immune effector cells without killing other normal cells, thereby having excellent differential toxicity.
  • the immune effector cells of the present invention can be specifically killed by an immunoconjugate comprising: an antibody that specifically binds to an antibody binding region in the fusion protein of the present invention, and a cell killing functional moiety.
  • the cytotoxic functional moiety comprises a cytotoxic molecule; preferably, the functional moiety is selected from the group consisting of MMAF, MMAE, Auristatin, calicheamicin, maytansine, maytansin, doxorubicin, paclitaxel, 5-fluorouracil, Methotrexate, DM1, DM4, MGBA, SN-38.
  • the antibody and the cytotoxic functional moiety may constitute a conjugate by covalent attachment, coupling, attachment, crosslinking, and the like.
  • the antibody that specifically binds to the antibody binding region of the fusion protein corresponds to an antibody binding region that is not present in normal cells in the fusion protein of the present invention.
  • the antibody that specifically binds to the antibody binding region of the fusion protein is a CH12 antibody, but is not limited thereto.
  • Those skilled in the art can prepare the immunoconjugates to have suitable sizes based on the knowledge in the prior art to facilitate endocytosis of the immune effector cells of the present invention in order to exert a killing effect.
  • ADC antibody drug conjugate or antibody drug conjugate
  • ADC antibody drug conjugate or antibody drug conjugate
  • the present invention also provides an immune effector cell expressing a chimeric antigen receptor, the immune effector cell expressing an endocytic functional region, the endocytic functional region capable of binding a substance to the endocytic functional region Transfer into the immune effector cells as described.
  • the substance is transported into the immune effector cells to initiate killing of the immune effector cells.
  • the endocytic domain described herein is capable of transporting a substance that binds to the endocytic domain or a substance that binds to the antibody binding region into the immune effector cell.
  • the substance is an antibody drug conjugate or an antibody drug conjugate (ADC).
  • ADC antibody drug conjugate
  • the endocytic functional region and the chimeric antigen receptor are expressed separately or fused on the surface of the immune effector cell, preferably expressed separately.
  • the present invention also provides a nucleic acid encoding the fusion protein of the present invention, an expression vector comprising the encoding nucleic acid, and a host cell comprising the expression vector or the genome in which the encoding nucleic acid is integrated .
  • the present invention also provides a kit comprising the immune effector cell or immunoconjugate of the present invention for use in treating or killing an immune effector cell; that is, by killing the immune conjugate of the present invention.
  • Immune effector cells are used in treating or killing an immune effector cell; that is, by killing the immune conjugate of the present invention.
  • the immune effector cell of the present invention can be recognized by a specific antibody, and can be killed by the antibody-conjugated drug derived from the antibody, and has less influence on other normal cells, and therefore has excellent differential toxicity;
  • the fusion protein surface-expressed by the immune effector cell of the present invention is capable of causing the fusion protein and the antibody-conjugated drug to be endocytosed into the inside of the immune cell after binding to the specific antibody, thereby utilizing the inside of the cell membrane
  • the toxic and powerful toxin molecule is combined to kill the immune effector cells, so the killing ability is remarkable;
  • the killing of immune effector cells by the technical scheme of the present invention is mainly accomplished in cells, and is less affected by other factors (such as the complement system and NK cell activity in vivo depending on the action of CDC and ADCC), thereby being able to The immune effector cells expressing the fusion protein provided by the present application in the environment achieve killing.
  • eGFP was selected as a fluorescent marker for analysis and the eGFP was an enhanced green fluorescent protein.
  • F2A was selected as a self-shearing sequence, and F2A is a core sequence derived from foot-and-mouth disease virus 2A (or "self-cleaving polypeptide 2A"), which has a "self-shearing" function of 2A; selection of human folate receptor subunits
  • the partial amino acid sequence of type 1 (FOLR1) (SEQ ID NO: 32) and the partial sequence of EGFR (SEQ ID NO: 28) were expressed as fusion protein FR806 (SEQ ID NO: 44), and the signal peptide was selected as the signal peptide of FOLR1.
  • the following genetic engineering operations were performed using standard methods known to those skilled in the art.
  • the nucleotide (SEQ ID NO: 1) of eGFP-F2A-FR806 was prepared as follows:
  • eGFP is shown in bold, F2A is underlined, FR SP (folate receptor signal peptide) is shown in bold underline, 806 epitope is shown in italics, and the rest is the rest of the folate receptor
  • amino acid sequence of eGFP-F2A-FR806 (SEQ ID NO: 2) is:
  • nucleotide sequence of the 284-304 epitope of EGFR was prepared according to the experimental procedure in Journal of Biological Chemistry, 2004, 279(29), 30375-30384 and the sequence of Genebank Accession No. X00588.1 (SEQ ID NO: 5) ).
  • nucleotide sequence SEQ ID NO: 3 The nucleotide sequence SEQ ID NO: 4, and the nucleotide sequence SEQ ID NO: 5 were combined in order, and then Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to complete the whole gene combination.
  • a gene fragment of the nucleotide sequence of FR806 SEQ ID NO: 6).
  • PCR amplification was carried out with the upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 7) and the downstream primer 5'-gttgtcatccgctgagccatgggcccagggttggactc-3' (SEQ ID NO: 8) to obtain a 3' end comprising F2A (66 bp) and A small amount of nucleic acid (20 bp) eGFP nucleic acid fragment that was assembled downstream. .
  • FR SP in Figure 1 expresses the signal peptide of folate receptor (SEQ ID NO: 3), 806 epitope represents EGFR284-304 epitope (SEQ ID NO: 5), and FR represents folate receptor except signal peptide Other parts (SEQ ID NO: 4). .
  • the DNA polymerase was supplemented, and the upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 7) was added, and the downstream primer 5'-ctcgaggtcgacctagctgagcagccacagc-3' (SEQ ID NO: 9) was subjected to PCR to obtain MulI SalI enzymes at both ends.
  • the gene fragment of the nucleotide sequence of eGFP-F2A-FR806 at the cleavage site was theoretically 2047 bp, and the amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • the vector system used in the lentiviral plasmid vector used in this embodiment belongs to the third generation auto-inactivated lentiviral vector system, and the system comprises: a protein encoding Gag/Pol, a packaging plasmid encoding the Rev protein, psPAX2, and a package encoding the VSV-G protein.
  • the promoter of elongation factor-1 ⁇ regulates the expression of enhanced green fluorescent protein (eGFP), while encoding the target gene eGFP-
  • eGFP was co-expressed with the target gene FR806 by a food and mouth disease virus (FMDV, ribosomal skipping sequence, F2A).
  • FMDV food and mouth disease virus
  • the gene fragment of the nucleotide sequence of eGFP-F2A-FR806 containing MulI SalI restriction sites at both ends obtained in the above Example 1.1 was digested by MluI and SalI restriction enzymes, and ligated into the same double In the pWPT vector digested, a plasmid pWPT-eGFP-F2A-FR806 co-expressing eGFP and FR806 linked by F2A was constructed.
  • 293T cells (ATCC) were seeded in a 15 cm culture dish at a density of 1.25 ⁇ 10 7 in a medium of 10% fetal bovine serum (Gbico) in L110DMEM medium (Gbico).
  • Human peripheral blood mononuclear cells were added to the lymphocyte culture medium at a density of about 1 ⁇ 10 6 /mL, and magnetic beads coated with anti-CD3 and CD28 antibodies were added according to the magnetic bead: cell ratio of 1:1 (Invitrogen) The company) and recombinant human IL-2 (Shanghai Huaxin Biotech Co., Ltd.) with a final concentration of 300 U/mL were activated for 48 h.
  • the activated T cells were added to a plate (24-well plate) coated with Retronectin (purchased from takara) at a concentration of 1 ⁇ 10 6 cells/ml, and the virus concentrate (MOI ⁇ 10) obtained in the step 3 was added and centrifuged. , cultured in an incubator to obtain T cells (CAR-FR806-T cells) expressing the fusion proteins FR806 and eGFP, and Mock T cells, wherein the FR806 fusion protein sequence further contains a signal peptide, as shown in SEQ ID NO: 10. :
  • the primary antibody was incubated with CH12 antibody (10 ⁇ g/ml) as disclosed in CN 200810038848.8 for 45 min, followed by washing with 1% FBS in PBS twice.
  • the secondary antibody was PE-labeled goat anti-human IgG (Santa), incubated for 45 min at 1:50 dilution. After 1% FBS PBS was washed twice and resuspended, flow analysis, the results shown in Figure 2A, indicating that T cells expressing FR806 fusion protein can effectively bind to CH12 antibody, and can co-express with eGFP in T cells.
  • the light chain of the CH12 antibody is set forth in SEQ ID NO: 46 and the heavy chain is set forth in SEQ ID NO:45.
  • Keratinocyte cells expressing EGFR and HEK-293T cells were selected, and the binding of CH12 antibody to both was analyzed by FACS. The results showed that the CH12 antibody did not bind to both EGFR-expressing Keratinocyte cells and HEK-293T cells (Fig. 2B).
  • the CH12 antibody was labeled with biotin.
  • the CH12 antibody was diluted to 2.5 mg/ml, PBS pH 7.4, and the labeled volume was 1.6 ml; 1 mg of Sulfo-NHS-LC-Biotin (Thermo) was added, and 180 ul of ultrapure water was added to dissolve; 79 ul of Biotin was added to 1.6.
  • the reaction was overnight.
  • the mixture was desalted using a PD-10 desalting column (GE Corporation, USA), and replaced with PBS 5% glycerol buffer to obtain CH12-Biotin, and the concentration of OD280/1.45 was 0.77 mg/ml.
  • CH12-biotin was diluted to different concentrations (100 ⁇ g/ml, 10 ⁇ g/ml, 1 ⁇ g/ml, 0.1 ⁇ g/ml, 0.01 ⁇ g/ml, 0 ⁇ g/ml) in PBS containing 1% FBS, respectively, and expressed with eGFP-F2A-
  • the T cells of FR806 were incubated for 45 min, followed by PBS washing, and the secondary antibody was diluted 1:300 in PE-SA (ebioscience), and after resuspending the cells, it was incubated for 45 min. After washing twice with PBS, flow analysis, the results are shown in Figure 3, showing that the higher the concentration of CH12-biotin, the affinity The stronger the force, the similarity of the binding level of 10 ⁇ g/ml to 100 ⁇ g/ml.
  • Example 3 Sorting FR806-positive T cells with CH12-biotin
  • T cells expressing eGFP-F2A-FR806 were washed with PBS, diluted with CH12-biotin (10 ⁇ g/ml, diluted with PBS containing 1% FBS) for 45 min at 4 ° C, followed by washing with PBS, and anti-Biotin sorting beads were added. (purchased from Meitian Company), the T cells of FR806 were sorted according to the procedure given by the sorted magnetic bead product. The cells before and after sorting were flowed and analyzed. The results are shown in Figure 4. It is shown that the T cells expressing FR806 can be effectively sorted by anti-Biotin sorting magnetic beads after binding to CH12-biotin, and the positive rate of sorting is up. 95%.
  • the T cells of the lentiviral vectors pWPT-eGFP-F2A-FR806 and pWPT-eGFP (Mock) obtained in Example 1 were washed with PBS; the CH12-biotin synthesized in Example 2 (10 ⁇ g/ml, diluted with Petri) was taken.
  • the secondary antibody was diluted 1:300 in PE-SA (ebioscience), added to the resuspended cells, incubated for 45 min, washed twice with PBS and incubated for 4 h. Subsequently, paraformaldehyde was fixed, stained with DAPI staining solution (Roche), and observed under a confocal microscope. The results are shown in Fig. 5.
  • CH12-biotin represented by red fluorescence
  • T cells infected with pWPT-eGFP-F2A-FR806 and pWPT-eGFP were washed with PBS; CH12-MMAF (10 ⁇ g/ml, cultured diluted) was incubated at 4 ° C for 45 min, washed with PBS.
  • the second antibody was goat anti-human PE (Shanghai Lianke Biotechnology Co., Ltd.), diluted 1:50, and resuspended with cells for 45 min. After washing twice with PBS, the incubator was incubated for 4 h.
  • the positive rate of T cells of Mock and eGFP-FR806 was adjusted by adding appropriate proportion of uninfected T cells. 50%, plated in 6-well plates, 2 x 10 6 cells per well, 2 ml medium (AIM-V Petri + 2% human AB serum, IL-2 500 U/ml).
  • the CH12-MMAF drugs were diluted to 0.01, 0.1, 1, 10, and 100 ⁇ g/ml with PBS, and then added to the experimental group and the control group.
  • the eGFP positive rate was detected every 24 hours, and the results were determined for 96 hours. As shown in Fig.
  • CH12-MMAF did not kill human Keratinocy cells, indicating that CH12-MMAF was safe.
  • mice The T cells expressing eGFP-FR806 after sorting in Example 3 were plated in a 96-well plate, 3 ⁇ 10 4 cells per well, 100 ul of culture medium, and 5 replicate wells per drug concentration. Then set a blank group with only Peiji. Control group: T cells that were not infected with the virus were plated in a 96-well plate with reference to the operation of the experimental group.
  • cell viability (%) [A (dosing) - A (blank)] / [A (0 dosing) - A (blank)]
  • eGFP was selected as a fluorescent marker, and eGFP was an enhanced green fluorescent protein.
  • the following genetic engineering operations were performed using standard methods known to those skilled in the art.
  • nucleotide fragment of single-chain antibody of ⁇ CD19 disclosed in US20060193852A1 was selected as the anti-CD19 antibody sequence of CAR
  • CD8-CD137-CD3 ⁇ was selected as the transmembrane domain and intracellular domain of CAR.
  • SEQ ID NO: 12 (the bold portion is the CD8 ⁇ signal peptide sequence, the underlined is the ⁇ CD19CAR nucleotide sequence, and the italicized bold is the CD8-CD137-CD3 ⁇ nucleotide sequence)
  • SEQ ID NO: 15 (FR806 is underlined, ⁇ CD19CAR is shown in bold underline, F2A is shown in bold, eGFP is normally displayed)
  • the primer pair used for amplification is the upstream primer 5'-cttacgcgtcctagcgctaccggtcgccaccatggctcagcggatg-3' (SEQ ID NO: 16), downstream primer 5'-gtctcctgccaacttcagaaggtcaaaattcaaagtctgtttcacgctgagcagccac-3' (SEQ ID NO: 17).
  • the size of the amplified band was 910 bp.
  • the PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 1 min; 25 cycles followed by a total extension of 68 ° C, 10 min.
  • the PCR amplified bands were determined by agarose gel electrophoresis to determine the size of the amplified bands of interest.
  • the primer pair taken for amplification was the upstream primer 5'-accttctgaagttggcaggagacgttgagtccaaccctgggcccatggtgagcaagggc-3' (SEQ ID NO: 18), and the downstream primer 5'-ctcgaggtcgacctacttgtacagctcg-3 '(SEQ ID NO: 19).
  • An eGFP-F2A-FR806 nucleic acid fragment having a partial F2A fragment at the 5' end was obtained.
  • the PCR amplified bands were determined by agarose gel electrophoresis to match the expected fragment size.
  • the DNA polymerase was supplemented, and the upstream primer 5'-cttacgcccctagcgctaccggtcgccaccatggctcagcggatg-3' (SEQ ID NO: 16) was added, and the downstream primer 5'-tcctgccaacttcagaaggtcaaaattcaaagtctgtttcacgcgagggggcagggc-3' (SEQ ID NO: 14) was PCR for 25 cycles to obtain the nucleus of FR806 and ⁇ CD19CAR. A spliced fragment of a nucleotide sequence. The theoretical size is 2458 bp, and the amplified product is confirmed by agarose gel electrophoresis to be consistent with the theoretical size.
  • the DNA polymerase was supplemented, and the upstream primer 5'-cttacgcccctagcgctaccggtcgccaccatggctcagcggatg-3' (SEQ ID NO: 16) was added, and the downstream primer 5'-ctcgaggtcgacctacttgtacagctcg-3' (SEQ ID NO: 19) was PCR for 25 cycles, and MluI was obtained at both ends. And the FR806 of the SalI restriction endonuclease site and the spliced fragment of FR806-F2A-CAR19-F2A-eGFP of ⁇ CD19CAR and eGFP. The theoretical size is 3214 bp, and the amplified product is confirmed by agarose gel electrophoresis to be consistent with the theoretical size.
  • the nucleotide sequence of the obtained FR806-F2A-CAR19-F2A-eGFP was digested with MluI and SalI restriction enzymes by the operation of the construction of the lentiviral vector in Example 1, and ligated into the same double digestion.
  • a lentiviral expression vector in which F2A-linked FR806, ⁇ CD19CAR and eGFP were co-expressed was constructed.
  • the lentiviral expression vector obtained in step 2 of the present example, the pWPT-eGFP control plasmid, the packaging plasmid PAX2, and the envelope plasmid pMD2.G were dissolved in 2200 ul of serum-free DMEM medium according to the procedure of step 3 in Example 1. , for slow virus packaging.
  • step 4 in Example 1 the packaged lentivirus obtained in step 3 of the present example was transfected into T cells to obtain CAR-T cells with CAR19 and FR806 surface expression, namely FR806-CAR19T cells, and FR806- Flow cytometry analysis of CAR19T cells showed that the three proteins FR806, eGFP, and ⁇ CD19CAR were efficiently expressed in T cells.
  • FR806-CAR19T cells were sorted using CH12-biotin and anti-biotin magnetic beads.
  • the results are shown in Figure 8C, showing that FR806-CAR19T cells can be sorted by anti-Biotin after binding to CH12-biotin.
  • the beads were effectively sorted, and the positive rate of sorting was 94.3%.
  • splicing and PCR were carried out in accordance with the mode shown in Fig. 9A to obtain T cells (FR806-CAR19T cells) expressing FR806 and CAR19, and flow cytometry, and the results are shown in Fig. 9B.
  • Example 8 Killing of tumor cells by FR806-CAR19T cells and release of cytokines
  • T cells expressing CAR19 and not expressing FR806, namely, CAR19T cells were prepared.
  • the resulting FR806-CAR19T cells were spliced with reference to Figure 9A for cell killing experiments.
  • Daudi cells were used as target cells, and the effector cells were FR806-CAR19T cells and CAR19T cells.
  • the target ratios were 20:1, 10:1, 5:1, 2.5:1, and the target cell number was 10000/well, depending on the effect. Target ratio setting different quantity effect Cell.
  • Each group has 5 duplicate holes.
  • FR806-CAR19T cells and CAR19T cells were co-incubated with Daudi cells, and the control group was co-incubated with Daudi cells infected with Mock virus. After 4 hours of incubation, the LDH content in the supernatant was determined by CytoTox96 non-radioactive cytotoxicity kit (Promega), and its killing activity was calculated.
  • the initial positive rate of the FR806-CAR19T cells and the control mock spliced with reference to FIG. 8A was adjusted to 50%, and 10 ⁇ g/ml of CH12-MMAF was added, and the positive rate of eGFP was detected by flow detection every 24 hours for 96 hours.
  • the number of T cells of FR806-CAR19 had decreased, and at 72 h, the number of T cells of FR806-CAR19 was reduced by about 80%.
  • FR806-CAR19T cells were plated in 96-well plates at 3 x 10 4 cells per well, 100 ul of phage, 5 replicate wells per drug concentration, and a set of blanks with only peper was set.
  • Control group T cells that were not infected with the virus were plated in a 96-well plate with reference to the operation of the experimental group.
  • cell viability (%) [A (dosing) - A (blank)] / [A (0 dosing) - A (blank)]
  • Example 10 Determination of the in vivo killing effect of CH12-MMAF on FR806-CAR19T cells
  • NOD/SCID mice were inoculated with 3 ⁇ 10 6 Daudi cells, and on day 12, NOD/SCID mice were exposed to cyclophosphamide (100 mg/kg).
  • mice were injected with FR806-CAR19T cells (3 x 10 7 cells/cell) in the tail vein.
  • the experimental group was administered CH12-MMAF, 0.1 mg/head, and the control group was given physiological saline.
  • the peripheral blood, bone marrow, and spleen of the mice were taken, and the red blood cells were lysed by erythrocyte lysate (ebioscience).
  • PE-labeled goat anti-human CD3 antibody (1:50, diluted with PBS containing 1% FBS) was added. After 45 minutes of incubation at 4 degrees, PBS containing 1% FBS was washed, and the eGFP positive rate was analyzed by flow cytometry as shown in Fig. 12A.
  • eGFP was selected as a fluorescent marker for analysis and the eGFP was an enhanced green fluorescent protein.
  • F2A is selected as a self-shearing sequence.
  • F2A is a core sequence derived from foot-and-mouth disease virus 2A (or "self-cleaving polypeptide 2A"). It has a "self-shearing" function of 2A and can achieve a total of upstream and downstream genes. expression.
  • the partial amino acid sequence of CD30 (SEQ ID NO: 44) and the partial sequence of EGFR (SEQ ID NO: 28) were selected to be expressed as fusion protein CD30806, and the signal peptide was selected as the signal peptide of CD30.
  • the following genetic engineering operations were performed using standard methods known to those skilled in the art.
  • the nucleotide (SEQ ID NO: 20) of eGFP-F2A-CD30806 was prepared as follows:
  • eGFP is boldly displayed, F2A is underlined, CD30SP is shown in bold underline, 806 is shown in italics, linker is underlined in italics, and the rest are CD30 receptor transmembrane and intracellular segments.
  • amino acid sequence of eGFP-F2A-CD30806 (SEQ ID NO: 21) is:
  • the nucleotide sequence of the epidermal growth factor receptor 284-304 epitope was prepared by following the experimental procedure in Journal of Biological Chemistry, 2004, 279(29), 30375-30384 and the sequence of Genebank Accession No. X00588.1 (SEQ ID NO: 5).
  • nucleic acid encoding the GPC-3 chimeric antigen receptor protein and the sequence of GPC3-Z (SEQ ID NO: CN201310164725.X) expressing the GPC-3 chimeric antigen receptor protein.
  • ID NO: 18 The nucleotide sequence (SEQ ID NO: 24) of the linker joining the 806 epitope and the CD30 transmembrane and intracellular segments was obtained.
  • nucleotide sequence SEQ ID NO: 22 was sequentially combined and entrusted to Suzhou Jinweizhi Bio After the whole gene was combined, the Science and Technology Co., Ltd. obtained a gene fragment of the nucleotide sequence of CD30806 (SEQ ID NO: 25).
  • PCR amplification was carried out with the upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 7) and the downstream primer 5'-gcggcgaggaggacgcgcatgggcccagggtgggactc-3' (SEQ ID NO: 26) to obtain a 3' end comprising F2A (66 bp) and A small amount of nucleic acid (20 bp) eGFP nucleic acid fragment that was assembled downstream.
  • the vector system used in the lentiviral plasmid vector used in this embodiment belongs to the third generation auto-inactivated lentiviral vector system, and the system comprises: a protein encoding Gag/Pol, a packaging plasmid encoding the Rev protein, psPAX2, and a package encoding the VSV-G protein.
  • the promoter of elongation factor-1 ⁇ regulates the expression of enhanced green fluorescent protein (eGFP), while encoding the target gene eGFP-
  • eGFP was co-expressed with the target gene FR806 by a food and mouth disease virus (FMDV, ribosomal skipping sequence, F2A).
  • FMDV food and mouth disease virus
  • the gene fragment of the nucleotide sequence of eGFP-F2A-CD30806 containing MulI SalI cleavage sites obtained in the above Example 1.1 was digested by MluI and SalI restriction enzymes, and ligated into the same double In the pWPT vector, the plasmid pWPT-eGFP-F2A-CD30806 co-expressed by F2A-linked eGFP and CD30806 was constructed, and subjected to virus packaging and T cell transfection to obtain T cells expressing CD30-806 fusion protein and eGFP.
  • CAR-T cell killing activity experiment T cells infected with eGFP-CD30806 (CD30-806 for short), 3 ⁇ 10 5 density plating, different concentrations of CH12-MMAF were added to each well, and cells were collected after 72 hours.
  • the proportion of eGFP-positive (ie, CD30-806 positive cells) cells in each well was observed by cytometry. The results are shown in Fig. 13. With the increase of the concentration of CH12-MMAF, the proportion of CD30-806 positive cells decreased gradually, indicating that CH12-MMAF has strong killing toxicity against CD30-806 positive cells.

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Abstract

提供了一种包含抗体结合区和内吞功能区的融合蛋白及其编码核酸、表达载体和宿主细胞，表达该融合蛋白或内吞功能区或进一步表达嵌合抗原受体的免疫效应细胞。还提供了包括细胞杀伤性部分和特异性结合免疫效应细胞中的抗体结合物或内吞功能区的抗体的免疫缀合物及其试剂盒和用途，特异性清除、分选或富集以及检测免疫效应细胞的方法。

Description

融合蛋白及其应用 技术领域
本发明涉及免疫治疗领域。更具体地，本发明涉及控制嵌合抗原受体免疫效应细胞或TCR-T细胞的融合蛋白及其应用。
背景技术
近年来，针对恶性肿瘤的过继免疫疗法(adoptive cell therapy，ACT)，如CAR-T、TCR-T等取得了长足的进展，其中CAR-T疗法的发展最为显著。
然而，伴随着CAR-T细胞疗法临床实验的开展，也出现了很多严重的副反应，如细胞因子风暴、脱靶效应等，当出现严重不良反应时，如果不能及时抑制CAR-T细胞，会导致严重的不良反应甚至危及患者生命。因此，在使用CAR-T治疗时，有必要同时引入一种安全开关，当患者使用CAR-T细胞后产生严重不良反应危机生命时，体内的CAR-T细胞能被有效且特异性的清除。
目前应用于细胞治疗的安全开关主要包括两种形式：自杀基因和标记基因。
自杀基因主要包括单纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase,HSV-TK)以及可诱导的含半胱氨酸的天冬氨酸蛋白水解酶9(inducible caspase-9,iCasp9)。HSV-TK自杀基因是通过在T细胞上表达HSV-TK，大大增强了T细胞对更昔洛韦药物的敏感性。然而，由于HSV-TK会在病人体内产生免疫原性，并且接受细胞治疗的病人将不能继续使用更昔洛韦作为抗病毒药物，这两点极大地限制了HSV-TK在临床上的使用。iCasp9是通过在病人体内施加小分子药物(AP20187)，从而诱导表达iCasp9自杀基因的T细胞的发生凋亡。只是AP20187尚未完成商业化，限制了iCasp9自杀基因的普及。
标记基因通过在T细胞表面表达一种能被抗体识别的特异性标志物，使得T细胞具备了能够被分选、检测以及清除。如Hum Gene Ther,11(4):611-20报导了在T细胞表面表达CD20受体，使得T细胞能够被抗CD20单克隆抗体识别并被杀死；Blood,118(5):1255-1263报导了在CAR-T细胞上共表达一段能够被抗EGFR单克隆抗体识别的截短的EGFR受体。
标志基因的发展拓宽了安全开关的应用范围，但是由于其杀伤效果往往是由补体依赖的细胞毒性(complement dependent cytotoxicity,CDC)以及抗体依赖的细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity，ADCC)所介导的，其杀伤效果依赖于补体系统和体内NK细胞的活性。当病人体内补体系统或NK细胞活性出现缺陷时，其杀伤能力往往受限。这些缺点限制了这些标志基因的应用。
因此，伴随着细胞治疗快速发展及临床应用，本领域迫切需要一种能够有效且特异地杀伤T细胞的技术手段。
发明内容
本发明的目的在于提供一种表达嵌合抗原受体的免疫效应细胞，所述免疫效应细胞的表面同时表达有一种融合蛋白，借助该融合蛋白，可以利用特异性抗体偶联药物对该免疫效应细胞进行高效杀伤。
在第一方面，本发明提供一种在表面表达嵌合抗原受体的免疫效应细胞，所述免疫细胞还表达式I所示的融合蛋白，
Z-A-L-B
I
其中，Z是任选的信号肽；
A是抗体结合区；
L是任选的接头部分；
B是内吞功能区。
本发明还提供这样的表达嵌合抗原受体的免疫效应细胞，所述免疫细胞还表达一融合蛋白，该融合蛋白含有抗体结合区和内吞功能区。
在优选的实施方式中，所述抗体结合区为在正常细胞中不存在、或者在正常细胞中为隐蔽状态、或者在正常细胞中低表达的多肽。
在具体的实施方式中，所述的抗体结合区选自以下抗原或其片段：EGFRvIII、EGFR、CD20、CD22、CD19、BCMA、proBDNF前体蛋白、GPC3、CLD18.2、CLD6、间皮素、PD-L1、PD-1、WT-1、IL13Ra2、Her-2、Her-1、Her-3；
优选的，所述的抗体结合区含有以下任一氨基酸序列或者含有与以下氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列：SEQ ID NO:28、29、33、34、35、36、37、38、39、40、41、42、43；
更优的，所述的抗体结合区含有以下任一氨基酸序列的活性片段：SEQ ID NO:28、29、33、34、35、36、37、38、39、40、41、42、43。
在具体的实施方式中，所述抗体结合区与EGFR抗体特异性结合。
在优选的实施方式中，所述嵌合抗原受体的胞外部分与所述的融合蛋白不具有结合能力。
在具体的实施方式中，所述内吞功能区衍生自叶酸受体、LDL、CD30、CD33、CD3、EGFR、TFR1；优选衍生自叶酸受体和CD30；更优地，所述内吞功能区具有SEQ ID NO：32或44所示氨基酸序列，或者与SEQ ID NO：32或44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列，或者为SEQ ID NO:32或44所示的氨基酸序列的活性片段。
在具体的实施方式中，所述信号肽为叶酸受体信号肽。
在具体的实施方式中，所述融合蛋白具有SEQ ID NO:10所示的氨基酸序列或者含有与SEQ ID NO:10具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列或其活性片段。
在具体的实施方式中，所述融合蛋白与嵌合抗原受体在免疫效应细胞表面单独表达或融合表达，优选单独表达。
在优选的实施方式中，所述的内吞功能区能将与所述的抗体结合区或内吞功能区结合的物质转运入所述的免疫效应细胞内。
在优选的实施方式中，所述物质转运入所述的免疫效应细胞后启动对所述免疫效应细胞的杀伤。
在优选的实施方式中，所述物质是抗体药物偶联物或抗体药物缀合物(ADC)。
在第二方面，本发明提供一种表达嵌合抗原受体的免疫效应细胞，所述细胞还表达内吞功能区，所述的内吞功能区能将与所述内吞功能区结合的物质转运入所述的免疫效应细胞内。
在优选的实施方式中，所述物质转运入所述的免疫效应细胞后启动对所述免疫效应细胞的杀伤。
在优选的实施方式中，所述物质是抗体药物偶联物或抗体药物缀合物(ADC)。
在具体的实施方式中，所述内吞功能区衍生自叶酸受体、LDL、CD30、CD33、CD3、EGFR、TFR1；优选衍生自叶酸受体和CD30；更优地，所述内吞功能区具有SEQ ID NO:32或44所示氨基酸序列，或者与SEQ ID NO:32或44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列，或者为SEQ ID NO:32或44所示的氨基酸序列的活性片段。
在具体的实施方式中，所述内吞功能区与嵌合抗原受体在免疫效应细胞表面单独表达或融合表达，优选单独表达。
在第三方面，本发明提供式I所示的融合蛋白，
Z-A-L-B
I
其中，Z是任选的信号肽；
A是抗体结合区；
L是任选的接头部分；
B是内吞功能区。
本发明还提供这样的融合蛋白，所述融合蛋白包含抗体结合区和内吞功能区。
在优选的实施方式中，所述抗体结合区为在正常细胞中不存在、或者在正常细胞中为隐蔽状态、或者在正常细胞中低表达的多肽。
在具体的实施方式中，所述的抗体结合区选自以下抗原或其片段：EGFRvIII、EGFR、CD20、CD22、CD19、BCMA、proBDNF前体蛋白、GPC3、CLD18.2、CLD6、间皮素、PD-L1、PD-1、WT-1、IL13Ra2、Her-2、Her-1、Her-3；
优选的，所述的抗体结合区含有以下任一氨基酸序列或者含有与以下氨基酸序列具有 至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列：SEQ ID NO:28、29、33、34、35、36、37、38、39、40、41、42、43；
更优的，所述的抗体结合区含有以下任一氨基酸序列的活性片段：SEQ ID NO:28、29、33、34、35、36、37、38、39、40、41、42、43。
在具体的实施方式中，所述抗体结合区与EGFR抗体特异性结合。
在具体的实施方式中，所述内吞功能区衍生自叶酸受体、LDL、CD30、CD33、CD3、EGFR、TFR1；优选衍生自叶酸受体和CD30；更优地，所述内吞功能区具有SEQ ID NO:32或44所示氨基酸序列，或者与SEQ ID NO：32或44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列，或者为SEQ ID NO:32或44所示氨基酸序列的活性片段。
在具体的实施方式中，所述信号肽为叶酸受体信号肽。
在具体的实施方式中，所述融合蛋白具有SEQ ID NO:10所示的氨基酸序列或者含有与SEQ ID NO:10具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列或其活性片段。
在第四方面，本发明提供本发明第三方面所述的融合蛋白的编码核酸。
在第五方面，本发明提供一种表达载体，包含本发明第三方面所述的编码核酸。
在第六方面，本发明提供一种宿主细胞，包含本发明第五方面所述的表达载体或基因组中整合有本发明第四方面所述的编码核酸。
在第七方面，本发明提供一种免疫辍合物，所述免疫辍合物包括：
细胞杀伤性功能部分；和
特异性结合本发明第一方面所述免疫效应细胞中的抗体结合区或内吞功能区的抗体或者特异性结合本发明第二方面所述免疫效应细胞中的内吞功能区的抗体。
在优选的实施方式中，所述细胞杀伤性功能部分为小分子药物或杀伤性细胞因子，包括但不限于MMAF、Auristatin、卡奇霉素、美登素、美坦辛、阿霉素、紫杉醇、5-氟尿嘧啶、甲氨蝶呤、DM1、DM4、MGBA、SN-38(参见：Sassoon I,Blanc V.Antibody-Drug Conjugate(ADC)Clinical Pipeline:A Review[M]//Antibody-Drug Conjugates.Humana Press,2013:1-27)。
在第八方面，本发明提供本发明第七方面所述免疫辍合物在特异性杀伤本发明第一或第二方面所述免疫效应细胞中的用途。
在第九方面，本发明提供一种试剂盒，包含本发明第一或第二方面所述的免疫效应细胞或本发明第七方面所述的免疫缀合物。
在第十方面，本发明提供特异性清除本发明第一或第二方面所述免疫效应细胞的方法，所述方法包括给予本发明第七方面所述的免疫缀合物的步骤。
在优选的实施方式中，所述免疫缀合物的给予浓度不低于0.1μg/ml；优选0.1μg/ml～100μg/ml；更优的，为1μg/ml-100μg/ml；更优的，为10μg/ml。
在优选的实施方式中，所述物质对不表达本发明第三方面所述融合蛋白的细胞几乎没有杀伤。
在第十一方面，本发明提供一种分选或富集本发明第一或第二方面所述的免疫效应细胞的方法，所述方法包括以下步骤：
将分选试剂加入包含所述免疫效应细胞的体系，所述分选试剂包含能够特异性结合本发明第一方面所述免疫效应细胞中的抗体结合区或内吞功能区的物质或者特异性结合本发明第二方面所述免疫效应细胞中的内吞功能区的物质；和
将结合有所述免疫效应细胞的物质从所述体系分离的步骤。
在优选的实施方式中，所述物质是抗体或其活性片段。
在具体的实施方式中，能够特异性结合本发明第一方面所述免疫效应细胞中的抗体结合区或内吞功能区的物质或者特异性结合本发明第二方面所述免疫效应细胞中的内吞功能区的物质固定于固相载体，从而能够实现结合有所述免疫效应细胞的物质从所述体系分离。
在优选的实施方式中，所述固相载体是磁珠或树脂。
在优选的实施方式中，所述物质是抗体或其活性片段。
在优选的实施方式中，所述分选试剂的浓度不低于0.01μg/ml；优选0.01μg/ml～100μg/ml；更优的，为0.1μg/ml-10μg/ml；更优的，为10μg/ml。
在优选的实施方式中，所述分选试剂对所述免疫效应细胞的分选效率大于80％。
在第十二方面，本发明提供检测本发明第一或第二方面所述的免疫效应细胞的方法，所述方法包括：
给予特异性结合本发明第一方面所述免疫效应细胞中的抗体结合区或内吞功能区的检测试剂或者特异性结合本发明第二方面所述免疫效应细胞中的内吞功能区的检测试剂，所述检测试剂与可检测标记物相连；和
检测所述检测试剂与所述免疫效应细胞形成的复合物。
在优选的实施方式中，所述检测试剂是抗体或其活性片段。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了构建本发明融合蛋白的示意图；
图2A显示了表达FR806融合蛋白的T细胞与CH12抗体的流式测试图；图2B显示了表达EGFR的Keratinocyte细胞和HEK-293T细胞与CH12抗体的流式测试图；
图3显示了CH12-biotin对FR806的亲和力；
图4显示了利用CH12-biotin分选FR806阳性细胞的结果；
图5显示了FR806融合受体介导CH12抗体的内吞；
图6A显示了CH12-MMAF和CH12与表达FR806的T细胞的结合能力；图6B显示了CH12-MMAF被FR806+T-cells的内吞情况；图6C显示了不同浓度的CH12-MMAF在不同的时间对表达FR806的T细胞的杀伤；图6D显示了CH12-MMAF对人Keratinocy细胞的杀伤作用；
图7A显示了CCK8检测的CH12-MMAF及游离MMAF对FR806阳性和阴性T细胞的杀伤效果；图7B显示了CH12-MMAF及游离MMAF对FR806阳性和阴性的293T细胞的杀伤效果；
图8A显示了FR806与αCD19CAR及与eGFP的拼接模式；图8B显示了表面表达有CAR19及FR806的CAR-T细胞的流式分析结果；图8C显示了采用CH12-biotin对FR806-CAR19的T细胞的分选；
图9A显示了FR806与αCD19CAR的拼接方式，图9B显示了表达有按照CAR19和FR806的T细胞的流式分析结果；
图10A显示了表达FR806和不表达FR806的CAR-T细胞对肿瘤细胞的杀伤结果；图10B显示了表达FR806和不表达FR806的CAR-T细胞的细胞因子释放结果；
图11A显示了CH12-MMAF对共表达FR806和CAR的T细胞的杀伤；图11B为CH12-MMAF的浓度对共表达FR806和CAR的T细胞的杀伤作用；
图12A为人CD3+细胞圈门分析eGFP阳性率的图；图12B显示了CH12-MMAF及生理盐水对表达FR806-CAR19-eGFP的CAR-T细胞的体内杀伤效果；图12C显示了给予CH12-MMAF和生理盐水后，CD3+/eGFP+在小鼠血液、脾脏、及骨髓中的检出率，n＝6；图12B显示了表面表达有CAR19及FR806的CAR-T细胞的流式分析结果；
图13显示了CH12-MMAF对共表达CD30806和CAR的T细胞的杀伤。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现在表达嵌合抗原受体的免疫效应细胞表面表达包含抗体结合区、任选的接头部分以及内吞功能区构成的融合蛋白，得到的免疫效应细胞可以用所述抗体结合区的特异性抗体杀伤。所述抗体结合区优选在正常细胞中不存在，当施予特异性结合该抗体结合区的抗体时，不会和正常细胞结合，因此，不会杀伤正常细胞；而即便在正常细胞上有暴露，由于杀伤免疫效应细胞的给药量很少，也不会对正常细胞造成太大影响。并且，由于该融合蛋白能够介导内吞，使得对细胞的杀伤在细胞膜内部完 成，杀伤能力显著。本发明还提供仅表达内吞功能区的表达嵌合抗原受体的免疫效应细胞，所述的内吞功能区能将与所述内吞功能区结合的物质或与所述免疫效应细胞表面的抗原结合的物质转运入所述的免疫效应细胞内。由于所述物质在内吞后对所述免疫效应细胞的杀伤作用也在细胞膜内完成，杀伤能力显著。在此基础上完成了本发明。
本发明的融合蛋白及免疫效应细胞
为特异性地杀伤免疫效应细胞，发明人在表达嵌合抗原受体的免疫效应细胞表面表达由抗体结合区、任选的接头部分以及内吞功能区构成的融合蛋白，即，安全开关。在本发明中，“本发明的融合蛋白”与“安全开关”具有相同的意义。在具体的实施方式中，所述免疫效应细胞包括但不限于T细胞或NK细胞。此外，在本文中，所用的术语“活性片段”是指具有某种活性的蛋白或多肽的一部分，即，活性片段并非全长的蛋白或多肽，但具有与该蛋白或多肽相同或相似的活性。
在具体的实施方式中，本发明的融合蛋白如式I所示
Z-A-L-B
I
其中，Z是任选的信号肽；
A是抗体结合区；
L是任选的接头部分；
B是内吞功能区。
基于本发明的教导，本领域技术人员可以想到并测试用于本发明融合蛋白中的各种合适接头，所述接头可以是本领域任何合适的接头，只要该接头能够起到连接本发明融合蛋白的各部分并且对最终的融合蛋白的功能不会产生不利影响。上述任选的接头包括含有接头和不含有接头，因此，在具体的实施方式中，本发明的融合蛋白可以仅包含抗体结合区和内吞功能区。
本发明的融合蛋白通过抗体结合区与特异性抗体结合，然后内吞功能区使得所述融合蛋白和抗体被内吞入免疫细胞内部。因此，基于本发明的教导，本领域技术人员可自主选择本文所述的“抗体结合区”。本发明融合蛋白中的抗体结合区优选是正常细胞中不存在的，或者，是正常细胞中为隐蔽状态的或低表达的多肽。例如，所述抗体结合区表位在正常细胞(包括但不限于表达EGFR的正常细胞)中为表位隐蔽状态。
在具体的实施方式中，所述抗体可以是但不限于EGFR抗体、GPC3抗体、间皮素抗体等特异性结合，例如CH12抗体。所述抗体结合区具体选自以下抗原或其片段：EGFRvIII、EGFR、CD20、CD22、CD19、BCMA、proBDNF前体蛋白、GPC3、CLD18.2、CLD6、间皮素、PD-L1、PD-1、WT-1、IL13Ra2、Her-2、Her-1、Her-3；优选地，所述的抗体结合区含有以下任一氨基酸序列或者含有与以下氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列：SEQ ID NO:28、29、33、34、35、 36、37、38、39、40、41、42、43；更优地，所述的抗体结合区含有以下任一氨基酸序列的活性片段：SEQ ID NO:28、29、33、34、35、36、37、38、39、40、41、42、43。在具体的实施方式中，所述抗体结合区与EGFR抗体特异性结合。
本文所用的术语“内吞功能区”是指当所述融合蛋白与所述抗体结合区的特异性结合物质，例如抗体结合后，使得所述融合蛋白和所述物质被内吞入所述免疫细胞内部的功能部分。所述内吞功能区可以衍生自叶酸受体、LDL、CD30、CD33、CD3、EGFR、TFR1；优选衍生自叶酸受体和CD30；更优地，所述内吞功能区具有SEQ ID NO:32或44所示氨基酸序列，或者与SEQ ID NO：32或44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列，或者为SEQ ID NO:32或44所示氨基酸序列的活性片段。
本领域技术人员知晓，本发明的融合蛋白中的信号肽是起到帮助融合蛋白牵出细胞膜的功能。本领域技术人员可以自主决定具体的信号肽。例如，所述信号肽可以是叶酸受体信号肽、CD30受体信号肽、CD33信号肽、CD8信号肽，优选叶酸受体信号肽。本发明的融合蛋白中的信号肽和内吞功能区可以来自相同或不同的蛋白。
在具体的实施方式中，本发明的融合蛋白可以具有SEQ ID NO:10所示的氨基酸序列或者含有与SEQ ID NO:10具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列或其活性片段。
基于本发明的教导，本领域技术人员应理解，本发明的融合蛋白可以与嵌合抗原受体在免疫效应细胞表面单独表达或融合表达。在具体的实施方式中，本发明的融合蛋白与嵌合抗原受体在免疫效应细胞表面单独表达。在本文中，所述“单独表达”是指融合蛋白和嵌合抗原受体分别表达在免疫效应细胞表面，二者不为融合状态；而“融合表达”是指融合蛋白和嵌合抗原以融合蛋白的形式表达在免疫效应细胞表面。
在具体的实施方式中，本发明的融合蛋白与嵌合抗原受体在免疫效应细胞表面融合表达。
基于本发明的教导，本领域技术人员可以选择针对不同肿瘤抗原的嵌合抗原受体，例如，CD19-CAR、GPC3-CAR、CD30-CAR、Mesothelin-CAR等。在具体的实施方式中，所述嵌合抗原受体的编码核苷酸序列如SEQ ID NO:12所示。本领域技术人员还可利用本领域已知的技术手段促进本发明的融合蛋白与嵌合抗原受体在免疫效应细胞表面的融合表达，包括但不限于利用自剪切序列进行融合蛋白与嵌合抗原受体的融合表达。在具体的实施方式中，所述自剪切序列优选F2A或P2A。其中，F2A是来自口蹄疫病毒的2A(或称为“自剪切多肽2A”)的一段核心序列，具备2A的“自剪切”功能，可以实现上游和下游基因的共表达。2A由于其剪切效率高、上下游基因表达平衡性高及自身序列短小的优点为构建基因治疗多顺反子载体提供了一种有效的可行策略。在优选的实施方式中，所述自剪切序列是 vkqtlnfdllklagdvesnpgp(SEQ ID NO:30)。
在具体的实施方式中，本发明的融合蛋白如SEQ ID NO:31所示。
表达本发明融合蛋白的免疫效应细胞可以利用所述抗体结合区的特异性抗体实现高效杀伤，特别是当所述融合蛋白中抗体结合区在正常细胞中不存在或者隐蔽表达时，利用所述抗体结合区的特异性抗体杀伤免疫效应细胞时不会杀伤其它正常细胞，从而具备优异的差异毒性。
本发明的免疫效应细胞可以利用免疫辍合物特异性杀伤，所述免疫辍合物包括：特异性结合本发明融合蛋白中的抗体结合区的抗体，和细胞杀伤性功能部分。所述细胞杀伤性功能部分包括细胞毒性分子；优选地，所述功能部分选自MMAF、MMAE、Auristatin、卡奇霉素、美登素、美坦辛、阿霉素、紫杉醇、5-氟尿嘧啶、甲氨蝶呤、DM1、DM4、MGBA、SN-38。所述抗体与所述细胞杀伤性功能部分可以通过共价连接、偶联、附着、交联等方式构成辍合物。
本领域技术人员知晓，所述特异性结合所述融合蛋白的抗体结合区的抗体是与本发明融合蛋白中的正常细胞中不存在的抗体结合区相对应的。在具体的实施方式中，所述特异性结合所述融合蛋白的抗体结合区的抗体是CH12抗体，但不限于此。本领域技术人员可以基于现有技术中的知识将所述免疫缀合物制备成具有合适的尺寸，从而利于内吞入本发明的免疫效应细胞以便发挥杀伤作用。
本领域技术人员知晓，所述免疫辍合物的一种具体形式即是抗体药物偶联物或抗体药物缀合物(ADC)。在抗体药物偶联物或抗体药物缀合物(ADC)进入细胞后，其偶联的毒性药物在细胞内酸性环境下释放，并在细胞内发生毒性作用。因此，细胞上仅具有内吞功能区的受体与其相应的抗体药物偶联物或抗体药物缀合物(ADC)结合后，介导抗体药物偶联物或抗体药物缀合物(ADC)内吞，抗体药物偶联物或抗体药物缀合物(ADC)进入细胞后，其偶联的毒性药物在细胞内酸性环境下释放，并在细胞内发生毒性作用。
因此，本发明还提供这样一种表达嵌合抗原受体的免疫效应细胞，所述免疫效应细胞表达内吞功能区，所述的内吞功能区能将与所述内吞功能区结合的物质转运入所述的免疫效应细胞内。所述物质转运入所述的免疫效应细胞后启动对所述免疫效应细胞的杀伤。因此，本文所述的内吞功能区能将与所述内吞功能区结合的物质或与所述抗体结合区结合的物质转运入所述的免疫效应细胞内。
优选地，所述物质是抗体药物偶联物或抗体药物缀合物(ADC)。在具体的实施方式中，所述内吞功能区与嵌合抗原受体在免疫效应细胞表面单独表达或融合表达，优选单独表达。
在本发明的融合蛋白的基础上，本发明还提供了本发明的融合蛋白的编码核酸，包含所述编码核酸的表达载体以及包含所述表达载体或基因组中整合有所述编码核酸的宿主细胞。
本发明还提供了一种试剂盒，其包含本发明的免疫效应细胞或免疫缀合物用来进行治疗或对免疫效应细胞进行杀伤；即，通过给予本发明的免疫缀合物来杀伤所述免疫效应细胞。
本发明的优点：
1.本发明的免疫效应细胞可以被特异性抗体识别，并且能被该抗体衍生出的抗体偶联药物杀伤，同时对其它正常细胞的影响较小，因此，具备优异的差异毒性；
2.本发明的免疫效应细胞表面表达的融合蛋白能够在与特异性抗体结合后，使得所述融合蛋白和所述抗体偶联药物被内吞入所述免疫细胞内部，从而在细胞膜内部利用偶联的毒性强力的毒素分子完成对所述免疫效应细胞的杀伤，因此杀伤能力显著；和
3.本发明的技术方案对免疫效应细胞的杀伤主要是在细胞内完成的，受其他因素(如CDC和ADCC作用所依赖的补体系统和体内NK细胞活性)的影响少，从而能够对多种环境下的表达有本申请提供的融合蛋白的免疫效应细胞实现杀伤。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。例如，实施例中涉及的流式分析采用Beckman流式分析仪测定，测定结果采用FlowJo软件处理。以下实施例中所用的材料也均是市售可得的。
实施例1.融合蛋白FR806的表达
在本实施例中，选择eGFP作为荧光标记物进行分析测试，eGFP为增强型绿色荧光蛋白。选择F2A作为自剪切序列，F2A是来自口蹄疫病毒的2A(或称为“自剪切多肽2A”)的一段核心序列，具备2A的“自剪切”功能；选择人的叶酸受体的亚型1(FOLR1)的部分氨基酸序列(SEQ ID NO:32)和EGFR的部分序列(SEQ ID NO:28)表达为融合蛋白FR806(SEQ ID NO:44)，信号肽选择FOLR1的信号肽。用本领域技术人员已知的标准方法进行以下基因工程操作。eGFP-F2A-FR806的核苷酸(SEQ ID NO:1)的制备方法如下：
SEQ ID NO:1
(eGFP加粗显示，F2A以下划线显示，FR SP(叶酸受体信号肽)以加粗下划线显示，806表位以斜体显示，其余为叶酸受体剩余部分)
Figure PCTCN2017092108-appb-000001
Figure PCTCN2017092108-appb-000002
eGFP-F2A-FR806的氨基酸序列(SEQ ID NO:2)为：
Figure PCTCN2017092108-appb-000003
1、eGFP-F2A-FR806核苷酸序列的制备
1.1、参照J.Biol.Chem.264:14893-14901(1989)中的实验操作及Genebank登录号NM_016729.2的序列，制备得到如SEQ ID NO：3所示的FOLR1信号肽以及如SEQ ID NO：4所示的FOLR1其余部分的核苷酸序列。
SEQ ID NO：3
Figure PCTCN2017092108-appb-000004
SEQ ID NO：4
Figure PCTCN2017092108-appb-000005
参照Journal of Biological Chemistry,2004,279(29),30375-30384中的实验操作及Genebank登录号X00588.1的序列，制备得到EGFR的284-304表位的核苷酸序列(SEQ ID NO：5)。
SEQ ID NO：5
Figure PCTCN2017092108-appb-000006
将核苷酸序列SEQ ID NO：3、核苷酸序列SEQ ID NO：4、以及核苷酸序列SEQ ID NO：5按顺序组合后委托苏州金唯智生物科技有限公司进行全基因组合成后，得到FR806的核苷酸序列(SEQ ID NO:6)的基因片段。
SEQ ID NO:6
Figure PCTCN2017092108-appb-000007
Figure PCTCN2017092108-appb-000008
1.2、为得到3’端包含F2A(66bp)及和下游拼配的少许核酸(20bp)的eGFP核酸片段，以CN201310164725.X所采用的pWPT-eGFP-F2A-GPC3-BBZ为模板，模板的序列参见CN201310164725.X中的SEQ ID NO：28。
以上游引物5’-gcaggggaaagaatagtagaca-3’(SEQ ID NO:7)和下游引物5’-gttgtcatccgctgagccatgggcccagggttggactc-3’(SEQ ID NO:8)进行PCR扩增，得到3’端包含F2A(66bp)及和下游拼配的少许核酸(20bp)的eGFP核酸片段。。
1.3将等摩尔的步骤1.2获得的3’端包含F2A(66bp)及和下游拼配的少许核酸(20bp)的eGFP核酸片段与步骤1.1获得的FR806核苷酸序列片段按图1所示的模式进行拼接并PCR，图1中FR SP表述叶酸受体的信号肽(SEQ ID NO:3)，806epitope表示EGFR284-304表位(SEQ ID NO:5)，FR表示叶酸受体除信号肽外的其他部分(SEQ ID NO:4)。。补充DNA聚合酶，加入上游引物5’-gcaggggaaagaatagtagaca-3’(SEQ ID NO:7)，下游引物5’-ctcgaggtcgacctagctgagcagccacagc-3‘(SEQ ID NO:9)后PCR，得到两端包含MulⅠ个SalⅠ酶切位点的eGFP-F2A-FR806的核苷酸序列的基因片段，理论大小为2047bp，扩增产物经琼脂糖电泳确认与理论大小一致。
2、eGFP-F2A-FR806慢病毒载体的构建
本实施例采用的慢病毒质粒载体使用的载体系统属于第三代自灭活慢病毒载体系统，该系统包括：编码蛋白Gag/Pol、编码Rev蛋白的包装质粒psPAX2、编码VSV-G蛋白的包膜质粒PMD2.G及基于空载体pWPT-eGFP的编码目的基因eGFP-F2A-FR806的重组表达载体。
在空载体中pWPT-eGFP中，延长因子-1α(elongation factor-1α,EF-1α)的启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的表达，而在编码目的基因eGFP-F2A-FR806的重组表达载体中通过来自口蹄疫病毒的核糖体跳跃序列(food and mouth disease virus，FMDV,ribosomal skipping sequence，F2A)实现eGFP与目的基因FR806共表达。
将上述实施例1.1中所得的两端包含MulⅠ个SalⅠ酶切位点的eGFP-F2A-FR806的核苷酸序列的基因片段，通过MluⅠ和SalⅠ限制性内切酶双酶切，连入同样双酶切的pWPT载体中，构建得到由F2A相连的eGFP与FR806共表达的质粒pWPT-eGFP-F2A-FR806。
3、慢病毒包装与浓缩
以1.25×107的密度将293T细胞(ATCC)接种于15cm培养皿中，培养基为含10％胎牛血清(Gbico公司)的L110DMEM培养基(Gbico)。
将27.5μg的pWPT-eGFP-F2A-FR806的质粒和27.5μg的pWPT-eGFP(Mock)对照质粒分别和20.7μg的包装质粒PAX2、8.3μg包膜质粒pMD2.G溶于2200ul不含血清的DMEM培养基中，将165μg PEI(polyscience公司)溶于2200ul不含血清的DMEM培养基中，再将两者混匀，加入293T中，72h后收集包含病毒的上清液过滤，纯化后收集病毒浓缩液。
4、慢病毒转导T淋巴细胞
将人外周血单个核细胞以约1×106/mL密度加入淋巴细胞培养基液培养，并按照磁珠:细胞比例为1:1加入同时包被有抗CD3和CD28抗体的磁珠(Invitrogen公司)和终浓度300U/mL的重组人IL-2(上海华新生物高技术有限公司)活化48h。
将活化好的T细胞按照1×106cell/ml的浓度加入Retronectin(购买自takara公司)包被的板(24孔板)中，加入步骤3所得的病毒浓缩液(MOI≈10)，离心，放入培养箱中培养，得到表达融合蛋白FR806及eGFP的T细胞(CAR-FR806-T细胞)以及Mock T细胞，其中，FR806融合蛋白序列还含有信号肽，如SEQ ID NO:10所示：
SEQ ID NO：10
Figure PCTCN2017092108-appb-000009
5、流式检测T细胞中融合受体FR806及eGFP的表达
取步骤4得到的CAR-FR806-T细胞和Mock T细胞，。一抗用CN 200810038848.8所公开的CH12抗体(10μg/ml)孵育45min，随后1％FBS的PBS洗两遍。二抗为PE标记的羊抗人IgG(Santa公司)，1:50稀释孵育45min。其后1％FBS的PBS洗两遍重悬后，流式分析，结果如图2A所示，说明表达了FR806融合蛋白的T细胞能够与CH12抗体有效结合，并且能与eGFP在T细胞中共表达。CH12抗体的轻链如SEQ ID NO:46所示，重链如SEQ ID NO:45所示。
选择表达EGFR的Keratinocyte细胞和HEK-293T细胞，FACS分析CH12抗体与二者的结合情况，结果显示CH12抗体与表达EGFR的Keratinocyte细胞和HEK-293T细胞均不结合(图2B)。
实施例2、CH12-biotin的合成及滴定
将CH12抗体用biotin进行标记。将CH12抗体稀释到2.5mg/ml，PBS pH7.4，标记体积1.6ml；取1mg的Sulfo-NHS-LC-Biotin(Thermo公司)，加入180ul的超纯水溶解；取79ul的Biotin加入到1.6ml的CH12抗体中，反应过夜。使用PD-10脱盐柱(美国GE公司)脱盐，置换到PBS 5％甘油缓冲液中，得到CH12-Biotin，OD280/1.45测浓度为0.77mg/ml。
用含有1％FBS的PBS将CH12-biotin稀释成不同浓度(100μg/ml、10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml、0μg/ml)，分别与表达eGFP-F2A-FR806的T细胞孵育45min，随后PBS洗涤，二抗为PE-SA(ebioscience公司)1∶300培基稀释，加入重悬细胞后，孵育45min。PBS洗两次后，流式分析，结果如图3所示，显示随着CH12-biotin浓度越高，亲和 力越强，10μg/ml与100μg/ml的结合水平相似。
实施例3、利用CH12-biotin分选FR806阳性的T细胞
取1×107表达eGFP-F2A-FR806的T细胞，PBS洗涤，CH12-biotin(10μg/ml，含有1％FBS的PBS稀释)4℃孵育45min，随后PBS洗涤，加入抗Biotin分选磁珠(购买自美天旎公司)，按照该分选磁珠产品所给出的步骤分选出FR806的T细胞。取适量分选前后的细胞，流式分析，结果如图4所示，显示表达FR806的T细胞在与CH12-biotin结合后能被抗Biotin分选磁珠有效分选出，分选阳性率达95％。
实施例4、表达FR806的T细胞的内吞实验
取实施例1所得的感染慢病毒载体pWPT-eGFP-F2A-FR806及pWPT-eGFP(Mock)的T细胞，PBS洗涤；取实施例2中合成的CH12-biotin(10μg/ml，培基稀释)，二抗为PE-SA(ebioscience公司)1∶300培基稀释，加入重悬细胞后，孵育45min，PBS洗两次后孵育4h。随后，多聚甲醛固定，DAPI染色液(罗氏公司)染色，在共聚焦显微镜下观察，结果如图5所示，在表达FR806的T细胞中，CH12-biotin(红色荧光表示)出现在了细胞膜内部，显示其能被T细胞有效内吞。
实施例5、抗体偶联药物CH12-MMAF的合成及细胞杀伤活性测定
取1ml(0.033mM)CH12抗体，加入10ul DTPA(Thermo公司)和1ul 100mM的TCEP(Thermo公司)，，按照抗体:MMAF＝10:1的比例加入MMAF的DMSO溶液(浓度3.4mM)，4℃维持3h，除去多余的MMAF，得到抗体偶联药物CH12-MMAF。
流式检测CH12抗体和CH12-MMAF对表达FR806的T细胞的结合能力，结果如图6A所示。
参照实施例4的操作，取感染pWPT-eGFP-F2A-FR806及pWPT-eGFP(Mock)的T细胞，PBS洗涤；取CH12-MMAF(10μg/ml，培基稀释)4℃孵育45min，PBS洗涤；二抗为羊抗人PE(上海联科生物技术有限公司)，1：50稀释，加入重悬细胞后孵育45min。PBS洗两次后，培养箱孵育4h。随后多聚甲醛固定，DAPI染色液(罗氏公司)1∶500稀释，二抗染色2min，共聚焦显微镜下观察拍摄，结果如图6B所示，CH12-MMAF能够被表达FR806的T细胞内吞。
流式检测感染Mock、eGFP-FR806的T细胞的阳性率后，通过添加适当比例未感染病毒的T细胞，将Mock(对照组)与eGFP-FR806(实验组)的T细胞的阳性率调整至50％，铺于6孔板中，每孔2×106个细胞，2ml培养基(AIM-Ⅴ培基+2％human AB serum、IL-2 500U/ml)。用PBS将CH12-MMAF药物分别稀释到0.01、0.1、1、10、以及100μg/ml后加入到实验组和对照组细胞中，每24h流式检测eGFP阳性率变化，共测定96h，结果如图6C所示，显示在加入CH12-MMAF后，对表达FR806的T细胞的有明显的杀伤，且随着CH12-MMAF浓度的增加，对表达FR806的T细胞的杀伤增强，在用量为10μg/ml时，96h，对表达FR806 的T细胞的杀伤作用可达88％。对于不表达FR806的T细胞(Mock)，则CH12-MMAF不显出杀伤，说明CH12-MMAF的安全性好。
检测CH12-MMAF对人Keratinocy细胞的杀伤作用，结果如图6D所示，CH12-MMAF对人Keratinocy细胞没有杀伤，说明CH12-MMAF安全性好。
实施例6、CCK8测定CH12-MMAF药物及游离的MMAF对表达FR806的T细胞的杀伤
实验组：取实施例3分选后的表达eGFP-FR806的T细胞，将其铺于96孔板中，每孔3×104个细胞，100ul培基，每个药物浓度5个复孔，再设定一组只加培基的空白组。对照组：取未感染病毒的T细胞，参照实验组的操作铺于96孔板中。分别取100μg/ml、10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml、0μg/ml六种溶度的CH12-MMAF分别加入到实验组和对照组T细胞中，做成六个梯度(即，前述的100μg/ml、10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml、0μg/ml六种溶度)。72h后每孔加入10ul CCK8试剂(Dojindo公司)37℃孵育3h后酶标仪测定450nm处吸光度，分别计算细胞活力。
参照上述操作取分选后的感染eGFP-FR806的T细胞，将其铺于96孔板中，每孔3×104个细胞，100ul培基，每个药物浓度5个复孔，再设定一组只加培基的空白组。对照组为未感染病毒的T细胞，相同方法铺于96孔板中。将1000nM、500nM、100nM、50nM、10nM、0nM六种溶度的游离MMAF按照特定浓度加入到T细胞中，做成六个梯度(即，前述的六种溶度)。72h后每孔加入10ul CCK8试剂(Dojindo公司)37℃孵育3h后酶标仪测定450nm处吸光度，分别计算细胞活力。
计算公式为：细胞活力(％)＝[A(加药)-A(空白)]/[A(0加药)-A(空白)]
结果如图7A所示，显示CH12-MMAF能特异杀伤FR806阳性的T细胞。游离的MMAF对表达和不表达FR806的T细胞的杀伤水平相当。
进一步的，申请人选择EGFR+的HEK293T细胞，将其表达FR806，并进行细胞杀伤实验，结果如图7B所示，显示CH12-MMAF对FR806阳性的HEK293T有显著的细胞杀伤，而对FR806阴性的HEK293T无明显杀伤，MMAF对FR806阳性和阴性的HEK293T均有杀伤。说明即使细胞显示EGFR阳性，但不表达FR806时，CH12-MMAF不会产生杀伤。
实施例7、FR806-CAR19T细胞的制备
本实施例选择eGFP作为荧光标记物，eGFP为增强型绿色荧光蛋白。用本领域技术人员已知的标准方法进行以下基因工程操作。
本实施例中，选择US20060193852A1公开的αCD19的单链抗体的核苷酸片段(SEQ ID NO:11)作为CAR的抗CD19抗体序列，选择CD8-CD137-CD3ζ作为CAR的跨膜域和胞内域。
SEQ ID NO:11
Figure PCTCN2017092108-appb-000010
Figure PCTCN2017092108-appb-000011
1、FR806-F2A-CAR(CD19)-F2A-eGFP的核苷酸序列的制备
1.1、3’端及5’端分别带有部分F2A片段的αCD19CAR核苷酸序列
委托苏州金唯智生物科技有限公司进行全基因组合成，得到αCD19CAR的核苷酸序列的基因片段(SEQ ID NO:12)，含有CD8α信号肽序列、αCD19的单链抗体的核苷酸片段、以及包含有铰链区、跨膜区、胞内段的序列的CD8-CD137-CD3ζ核酸片段。
SEQ ID NO:12(加粗部分为CD8α信号肽序列，下划线为αCD19CAR核苷酸序列，斜体加粗为CD8-CD137-CD3ζ核苷酸序列)
Figure PCTCN2017092108-appb-000012
1.2、以上述合成的αCD19CAR(SEQ ID NO:12)的核苷酸序列的基因片段为模板，扩增采取的引物对为上游引物5’-ccttctgaagttggcaggagacgttgagtccaaccctgggcccatggccttaccagtg-3‘(SEQ ID NO:13)，下游引物5’-tcctgccaacttcagaaggtcaaaattcaaagtctgtttcacgcgagggggcagggc-3‘(SEQ ID NO:14)，得到3’端及5’端分别带有部分F2A片段的αCD19CAR核苷酸序列。PCR扩增条带通过琼脂糖凝胶电泳确定符合预计的片段大小。
2、FR806-F2A-CAR19-F2A-eGFP的核酸序列的制备
为制备FR806、αCD19CAR及eGFP的拼接序列FR806-F2A-CAR19-F2A-eGFP(SEQ ID NO:15)，使用以下操作：
SEQ ID NO:15(FR806以下划线显示，αCD19CAR以加粗下划线显示，F2A加粗显示，eGFP正常显示)
Figure PCTCN2017092108-appb-000013
Figure PCTCN2017092108-appb-000014
2.1、以实施例1中构建的eGFP-F2A-FR806慢病毒载体为模板进行PCR扩增，扩增采取的引物对为上游引物5’-cttacgcgtcctagcgctaccggtcgccaccatggctcagcggatg-3‘(SEQ ID NO:16)，下游引物5’-gtctcctgccaacttcagaaggtcaaaattcaaagtctgtttcacgctgagcagccac-3‘(SEQ ID NO:17)。目的扩增条带的大小为910bp。PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，1min；25个循环后再总延伸68℃，10min。PCR扩增条带通过琼脂糖凝胶电泳确定符合目的扩增条带的大小。
2.2、5’端带有部分F2A片段的eGFP-F2A-FR806序列的扩增
以实施例2中构建的eGFP-F2A-FR806慢病毒载体为模板，扩增采取的引物对为上游引物5’-accttctgaagttggcaggagacgttgagtccaaccctgggcccatggtgagcaagggc-3'(SEQ ID NO:18)，下游引物5’-ctcgaggtcgacctacttgtacagctcg-3’(SEQ ID NO:19)。得到5’端带有部分F2A片段的eGFP-F2A-FR806核酸片段。PCR扩增条带通过琼脂糖凝胶电泳确定符合预计的片段大小。
2.3、将等摩尔的3’端及5’端分别带有部分F2A片段的αCD19CAR的核苷酸序列与3’ 端带有部分F2A片段的FR806序列按图8A所示的模式进行拼接并PCR。补充DNA聚合酶，加入上游引物5’-cttacgcgtcctagcgctaccggtcgccaccatggctcagcggatg-3’(SEQ ID NO:16)，下游引物5’-tcctgccaacttcagaaggtcaaaattcaaagtctgtttcacgcgagggggcagggc-3’(SEQ ID NO:14)后PCR25个循环，得到FR806与αCD19CAR的核苷酸序列的拼接片段。理论大小为2458bp，扩增产物经琼脂糖电泳确认与理论大小一致。
2.4、将等摩尔的FR806与αCD19CAR的核苷酸序列的拼接片段与5’端带有部分F2A片段的eGFP序列按图8A所示的模式进行拼接并PCR。补充DNA聚合酶，加入上游引物5’-cttacgcgtcctagcgctaccggtcgccaccatggctcagcggatg-3‘(SEQ ID NO:16)，下游引物5’-ctcgaggtcgacctacttgtacagctcg-3‘(SEQ ID NO:19)后PCR25个循环，得到两端带有MluⅠ和SalⅠ限制性内切酶位点的FR806与αCD19CAR与eGFP的拼接片段FR806-F2A-CAR19-F2A-eGFP。理论大小为3214bp，扩增产物经琼脂糖电泳确认与理论大小一致。
3、FR806-F2A-CAR19-F2A-eGFP慢病毒载体的构建
参照实施例1中慢病毒载体的构建的操作，将所得的FR806-F2A-CAR19-F2A-eGFP的核苷酸序列通过MluⅠ和SalⅠ限制性内切酶双酶切，连入同样双酶切的pWPT载体中，构建得到由F2A相连的FR806、αCD19CAR与eGFP共表达的慢病毒表达载体。
4、质粒转染293T包装慢病毒
参照实施例1中步骤3的操作，将本实施例步骤2所得的慢病毒表达载体、pWPT-eGFP对照质粒、包装质粒PAX2、包膜质粒pMD2.G溶于2200ul不含血清的DMEM培养基中，进行慢病毒包装。
5、慢病毒转导T细胞
参照实施例1中步骤4的操作，将本实施例步骤3所得的包装后的慢病毒转染T细胞，得到表面表达有CAR19及FR806的CAR-T细胞，即FR806-CAR19T细胞，将FR806-CAR19T细胞进行流式分析，结果如图8B所示，显示三种蛋白FR806、eGFP、和αCD19CAR均能在T细胞中能有效的表达。
参照实施例3的操作，采用CH12-biotin和抗-biotin磁珠分选FR806-CAR19T细胞，结果如图8C所示，显示FR806-CAR19T细胞在与CH12-biotin结合后能被抗Biotin分选磁珠有效分选出，分选阳性率达94.3％。
参照上述操作，按照9A所示的模式进行拼接并PCR，得到表达FR806和CAR19的T细胞(FR806-CAR19T细胞)，流式分析，结果如图9B所示。
实施例8、FR806-CAR19T细胞对肿瘤细胞的杀伤以及细胞因子释放
参照实施例7的操作，制备得到表达CAR19，不表达FR806的T细胞，即CAR19T细胞。将参照图9A拼接所得的FR806-CAR19T细胞进行细胞杀伤实验。
以Daudi细胞为靶细胞，效应细胞为FR806-CAR19T细胞和CAR19T细胞，效靶比分别为20:1，10:1，5:1，2.5:1，靶细胞数量为10000/孔，根据不同效靶比设定不同数量效应细 胞。各组均设5个复孔。实验组为FR806-CAR19T细胞和CAR19T细胞与Daudi细胞共孵育，对照组为感染Mock病毒的T细胞与Daudi细胞共孵育。共孵育4h后通过CytoTox96非放射性细胞毒性试剂盒(Promega公司)检测上清中LDH含量，计算其杀伤活性。具体参照CytoTox96非放射性细胞毒性试剂盒说明书。结果如图10A显示，FR806-CAR19T细胞的细胞杀伤活性略好于CAR19T细胞。
按效靶比为1:1，将CAR19T细胞、CAR19-FR806T细胞以及空质粒转染的T细胞(Mock)与Daudi细胞共孵育24h，ELISA检测IFN-γ,IL-2and TNF-α的分泌水平，结果如图10B所示，显示表达FR806对CAR-T细胞的细胞因子释放水平基本无影响。
实施例9、CH12-MMAF对FR806-CAR19T细胞的体外杀伤效果
将参照图8A拼接所得的FR806-CAR19T细胞及对照组mock的初始阳性率调整为50％，加入10μg/ml的CH12-MMAF，每24h对eGFP的阳性率进行流式检测，共检测96h，结果如图11A所示，在24h，FR806-CAR19的T细胞数量已降低，72h，FR806-CAR19的T细胞数量降低约80％。
将FR806-CAR19T细胞铺于96孔板，每孔3×104个细胞，100ul培基，每个药物浓度5个复孔，再设定一组只加培基的空白组。对照组：取未感染病毒的T细胞，参照实验组的操作铺于96孔板中。分别取100μg/ml、10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml、0μg/ml六种溶度的CH12-MMAF分别加入到实验组和对照组T细胞中，做成六个梯度(即，前述的100μg/ml、10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml、0μg/ml六种溶度)。72h后每孔加入10ul CCK8试剂(Dojindo公司)37℃孵育3h后酶标仪测定450nm处吸光度，分别计算细胞活力。
计算公式为：细胞活力(％)＝[A(加药)-A(空白)]/[A(0加药)-A(空白)]
结果如图11B所示，显示CH12-MMAF能特异杀伤FR806阳性的CAR T细胞，而不杀伤Mock细胞。
实施例10、测定CH12-MMAF对FR806-CAR19T细胞的体内杀伤效果
将参照图8A拼接所得的FR806-CAR19T细胞进行下述实验。
在NOD/SCID小鼠接种3×106Daudi细胞，第12天，NOD/SCID小鼠环磷酰胺(100mg/kg)清淋。第十四天，小鼠尾静脉注射FR806-CAR19T细胞(3×107细胞/只)。第15天，实验组给药CH12-MMAF，0.1mg/只，对照组给生理盐水。第18天，取小鼠外周血、骨髓、脾脏，红细胞裂解液(ebioscience公司)裂解红细胞，PBS洗涤干净后，加入PE标记的羊抗人CD3抗体(1：50，含有1％FBS的PBS稀释)，4度孵育45分钟后，含有1％FBS的PBS洗涤，如图12A所示流式分析eGFP阳性率。
流式结果如11B和11C所示，给予CH12-MMAF后，人CD3+/eGFP+的细胞在血液中降低了93％，在脾脏中降低了94％，在骨髓中降低了64％，而对照组中，人CD3+/eGFP+的细胞在血液、脾脏、以及骨髓中的检出量分别是40.8％、37.7％、52.8％结果说明，CH12-MMAF能够有效清除小鼠体内的FR806-CAR19T细胞。
实施例11、eGFP-F2A-CD30806在T细胞的表达
在本实施例中，选择eGFP作为荧光标记物进行分析测试，eGFP为增强型绿色荧光蛋白。选择F2A作为自剪切序列，F2A是来自口蹄疫病毒的2A(或称为“自剪切多肽2A”)的一段核心序列，具备2A的“自剪切”功能，可以实现上游和下游基因的共表达。选择CD30的部分氨基酸序列(SEQ ID NO:44)和EGFR的部分序列(SEQ ID NO:28)表达为融合蛋白CD30806，信号肽选择CD30的信号肽。用本领域技术人员已知的标准方法进行以下基因工程操作。eGFP-F2A-CD30806的核苷酸(SEQ ID NO:20)的制备方法如下：
SEQ ID NO:20
其中，eGFP加粗显示，F2A以下划线显示，CD30SP以加粗下划线显示，806表位以斜体显示，linker以斜体下划线显示，其余为CD30受体跨膜区及胞内段。
Figure PCTCN2017092108-appb-000015
eGFP-F2A-CD30806的氨基酸序列(SEQ ID NO:21)为：
Figure PCTCN2017092108-appb-000016
1、eGFP-F2A-CD30806核苷酸序列的制备
1.1、参照Cell.1992Feb 7；68(3):421-7中的实验操作及Genebank登录号NM_001243.4的序列，制备得到如SEQ ID NO：22所示的CD30信号肽以及如SEQ ID NO：23所示的CD30受体跨膜区及胞内段核苷酸序列。
SEQ ID NO：22
Figure PCTCN2017092108-appb-000017
SEQ ID NO：23
Figure PCTCN2017092108-appb-000018
参照Journal of Biological Chemistry,2004,279(29),30375-30384中的实验操作及Genebank登录号X00588.1的序列，制备得到表皮生长因子受体284-304表位的核苷酸序列(SEQ ID NO：5)。
SEQ ID NO：5
Figure PCTCN2017092108-appb-000019
参照本实验室申请的编码GPC-3嵌合抗原受体蛋白的核酸及表达GPC-3嵌合抗原受体蛋白的T淋巴细胞专利(中国申请专利CN201310164725.X)中的序列GPC3-Z(SEQ ID NO：18)得到连接806表位和CD30跨膜段和胞内段的linker的核苷酸序列(SEQ ID NO：24)。
SEQ ID NO：24
ggtggaggcggttcaggcggaggtggctctggcggtggcggatcg(为GPC3-Z中的一段linker)
将核苷酸序列SEQ ID NO：22、核苷酸序列SEQ ID NO：23、核苷酸序列SEQ ID NO：24、以及核苷酸序列SEQ ID NO：5按顺序组合后委托苏州金唯智生物科技有限公司进行全基因组合成后，得到CD30806的核苷酸序列(SEQ ID NO:25)的基因片段。
SEQ ID NO:25
Figure PCTCN2017092108-appb-000020
1.2、为得到3’端包含F2A(66bp)及和下游拼配的少许核酸(20bp)的eGFP核酸片段，以CN201310164725.X所采用的pWPT-eGFP-F2A-GPC3-BBZ为模板，模板的序列参见CN201310164725.X中的SEQ ID NO：28。
以上游引物5’-gcaggggaaagaatagtagaca-3’(SEQ ID NO:7)和下游引物5’-gcggcgaggaggacgcgcatgggcccagggttggactc-3’(SEQ ID NO:26)进行PCR扩增，得到3’端包含F2A(66bp)及和下游拼配的少许核酸(20bp)的eGFP核酸片段。1.3将等摩尔的步骤1.2获得的3’端包含F2A(66bp)及和下游拼配的少许核酸(20bp)的eGFP核酸片段与步骤1.1获得的CD30806核苷酸序列片段进行拼接并PCR。补充DNA聚合酶，加入上游引物 5’-gcaggggaaagaatagtagaca-3’(SEQ ID NO:7)，下游引物5’-ctcgaggtcgacctactttccagaggcagctg-3‘(SEQ ID NO:27)后PCR25个循环，得到两端包含MulⅠ个SalⅠ酶切位点的eGFP-F2A-CD30806的核苷酸序列的基因片段。理论大小为2023bp，扩增产物经琼脂糖电泳确认与理论大小一致。
2、eGFP-F2A-CD30806慢病毒载体的构建
本实施例采用的慢病毒质粒载体使用的载体系统属于第三代自灭活慢病毒载体系统，该系统包括：编码蛋白Gag/Pol、编码Rev蛋白的包装质粒psPAX2、编码VSV-G蛋白的包膜质粒PMD2.G及基于空载体pWPT-eGFP的编码目的基因eGFP-F2A-FR806的重组表达载体。
在空载体中pWPT-eGFP中，延长因子-1α(elongation factor-1α,EF-1α)的启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的表达，而在编码目的基因eGFP-F2A-FR806的重组表达载体中通过来自口蹄疫病毒的核糖体跳跃序列(food and mouth disease virus，FMDV,ribosomal skipping sequence，F2A)实现eGFP与目的基因FR806共表达。
将上述实施例1.1中所得的两端包含MulⅠ个SalⅠ酶切位点的eGFP-F2A-CD30806的核苷酸序列的基因片段，通过MluⅠ和SalⅠ限制性内切酶双酶切，连入同样双酶切的pWPT载体中，构建得到由F2A相连的eGFP与CD30806共表达的质粒pWPT-eGFP-F2A-CD30806，进行病毒包装和T细胞转染，得到表达CD30-806融合蛋白及eGFP的T细胞。
CAR-T细胞杀伤活性实验：取感染了eGFP-CD30806的T细胞(简称CD30-806)，3×105密度铺板，分别在每孔中加入不同浓度的CH12-MMAF，72h后收集细胞，流式细胞术观察每孔中eGFP阳性(即CD30-806阳性的细胞)细胞的比例。结果如图13所示，随着CH12-MMAF加药浓度的提高，CD30-806阳性的细胞比例也逐渐减少，说明CH12-MMAF对CD30-806阳性的细胞有较强的杀伤毒性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (28)

  1. 一种在表面表达嵌合抗原受体的免疫效应细胞，其特征在于，所述免疫细胞还表达式I所示的融合蛋白，
    Z-A-L-B
    I
    其中，Z是任选的信号肽；
    A是抗体结合区；
    L是任选的接头部分；
    B是内吞功能区。
  2. 一种表达嵌合抗原受体的免疫效应细胞，其特征在于，所述免疫细胞还表达一融合蛋白，该融合蛋白含有抗体结合区和内吞功能区。
  3. 如权利要求1或2所述的免疫效应细胞，其特征在于，所述的抗体结合区选自以下抗原或其片段：EGFRvIII、EGFR、CD20、CD22、CD19、BCMA、proBDNF前体蛋白、GPC3、CLD18.2、CLD6、间皮素、PD-L1、PD-1、WT-1、IL13Ra2、Her-2、Her-1、Her-3；
    优选的，所述的抗体结合区含有以下任一氨基酸序列或者含有与以下氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列：SEQ ID NO:28、29、33、34、35、36、37、38、39、40、41、42、43；
    更优的，所述的抗体结合区含有以下任一氨基酸序列的活性片段：SEQ ID NO:28、29、33、34、35、36、37、38、39、40、41、42、43。
  4. 如权利要求3所述的免疫效应细胞，其特征在于，所述抗体结合区与EGFR抗体特异性结合。
  5. 如权利要求1或2所述的免疫效应细胞，其特征在于，所述内吞功能区衍生自叶酸受体、LDL、CD30、CD33、CD3、EGFR、TFR1；优选衍生自叶酸受体和CD30；更优地，所述内吞功能区具有SEQ ID NO：32或44所示氨基酸序列，或者与SEQ ID NO：32或44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列，或者为SEQ ID NO:32或44所示的氨基酸序列的活性片段。
  6. 如权利要求1所述的免疫效应细胞，其特征在于，所述信号肽为叶酸受体信号肽。
  7. 如权利要求6所述的免疫效应细胞，其特征在于，所述融合蛋白具有SEQ ID NO:10所示的氨基酸序列或者含有与SEQ ID NO:10具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列。
  8. 如权利要求1或2所述的免疫效应细胞，其特征在于，所述融合蛋白与嵌合抗原受体在免疫效应细胞表面单独表达或融合表达，优选单独表达。
  9. 一种表达嵌合抗原受体的免疫效应细胞，其特征在于，所述细胞还表达内吞功能区，所述的内吞功能区能将与所述内吞功能区结合的物质转运入所述的免疫效应细胞内。
  10. 如权利要求9所述的免疫效应细胞，其特征在于，所述内吞功能区衍生自叶酸受体、 LDL、CD30、CD33、CD3、EGFR、TFR1；优选衍生自叶酸受体和CD30；更优地，所述内吞功能区具有SEQ ID NO:32或44所示氨基酸序列，或者与SEQ ID NO:32或44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列，或者为SEQ ID NO:32或44所示的氨基酸序列的活性片段。
  11. 如权利要求9或者10所述的免疫效应细胞，其特征在于，所述内吞功能区与嵌合抗原受体在免疫效应细胞表面单独表达或融合表达，优选单独表达。
  12. 式I所示的融合蛋白，
    Z-A-L-B
    I
    其中，Z是任选的信号肽；
    A是抗体结合区；
    L是任选的接头部分；
    B是内吞功能区。
  13. 一种融合蛋白，所述融合蛋白包含抗体结合区和内吞功能区。
  14. 如权利要求12或13所述的融合蛋白，其特征在于，所述的抗体结合区选自以下抗原或其片段：EGFRvIII、EGFR、CD20、CD22、CD19、BCMA、proBDNF前体蛋白、GPC3、CLD18.2、CLD6、间皮素、PD-L1、PD-1、WT-1、IL13Ra2、Her-2、Her-1、Her-3；
    优选的，所述的抗体结合区含有以下任一氨基酸序列或者含有与以下氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列：SEQ ID NO:28、29、33、34、35、36、37、38、39、40、41、42、43；
    更优的，所述的抗体结合区含有以下任一氨基酸序列的活性片段：SEQ ID NO:28、29、33、34、35、36、37、38、39、40、41、42、43。
  15. 如权利要求14所述的融合蛋白，其特征在于，所述抗体结合区与EGFR抗体特异性结合。
  16. 如权利要求12或13所述的融合蛋白，其特征在于，所述内吞功能区衍生自叶酸受体、LDL、CD30、CD33、CD3、EGFR、TFR1；优选衍生自叶酸受体和CD30；更优地，所述内吞功能区具有SEQ ID NO:32或44所示氨基酸序列，或者与SEQ ID NO：32或44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列，或者为SEQ ID NO:32或44所示氨基酸序列的活性片段。
  17. 如权利要求12所述的融合蛋白，其特征在于，所述信号肽为叶酸受体信号肽。
  18. 如权利要求17所述的融合蛋白，其特征在于，所述融合蛋白具有SEQ ID NO:10所示的氨基酸序列或者含有与SEQ ID NO:10具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列。
  19. 如权利要求12-18中任一项所述的融合蛋白的编码核酸。
  20. 一种表达载体，包含权利要求19所述的编码核酸。
  21. 一种宿主细胞，包含权利要求20所述的表达载体或基因组中整合有权利要求19所 述的编码核酸。
  22. 一种免疫辍合物，所述免疫辍合物包括：
    细胞杀伤性功能部分；和
    特异性结合权利要求1-8所述免疫效应细胞中的抗体结合区或内吞功能区的抗体或者特异性结合权利要求9-11所述免疫效应细胞中的内吞功能区的抗体。
  23. 如权利要求22所述免疫辍合物在特异性杀伤权利要求1-11中任一项所述免疫效应细胞中的用途。
  24. 一种试剂盒，包含权利要求1-11中任一项所述的免疫效应细胞或权利要求22所述的免疫缀合物。
  25. 特异性清除权利要求1-11中任一项所述免疫效应细胞的方法，所述方法包括给予权利要求22所述的免疫缀合物的步骤。
  26. 一种分选或富集权利要求1-11中任一项所述的免疫效应细胞的方法，所述方法包括以下步骤：
    将分选试剂加入包含所述免疫效应细胞的体系，所述分选试剂包含能够特异性结合权利要求1-8所述免疫效应细胞中的抗体结合区或内吞功能区的物质或者特异性结合权利要求9-11所述免疫效应细胞中的内吞功能区的物质；和
    将结合有所述免疫效应细胞的物质从所述体系分离的步骤。
  27. 如权利要求26所述的方法，其特征在于，能够特异性结合权利要求1-8所述免疫效应细胞中的抗体结合区或内吞功能区的物质或者特异性结合权利要求9-11所述免疫效应细胞中的内吞功能区的物质固定于固相载体，从而能够实现结合有所述免疫效应细胞的物质从所述体系分离。
  28. 检测权利要求1-11中任一项所述的免疫效应细胞的方法，所述方法包括：
    给予特异性结合权利要求1-8所述免疫效应细胞中的抗体结合区或内吞功能区的检测试剂或者特异性结合权利要求9-11所述免疫效应细胞中的内吞功能区的检测试剂，所述检测试剂与可检测标记物相连；和
    检测所述检测试剂与所述免疫效应细胞形成的复合物。
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