WO2023143245A1 - Cd45作为生物标志物在筛查cd26抗体或其衍生物治疗肿瘤有效性和精准性中的应用 - Google Patents

Cd45作为生物标志物在筛查cd26抗体或其衍生物治疗肿瘤有效性和精准性中的应用 Download PDF

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WO2023143245A1
WO2023143245A1 PCT/CN2023/072600 CN2023072600W WO2023143245A1 WO 2023143245 A1 WO2023143245 A1 WO 2023143245A1 CN 2023072600 W CN2023072600 W CN 2023072600W WO 2023143245 A1 WO2023143245 A1 WO 2023143245A1
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antibody
cells
derivative
treatment
expression
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French (fr)
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马永
曹锫沛
葛晨楠
杭建花
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江苏众红生物工程创药研究院有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to a biomarker for predicting clinical curative effect, in particular to the application of CD45 as a biomarker for predicting clinical curative effect, and more specifically to the application of CD45 in screening the effectiveness and accuracy of treating tumors with CD26 antibody or its derivatives .
  • Precision medicine is based on clinicopathological and molecular characteristics, customized diagnosis, treatment and prognosis judgment strategies that can accurately meet the actual needs of different patients. Its scope includes precision prevention (prediction of disease risk and preventive intervention), precision diagnosis (early detection and diagnosis of disease, molecular typing), precision treatment (molecular targeted therapy, prediction and monitoring of curative effect, precision surgical technology, etc.) .
  • biomarkers can be divided into: diagnostic biomarkers to clarify disease state or type, prognostic biomarkers to reflect the prognosis of the disease, predictive biomarkers to predict the response to intervention measures, and reflect the biomarkers produced after treatment.
  • diagnostic biomarkers to clarify disease state or type
  • prognostic biomarkers to reflect the prognosis of the disease
  • predictive biomarkers to predict the response to intervention measures
  • reflect the biomarkers produced after treatment and reflect the biomarkers produced after treatment.
  • Pharmacodynamic biomarkers for drug response and safety biomarkers for monitoring to avoid or reduce safety risks.
  • predictive biomarkers can be used to screen people who are more likely to benefit from treatment and exclude patients who do not benefit clinically.
  • Immune checkpoint inhibitors such as PD-1/PD-L1 and CTLA-4 have been extensively studied in various solid tumors and have been used as first-line treatment for non-small cell lung cancer, but the overall objective response rate is still only 20%. %about.
  • the expression of PD-L1 can be used as a predictive marker for the response to anti-PD-1/PD-L1 therapy, and tumor mutation burden (TMB) has also been shown to correlate with the efficacy of immune checkpoint inhibitors in melanoma, lung adenocarcinoma, and bladder cancer. related.
  • CD26 is a multifunctional type II transmembrane glycoprotein that can also be present in plasma in a dissolved form.
  • CD26 often exists in the form of homodimers, and its monomer contains 766 amino acids with a relative molecular mass of about 110kDa.
  • CD26 amino acid residues are divided into 5 parts from inside to outside: intracellular region (1-6), transmembrane region (7-28), highly glycosylated region (29-323), cysteine-rich region ( 324-551) and the C-terminal catalytic domain (552-766), the molecular three-dimensional structure and function are closely related.
  • CD26 (DPP4) inhibitors have been clinically used in the treatment of type II diabetes for decades.
  • CD26 on the surface of various tumor cells
  • the expression level is significantly increased, for example, malignant mesothelioma, kidney cancer, prostate cancer, lung cancer, etc.
  • CD26 is a valuable target (CD26/DPP4-a potential biomarker and target for cancer therapy, Pharmacology & Therapeutics (2019), 198:135-159).
  • YS110 only It can reduce tumor volume to a certain extent, alleviate tumor growth, but cannot completely inhibit tumor growth to achieve tumor cure, so its activity in animal models is still not ideal.
  • the results of the Phase II clinical study of YS110 have shown that YS110 is well tolerated, but the disease control rate did not meet expectations, which corresponds to the low activity of YS110 shown in preclinical studies.
  • CD26 antibody During the research of CD26 antibody, the inventors found that some CD26+ cells are sensitive to CD26 antibody or its derivatives, but many CD26+ cells are not sensitive to CD26 antibody. Combined with the low in vivo and in vitro activity of YS110 in preclinical research and clinical research, the inventors speculate that the CD26 antibody has a selective effect on CD26+ cells. Screening out biomarkers that can effectively predict the therapeutic effectiveness of CD26 antibodies is an urgent problem to be solved for the clinical application of CD26 antibodies.
  • the present application provides a biomarker CD45 for screening the effectiveness of a subject receiving CD26 antibody or its derivative treatment.
  • the present application provides the use of CD26 antibody or derivatives thereof in the preparation of a drug for treating tumors expressing CD26 but not expressing CD45.
  • the tumors expressing CD26 and not expressing CD45 include solid tumors and hematological tumors.
  • the application also provides the use of the CD26 antibody or its derivatives in the preparation of drugs for treating tumors with CD26 expression and low CD45 expression.
  • the tumors with CD26 expression and CD45 low expression include solid tumors and hematological tumors.
  • the "antibody” of the present application can be a full-length antibody containing two heavy chains and two light chains; it can also be an antigen-binding fragment, that is, an antibody fragment that retains the ability to specifically bind to an antigen, such as retaining one or more CDRs Fragments of regions, including but not limited to Fab fragments, FV fragments, linear antibodies, single chain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, etc.
  • “Derivative” in this application means that the active drug molecule directly administered to the subject is not a traditional antibody (the above-mentioned complete structural antibody or an antigen-binding fragment of an antibody), but contains other forms of antibodies, including but not limited to CAR targeting CD26 -T, etc., or other forms capable of producing antibodies in vivo, including but not limited to rAAV vectors (adeno-associated virus vectors) containing anti-CD26 antibody coding genes, etc.
  • the present application provides the use of CD45 antigen or anti-CD45 antibody in the preparation of a kit for screening the therapeutic effectiveness of CD26 antibody or its derivatives in subjects.
  • the method of using the above kit is as follows: use the reagent to detect the expression of CD45 in the lesion; when the expression of CD45 is low, it is judged that the subject’s treatment with CD26 antibody or its derivatives is effective or the probability is high, and the expression of CD45 is high or relatively high. When , it is judged that the subject has a high probability of being ineffective or ineffective in receiving CD26 antibody or its derivative treatment.
  • the method of using the above kit is as follows: use the reagent to detect the expression of CD45 in the lesion; when CD45 is not expressed, it is judged that the subject has a high probability of being effective or effective in receiving the treatment of CD26 antibody or its derivatives; when CD45 is expressed, it is judged that The subject has a high probability of being ineffective or ineffective in receiving CD26 antibody or its derivative treatment.
  • the present application provides a method for screening the effectiveness of a subject receiving CD26 antibody or its derivative treatment: detecting the expression of CD45 in the lesion; The derivative treatment has a high probability of being effective or effective; when CD45 is highly expressed or highly expressed, it is judged that the subject has a high probability of being ineffective or ineffective in receiving CD26 antibody or its derivative treatment.
  • the method provided by the present invention for screening the therapeutic effectiveness of a subject receiving CD26 antibody or its derivatives detecting the expression of CD45 in the lesion; when CD45 is not expressed, it is judged that the subject receives CD26 antibody Antibody or its derivatives have a high probability of being effective or effective in treatment; when CD45 is expressed, it is judged that the subject has a high probability of being ineffective or ineffective for treatment with CD26 antibody or its derivatives.
  • CD45 is a transmembrane protein tyrosine phosphatase (PTPase), which is widely expressed in blood cells and consists of an extracellular domain, a transmembrane domain, and an intracellular domain.
  • PTPase transmembrane protein tyrosine phosphatase
  • the extracellular region of CD45 is a fragment of about 391-552 amino acids, with 11-15 N-glycosylation sites and multiple O-glycosylation sites.
  • CD45 The intracellular domain of CD45 is highly conserved and contains two repeated protein tyrosine phosphatase (PTPase) domains, one of which has PTPase activity, while the other has no significant PTPase due to changes in key amino acids necessary for catalytic activity activity, but the latter may regulate the former PTPase activity.
  • PTPase protein tyrosine phosphatase
  • CD45 plays a key role in the development and activation of lymphocytes by regulating the Src family protein kinases through its cytoplasmic PTPase activity. Whether CD45 is related to the efficacy of CD26 antibody was unknown before this application.
  • This application provides the application of CD45 screening for the effectiveness of CD26 antibody or its derivative treatment for subjects, which can effectively improve the accuracy and effectiveness of CD26 antibody or its derivative tumor immunotherapy, and improve the clinical benefits of patients;
  • the application discovered a new mechanism for the therapeutic effectiveness and precision of CD26 as a new target for tumor immunotherapy.
  • FIG. 1 CD45, CD4, CD26 expression flow cytometry diagram of CD4+ T cells
  • FIG. 1 Flow cytometric detection of CD45, CD8, CD26 expression in CD8+ T cells
  • Figure 4 Changes in animal tumor volume of 18G272 and 19G294 in the subcutaneous transplantation model of PC-3 cells in NOD-SCID mice.
  • Figure 5 Animal body weight changes of 18G272 and 19G294 in PC-3 cell NOD-SCID mouse subcutaneous transplantation model.
  • Figure 6 Changes in animal tumor volume of 18G272 in the subcutaneous transplantation model of NCI-H596 cells in NOD/SCID mice.
  • Figure 7 Animal tumor volume of 19G294 in NCI-H226 cell NOD/SCID mouse subcutaneous transplantation model Variety.
  • Figure 8 Animal body weight changes of 19G294 in NCI-H226 cell NOD/SCID mouse subcutaneous transplantation model.
  • Figure 9 Changes in animal tumor volume of 19G294 in the subcutaneous transplantation model of A498 cell NOD-SCID mice.
  • Figure 10 Changes in animal tumor volume of 18G272 in the subcutaneous transplantation model of OS-RC-2 cells in NOD-SCID mice.
  • Figure 11 Changes in animal tumor volume of 19G294 in the subcutaneous transplantation model of OS-RC-2 cells in NOD-SCID mice.
  • Figure 12 Schematic diagram of the skeleton of the AAV expression vector, including the plasmid replication origin pUC ori, Amp resistance gene, adeno-associated virus 5' terminal inverted repeat (ITR), CMV promoter, ⁇ intron enhancer sequence, and multiple cloning sites , polyA termination sequence and 3' terminal inverted repeat (ITR).
  • Figure 13 Changes in tumor volume in NOD/SCID mice bearing OSRC-2 (kidney cancer) xenografts by AAV delivery of BiTE genes.
  • Figure 14 Changes in mouse body weight in NOD/SCID mice bearing OSRC-2 (kidney cancer) xenograft tumors by AAV delivery of BiTE gene.
  • Figure 15 Changes in mouse survival in NOD/SCID mice bearing OSRC-2 (kidney cancer) xenografts by AAV delivery of BiTE genes.
  • Figure 16 The positive rate of CAR-T cells detected by flow cytometry.
  • Figure 17 Killing of tumor cells by CD26-targeted CAR-T cells.
  • CD26 is also called dipeptidyl peptidase (DPP4, dipeptidyl peptidase 4).
  • DPP4 dipeptidyl peptidase 4
  • the amino acid sequence of human CD26 can be found in Genbank with accession number NP_001926.2, and its cDNA sequence can be found in Genbank with accession number NM_001935.3.
  • antibody refers to the family of immunoglobulins that can bind a corresponding antigen non-covalently, reversibly and in a specific manner.
  • naturally occurring IgG antibodies are tetramers comprising at least two heavy chains and two light chains interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL can be further subdivided into highly variable complementarity determining regions (CDRs, also known as hypervariable regions), and more conserved framework regions (FRs).
  • CDRs also known as hypervariable regions
  • FRs conserved framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • HCDR1, HCDR2, and HCDR3 are three heavy chain complementarity determining regions
  • LCDR1, LCDR2, LCDR3 are three light chain complementarity determining regions.
  • the heavy chain variable region (VH) and the light chain variable region (VL) are responsible for antigen recognition, especially the complementarity determining regions (CDRs), which usually have specificity for different epitopes of the antigen.
  • CDRs complementarity determining regions
  • the constant regions are primarily responsible for effector functions.
  • BiTE is a bispecific antibody
  • the full name is "bispecific T cell adapter", which is composed of two single chain variable fragments (single chain variable fragment, scFv) with antigen specificity connected by a linker.
  • scFv single chain variable fragment
  • One of the scFvs targets tumor-associated surface antigens, and the other scFv targets CD3 on the surface of T cells.
  • Single-chain variable fragments (scFv) are formed by linking the variable region of the heavy chain and the variable region of the light chain of the antibody through a linker.
  • CD3 is an integral part of T cell signaling.
  • T cells When BiTE molecules combine with T cells and tumor cells at the same time, T cells are activated, promoting CD8+ T cells to directly secrete perforin and granzymes, and CD4+ T cells secrete cytokines to further recruit and activate killer T cells, thereby killing tumor cells .
  • bispecific antibodies such as 18G272 and 19G294 are bispecific T cell engagers (ZHBiTE) targeting CD26, and their sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively .
  • ZB ZHBody
  • the BiTE molecular fusion Fc fragment contains in the order from amino to carboxyl: BiTE-connecting peptide-CH2-CH3-peptide linker-CH2-CH3.
  • the amino acid sequence of ZB09 is shown in SEQ ID NO:4.
  • ZB antibodies can be prepared according to conventional methods, such as CHO cell codon optimization of the antibody sequence, adding AvrII restriction site and kozak sequence upstream of the gene, adding stop codon and BstZ17I restriction site downstream, after the whole gene synthesis, PCR amplification Amplify the target gene, and connect it into the AvrII and BstZ17I sites of the pCHO1.0 vector by enzyme-linking reaction to form an expression vector.
  • the expression vector was stably transformed into CHO-S cells, and MTX and puromycin were used to select stable cell lines with high expression.
  • the high-expression stable cell line was inoculated in Dynamis medium (A2617501, Thermo Fisher), fed-batch fed-batch culture at 37° C., 8% CO 2 , 130 rpm. Take the supernatant culture solution, centrifuge to collect the supernatant, filter through a 0.45 ⁇ m collection filter membrane to obtain the treated culture solution supernatant, and carry out chromatographic purification. Obtain the target antibody molecule.
  • ZA ZHBiAb, full-length bispecific IgG-like antibody, basically the same structure as a natural antibody, consisting of two heavy chains and two light chains, each light chain is connected to a heavy chain by a disulfide bond; two light chains - The heavy chain dimers are linked via disulfide bonds between the heavy chains, forming a Y-shaped molecule.
  • One of the Fab fragments of ZA16 and ZA23 recognizes CD26, and the other Fab fragment recognizes CD3.
  • the amino acid sequence of the heavy chain A of ZA16 is shown in SEQ ID NO: 5
  • the amino acid sequence of the light chain A is shown in SEQ ID NO: 6
  • the amino acid sequence of the heavy chain B is shown in SEQ ID NO: 7
  • the amino acid sequence of the light chain B is shown in SEQ ID NO: 7 ID NO: 8.
  • the amino acid sequence of ZA23 heavy chain A is shown in SEQ ID NO: 9
  • the amino acid sequence of light chain A is shown in SEQ ID NO: 10
  • the amino acid sequence of heavy chain B is shown in SEQ ID NO: 11
  • CD26-targeting CAR-T cells T cells expressing CD26CAR molecules.
  • the CD26CAR molecular structure of this application includes CD8 ⁇ signal peptide, anti-CD26 single-chain antibody linked by VL-Linker-VH, CD8 ⁇ hinge, CD8 ⁇ transmembrane region, CD28 co-stimulatory domain, CD3zeta intracellular domain.
  • the rAAV vector containing the gene encoding the anti-CD26 antibody is to introduce the gene encoding the anti-CD26 antibody into the adeno-associated virus (AAV), and deliver the anti-CD26 antibody gene to the body through the recombinant adeno-associated virus (rAAV) vector. Cells continue to synthesize anti-CD26 antibodies.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • Example 1 The effect of CD26 antibody on PBMC cells
  • PBMC Peripheral blood mononuclear cells
  • Cultivate target cells in T75 cell culture flask for adherent cells When the cells are more than 80% confluent, trypsinize the cells and collect them, wash them once with MACS buffer, count them on a hemocytometer, and divide them into 5 ⁇ 105 cells per copy. ; Use anti-CD26 monoclonal antibody as the primary antibody, and incubate with the target cells at room temperature for about 40 minutes.
  • target cells Cultivate target cells with T75 cell culture flasks, collect them, wash them once with MACS buffer, count them on a hemocytometer, and divide them into 5 ⁇ 10 5 cells per part; use anti-CD45-FITC monoclonal antibody (Cat. The manufacturer Miltenyi, which can identify all CD45 subtypes) (1:1000) was co-incubated with target cells at 2-8°C for about 10 minutes. After the incubation, washed twice with MACS buffer, centrifuged to discard the supernatant, and collected the cell pellet; 200ul of MACS buffer was used to resuspend the cell pellet, and the flow cytometer ACCURI C6 was used to detect and analyze the positive rate of CD45 within 1 hour. The results are shown in Table 1.
  • Human kidney cancer 786-0 cell and OS-RC-2 cell models were prepared according to the following methods.
  • PBMC cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM, 786-0 cells were inoculated into U-shaped 96-well cell culture plates at a cell concentration of 6 ⁇ 10 5 cells/ml, 50 ⁇ l per well, and reacted with corresponding samples
  • Add 50 ⁇ l of antibodies with a final concentration of 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml, and 0.001ng/ml to the wells, add 50 ⁇ l of medium to the blank control wells, and add 50 ⁇ l of medium to the wells of the positive control.
  • PBMC cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM, OS-RC-2 cells were inoculated into U-shaped 96-well cell culture plates at a cell concentration of 6 ⁇ 10 5 cells/ml, 50 ⁇ l per well, and placed in the corresponding Add 50 ⁇ l of antibodies with final concentrations of 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml, and 0.001ng/ml to the sample reaction wells, add 50 ⁇ l of medium to the blank control wells, and Add 50 ⁇ l final concentration of 1% TritonX-100 to the positive control well, then add 50 ⁇ l concentration of For 9 ⁇ 10 6 cells/ml of PBMC cells, incubate the reaction system under carbon dioxide culture at 37°C for 5 hours.
  • the formula for cell lysis rate is: (V sample -V vehicle control )/(V TritonX-100 -V vehicle control ) ⁇ 100%.
  • V sample is the average value of fluorescence signal readings of the drug treatment group
  • V vehicle control is the average value of fluorescence signal readings of the blank control group
  • V Triton-100 is the average value of fluorescence signal readings of the positive control group.
  • the cell lysis rate and sample concentration values were calculated with GraphPad Prism 7.00 software to calculate the IC50 value of PBMC cells to PBMC cells mediated by each sample.
  • Antibody 18G272-mediated PBMC (4#) has an IC 50 value of cytotoxic effect on PBMC (4#) cells of about 99730000pg/ml; Antibody 18G272-mediated PBMC (6#) has no cytotoxic effect on PBMC (6#) cells effect.
  • Antibody 18G272-mediated PBMC (1#) had no cytotoxic effect on PBMC (1#) cells; antibody 18G272-mediated PBMC (3#) had no cytotoxic effect on PBMC (3#) cells.
  • Antibody 19G294-mediated PBMC killing of target cell 786-0 it does not mediate PBMC killing of PBMC cells.
  • Antibody 19G294-mediated cytotoxic effect of PBMC (2#) on PBMC (2#) cells IC 50 value is about 1915313pg/ml; antibody 19G294-mediated cytotoxicity of PBMC (3#) on PBMC (3#) cells The effect IC 50 value is about 1947224 pg/ml.
  • Antibody 19G294-mediated cytotoxic effect of PBMC (2#) on PBMC (2#) cells IC 50 value is about 47920315pg/ml; antibody 19G294-mediated cytotoxicity of PBMC (3#) on PBMC (3#) cells The effect IC 50 value is about 1406115138pg/ml.
  • PBMC expresses CD26 and CD45 at the same time, and antibodies targeting CD26 will not mediate the cytotoxic effect of PBMC cells on PBMC cells.
  • CD4+ magnetic beads (product number 130-045-101, Miltenyi) were used to isolate CD4+ T cells from PBMC cells, and CD8 magnetic beads (product number 130-045-201, Miltenyi) were used to separate CD8+ T cells from PBMC cells.
  • CD45-FITC monoclonal antibody (Product No. 130-113-679, manufacturer Miltenyi, which can recognize all CD45 subtypes) was diluted according to the instructions, and incubated with target cells at 2-8°C for about 10 minutes. After the incubation, wash with MACS buffer Twice, the supernatant was discarded by centrifugation, and the cell pellet was collected; the cell pellet was resuspended with about 200ul of MACS buffer, and the flow cytometer ACCURI C6 was used for detection within 1 hour. The results are shown in Figure 1 and Table 2.
  • the isolated CD8+ T cells were collected, washed once with MACS buffer, counted on a hemocytometer, and divided into 5 ⁇ 10 5 cells each; anti-CD8 antibody (product number 130-098-075, Miltenyi), anti-CD45 -FITC monoclonal antibody (Product No. 130-113-679, manufacturer Miltenyi, can recognize all CD45 subtypes) was diluted according to the instructions, and incubated with target cells at 2-8°C for about 10 minutes. After the incubation, wash the two cells with MACS buffer.
  • CD4+T and CD8+T cells were labeled with green fluorescent signal by the fluorescent dye Calcein-AM, and seeded into U-shaped 96-well cell culture plate at a cell concentration of 3 ⁇ 10 5 cells/ml, 50 ⁇ l per well, in the corresponding Add 50 ⁇ l of antibodies with final concentrations of 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml, and 0.001ng/ml to the sample reaction wells, add 50 ⁇ l of medium to the blank control wells, and Add 50 ⁇ l of TritonX-100 with a final concentration of 1% to the positive control well, then add 50 ⁇ l of PBMC cells with a concentration of 4.5 ⁇ 10 6 cells/ml at a ratio of E/T of 15:1, and place the reaction system under carbon dioxide culture at 37°C Incubate for 5 hours.
  • the formula for cell lysis rate is: (V sample -V vehicle control )/(V TritonX-100 -V vehicle control ) ⁇ 100%.
  • V sample is the average value of fluorescence signal readings of the drug treatment group
  • V vehicle control is the average value of fluorescence signal readings of the blank control group
  • V Triton-100 is the average value of fluorescence signal readings of the positive control group.
  • the cell lysis rate and sample concentration values were calculated with GraphPad Prism 7.00 software to calculate the IC50 value of PBMC cells to PBMC cells mediated by each sample.
  • CD4+ T cells and CD8+ T cells highly express CD45, and the antibody 19G294 targeting CD26 does not mediate the cytotoxic effect of PBMC cells on CD4+ and CD8+ T cell subtypes.
  • the detection method is the same as in Example 1, and the expression of CD26 and CD45 in different lymphoma cells is shown in Table 5.
  • Human lymphoma cell models of Jurkat cells, U937 cells, Z-138 cells, NAMALWA cells, HL-60 cells, SNK-6 cells, and MOLT-4 cells were respectively prepared according to the following methods.
  • Tumor cells were marked with a green fluorescent signal by fluorescent dye Calcein-AM, and inoculated into U-shaped 96-well cell culture plates at a cell concentration of 6 ⁇ 10 5 cells/ml, 50 ⁇ l per well, and 50 ⁇ l were added to corresponding reaction wells
  • fluorescent dye Calcein-AM fluorescent dye Calcein-AM
  • the formula for cell lysis rate is: (V sample -V vehicle control )/(V Triton-100 -V vehicle control ) ⁇ 100%.
  • V sample is the average value of fluorescence signal readings of the drug treatment group
  • V vehicle control is the average value of fluorescence signal readings of the blank control group
  • V Triton-100 is the average value of fluorescence signal readings of the positive control group.
  • the cell lysis rate and sample concentration were calculated using GraphPad Prism 7.00 software to calculate the IC 50 value of the PBMCs mediated by the sample on the target cells.
  • bispecific antibodies ZA23, ZA16, ZB09, 19G294, etc. could not mediate PBMC cells against CD45+CD26+ tumor cell lines Jurkat, U937, Z-138, NAMALWA, HL-60, SNK-6, MOLT -4 etc. produced cytotoxic effects, the results are shown in Table 6, and the representative drawings are shown in Figure 3.
  • the fluorescence signal in the figure does not change with the increase of antibody concentration (no dose-dependent relationship with the antibody concentration), indicating that the antibody does not mediate the cytotoxic effect of PBMC cells on the corresponding cells.
  • Most lymphoma cells express CD26 and highly express CD45, and antibodies targeting CD26 will not mediate the cytotoxic effect of PBMC cells on lymphoma cells with high expression of CD45.
  • the detection method is the same as in Example 1, and the expression of CD26 and CD45 in different solid tumor cells is shown in Table 7.
  • the experimental method was the same as that in Example 2, and human kidney cancer cells OS-RC-2 cells, 786-0 cells, human lung squamous cell carcinoma NCI-H226 cells, and human prostate cancer PC-3 cell models were prepared.
  • bispecific antibodies ZA23, ZA16, ZB09, 19G294, etc. mediated the killing activity of PBMC cells against CD26+CD45- tumor cell OS-RC-2, as shown in Table 8.
  • Bispecific antibodies 19G294, 18G272, etc. can all mediate PBMC cells to produce strong cytotoxic effects on various CD26+CD45- tumor cells, as shown in Table 9.
  • CD26 antibodies have killing effects on a variety of solid tumor cell lines with no or low expression of CD45.
  • Method Choose NOD-SCID mice with a body weight of 18-22g and about 5-7 weeks old, and mix 5 ⁇ 10 6 cells/0.1ml PC-3 cell suspension and 1 ⁇ 10 7 cells/0.1ml PBMC cell suspension according to the The ratio of 1:1 (volume ratio) was fully mixed and inoculated subcutaneously in NOD-SCID mice, each inoculated with 0.2ml. Animals were randomly divided into 3 groups according to body weight, namely Model group (PBS), 19G294 group (30 ⁇ g/animal/time), 18G272 group (30 ⁇ g/animal/time), with 6 animals in each group. The Model group and each treatment group were administered intravenously, starting on the day of inoculation, and continued for five days.
  • PBS Model group
  • 19G294 group (30 ⁇ g/animal/time
  • 18G272 group (30 ⁇ g/animal/time
  • the time of the first administration is 1 hour after the mice are subcutaneously inoculated with cells, that is, the first course of treatment is D1, D2, D3, D4, D5; the second course of treatment is D8, D9, D10, D11, D12. Dosing frequency is 1 day/time.
  • TGI (%) (1-T/C) ⁇ 100%. Recognized that T represents the relative tumor volume at a certain time point of the administration group (ratio of the tumor volume measured at the time and the tumor volume at the time of grouping), and C represents the relative tumor volume at a certain time point of the model group (the tumor volume measured at the time and the tumor volume ratio at the time of grouping). Tumor volume ratio at the time of grouping). On the day of inoculation in the experiment of this application, the groups were grouped according to body weight, and when TGI was calculated, T and C represented the actual measured tumor volumes of the administration group and the model group respectively.
  • Tumor weight After the last test, the animals were euthanized, the tumor mass was peeled off, rinsed with normal saline and blotted dry with filter paper, the tumor mass was weighed, and photographed.
  • Relative tumor inhibition rate TGI (%) (1-T TW /C TW ) ⁇ 100%, T TW represents the average tumor weight at the end of the experiment in the treatment group, and C TW represents the average tumor weight at the end of the experiment in the model group.
  • Methods choose NOD/SCID mice with a body weight of 18-22g and about 5-7 weeks old, and mix 8 ⁇ 10 6 cells/0.1ml NCI-H596 cell suspension and 1.6 ⁇ 10 7 cells/0.1ml PBMC cell suspension according to the The ratio of 1:1 (volume ratio) was fully mixed and inoculated subcutaneously in NOD/SCID mice, each inoculated with 0.2ml.
  • the animals were randomly divided into 2 groups according to body weight, namely Model group (PBS, 6 animals/group) and 18G272 group (30 ⁇ g/time, 6 animals/group).
  • test product 18G272 can significantly inhibit the tumor growth of human lung adenosquamous carcinoma model mice at a dose of 30 ⁇ g/mouse; the test product 18G272 at a dose of 30 ⁇ g/mouse, the animal body weight tends to increase, and the resistance during treatment feel good.
  • the results are shown in Figure 6 and Table 11.
  • Method Choose NOD/SCID mice with a body weight of 18-22g and about 5-7 weeks old, and mix 5 ⁇ 10 6 cells/0.1ml NCI-H226 cell suspension and 1.5 ⁇ 10 7 cells/0.1ml PBMC cell suspension according to the The ratio of 1:1 (volume ratio) was fully mixed and inoculated subcutaneously in NOD/SCID mice, each inoculated with 0.2ml.
  • the animals were randomly divided into 2 groups according to body weight, respectively Model group (PBS, 5 animals/group) and 19G294 group (60 ⁇ g/time, 5 animals/group). The route of administration and the cycle of administration are the same as above.
  • the test product 19G294 can significantly inhibit the tumor growth of human lung adenosquamous carcinoma (mesothelioma) model mice at a dose of 60 ⁇ g/mouse, as shown in Figure 7; the test product 19G294 at a dose of 60 ⁇ g/mouse Under , the body weight of the animals showed an upward trend, and the treatment period was well tolerated.
  • the results are shown in Figure 8 and Table 12.
  • Method Choose NOD-SCID mice with a body weight of 18-22g and about 5-7 weeks old, and mix 1 ⁇ 10 7 cells/0.1ml A498 cell suspension and 1 ⁇ 10 7 cells/0.1ml PBMC cell suspension according to the ratio of 1: The ratio of 1 (volume ratio) was thoroughly mixed and inoculated subcutaneously in NOD-SCID mice, each inoculated with 0.2ml. The animals were randomly divided into 2 groups according to body weight, namely the Model group (PBS) and the 19G294 group (30 ⁇ g/animal/time), with 5 animals in each group. The route of administration and the cycle of administration are the same as above.
  • PBS Model group
  • 19G294 group 30 ⁇ g/animal/time
  • TGI was calculated based on the tumor weight on day 61 after cell inoculation.
  • Method select NOD-SCID mice weighing 18-22g and about 5-7 weeks old, 3 ⁇ 10 6 cells/0.1ml OS-RC-2 cell suspension and 6 ⁇ 10 6 cells/0.1ml PBMC cell suspension Mix thoroughly according to the ratio of 1:1 (volume ratio) and inoculate subcutaneously in NOD-SCID mice, each inoculate 0.2ml. Animals were randomly divided into 2 groups according to body weight, namely Model group (PBS) and 18G272 group (30 ⁇ g/animal/time); 6 animals in each group. The route of administration and the cycle of administration are the same as above.
  • PBS Model group
  • 18G272 group 30 ⁇ g/animal/time
  • 18G272 can significantly inhibit tumor growth in human kidney cancer model mice at a dose of 30 ⁇ g/mouse. The results are shown in Figure 10 and Table 14. The OS-RC-2 human kidney cancer model will cause animal death during the test period. Under the conditions of this test, 18G272 can reduce animal mortality and significantly increase the median survival period of OS-RC-2 human kidney cancer model mice ( MST), the animals did not lose body weight and were well tolerated during the treatment, the results are shown in 14.
  • TGI was calculated based on the tumor weight on day 29 after cell inoculation.
  • Method select NOD-SCID mice weighing 18-22g and about 5-7 weeks old, 3 ⁇ 10 6 cells/0.1ml OS-RC-2 cell suspension and 6 ⁇ 10 6 cells/0.1ml PBMC cell suspension Mix thoroughly according to the ratio of 1:1 (volume ratio) and inoculate subcutaneously in NOD-SCID mice, each inoculate 0.2ml.
  • the animals were randomly divided into 2 groups according to body weight, namely Model group (PBS) and 19G294 group (30 ⁇ g/animal/time); 5 animals in each group.
  • the route of administration and the cycle of administration are the same as above.
  • 19G294 can significantly inhibit tumor growth in human kidney cancer model mice at a dose of 30 ⁇ g/mouse, and the results are shown in Figure 11 and Table 15.
  • the OS-RC-2 human kidney cancer model will cause the death of animals during the test period.
  • 19G294 can reduce the death rate of animals and prolong the survival period of animals. The animals did not lose weight and were well tolerated during the treatment. The results are shown in the table 15.
  • Example 4 The drug effect of the rAAV vector containing the gene encoding anti-CD26 antibody in tumor-bearing mice
  • FIG. 12 The schematic diagram of the skeleton of the AAV expression vector is shown in FIG. 12 .
  • GFP is green fluorescent protein
  • the GFP gene sequence (SEQ ID NO: 13) is amplified and constructed into the multiple cloning site MCS (XbaI and BamHI restriction site) to form a pAAV-GFP expression vector as a control.
  • the signal peptide gene (SEQ ID NO: 14) was fused with the BiTE gene sequence (SEQ ID NO: 15) and synthesized and amplified to construct multiple clones between the ⁇ intron enhancer sequence and the polyA termination sequence of the pAAV-CMV vector Site MCS (between BamHI and EcoRI restriction sites), forming an AAV expression vector: pAAV-21R23 (containing CD26-CD3BiTE).
  • 293T cell culture After thawing, 293T cells are adherently cultured in DMEM medium containing 10% FBS and 1% Glutamax. After 2-3 passages, the cell state recovers and can be used for packaging. 150mm cell culture dishes were coated with 15ml 0.1% gelatin for 30min at room temperature. 293T cells were digested with trypsin, resuspended and counted, discarded the coating solution in the culture dish, inoculated 1.21.8 ⁇ 10e7 293T cells in each 150mm dish, supplemented with 293T medium to 30ml, and cultured overnight at 37°C and 5% CO 2 .
  • Transfection On the next morning, grow 293T cells to 70-80% confluence, replace with fresh 293T medium, 30ml per plate, put back into the incubator. Prepare the transfection mixture, add the two constructed AAV expression vector plasmids to the pRC6 and pHelper plasmids at a mass ratio of 1:1:1 and add serum-free DMEM medium, the total DNA amount in each 15cm dish is 30 ⁇ g, and then gently add Lipo8000 lipid Plasmid (Biotech product number: C0533), mixed gently, and allowed to stand at room temperature for 30 minutes. Add 1.5ml transfection mixture to each dish, mix gently, and return to the incubator. After 24 hours of transfection, replace with 30ml AAV harvest medium (DMEM medium containing 2% FBS, 1% Glutamax, 1% 1M HEPES), and put it back into the incubator.
  • AAV harvest medium DMEM medium containing 2% FBS, 1% Glutamax, 1% 1M HEPES
  • rAAV Collection and purification of rAAV: After 72 hours of infection, add 0.5M EDTA (pH 8.0) of 1/80 of the volume of the culture medium to the culture dish containing 293T cells, pipette to suspend the cells, and collect them into a 50ml sterile centrifuge tube. Centrifuge at 2000g at 4°C for 10 minutes, discard the supernatant, and collect the cell pellet after completely removing the supernatant, which is the host 293T cells containing AAV particles. Next steps follow The Purification Kit Maxi kit (TAKARA product number: 6666) is used to purify and concentrate AAV, and store at -80°C after aliquoting.
  • EDTA pH 8.0
  • Method select NOD-SCID mice with a body weight of 18-22 g and about 5-7 weeks old, and mix 4 ⁇ 10 7 cells/ml OS-RC-2 cell suspension and 6 ⁇ 10 7 cells/ml PBMC cell suspension according to the 1 : 1 (volume ratio) and mix well, and dilute GFP and 21R23AAV purified samples to 1 ⁇ 10 11 vg/ml.
  • the tumor volume and body weight of the mice were measured, and the survival status of the mice was observed.
  • NOD-SCID mice were subcutaneously injected with a mixture of OS-RC-2 and PBMC cells to establish a mouse xenograft model of human kidney cancer.
  • each mouse was treated once with 10e10vg of recombinant AAV expressing BiTE bispecific antibody. From the 8th day of modeling, the tumors in the model group and the GFP-A group began to increase significantly, and the tumor volume in group B was significantly suppressed by AAV drug treatment, and the tumor in group B completely regressed on the 12th day, the results are shown in Figure 13 and Table 16 Show.
  • OS-RC-2 human kidney cancer model caused animal death during the test period, while the animals in the AAV treatment group did not lose weight, and the treatment was well tolerated, as shown in Figures 14 and 15 .
  • the BiTE bispecific antibody delivered by recombinant AAV can completely inhibit the renal cancer cells with CD26 expression and CD45 non-expression or low expression after only one administration, and has a significant therapeutic effect.
  • the recombinant CAR gene contains BamHI restriction site, Kozak sequence, CD8 ⁇ signal peptide, anti-CD26 single-chain antibody linked by VL-Linker-VH, CD8 ⁇ hinge, CD8 ⁇ transmembrane region, CD28 co-stimulatory domain, CD3zeta intracellular domain, terminator Codon, SalI restriction site, after the complete sequence of the recombinant CAR gene was synthesized (SEQ ID NO: 3), it was connected into the lentiviral plasmid vector pWPT-GFP through the BamHI and SalI double restriction site to replace the GFP gene, forming a slow Viral expression vector pWPT-44529.
  • the constructed PWPT-44529 plasmid was packaged and purified with lentivirus, and the recombinant CAR lentivirus was used to infect healthy donor T cells, IL2 and activator were used to stimulate and expand CAR-T cells, and the expanded CAR-T cells were harvested and used
  • the positive rate of CAR detected by flow cytometry can be seen from Figure 16 that the positive rate of lentivirus-infected T cells reached 40.7%.
  • the transfected effector cells CAR-T cells (44529), untransfected T cells (NTD) were co-cultured with different target cells, and the selection of target cells included: CD26+CD45- kidney Cancer cell A498 and 769-P; CD26- lymphoma cell Raji and kidney cancer cell G401; CD26+CD45+ lymphoma cell U937 and Jurkat.
  • target cells included: CD26+CD45- kidney Cancer cell A498 and 769-P; CD26- lymphoma cell Raji and kidney cancer cell G401; CD26+CD45+ lymphoma cell U937 and Jurkat.
  • DOJINDO company LDH detection kit DOJINDO company LDH detection kit
  • CD26-targeted CAR-T cells have no specific killing effect on CD26-negative target cells Raji and G401, and CD26+CD45- cells (A498, 769-P), CD26 CAR-T showed higher killing effect than control T cells.
  • the obvious killing effect proves the target-specific killing of CD26CAR-T.
  • CD26+CD45+ double-positive cells U937, Jurkat
  • the killing effect of CD26CAR-T was significantly inhibited, and no specific killing was detected.
  • CD26 antibody other gene and cell therapy methods related to CD26 antibody (such as AAV gene therapy targeting CD26, CAR-T cell therapy targeting CD26) all showed no or low expression of CD45.
  • Tumor cells have significant killing activity in vivo and in vitro; but have no significant killing activity on tumor cells with high expression of CD45.
  • this application provides the application of CD45 screening for the effectiveness of CD26 antibody or its derivative treatment for subjects, which can effectively improve the accuracy and effectiveness of CD26 antibody or its derivative tumor immunotherapy, and improve the clinical efficacy of patients.
  • Benefit discovered a new mechanism for the therapeutic effectiveness and precision of CD26 as a new target for tumor immunotherapy.

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Abstract

提供CD45作为生物标志物在筛查受试者对CD26抗体或其衍生物治疗肿瘤有效性中的应用。具体地，当CD45不表达或低表达时，判断该受试者接受CD26抗体或其衍生物治疗有效或有效的概率高，CD45高表达或较高表达时，判断该受试者接受CD26抗体或其衍生物治疗无效或无效的概率高。筛选出了能够有效筛查对CD26抗体或其衍生物肿瘤治疗有效性的生物标记物，能显著改善CD26抗体或其衍生物肿瘤治疗的精准性和有效性，提高患者的临床获益；发现了CD26作为肿瘤免疫治疗新靶点的治疗有效性和精准性的新机制。

Description

CD45作为生物标志物在筛查CD26抗体或其衍生物治疗肿瘤有效性和精准性中的应用 技术领域
本发明涉及预测临床疗效的生物标志物，具体涉及CD45作为预测临床疗效的生物标志物的应用，更具体地涉及CD45在筛查对CD26抗体或其衍生物治疗肿瘤有效性和精准性中的应用。
背景技术
精准医学是基于临床病理特征和分子特征，定制可精确满足不同患者实际需求的诊断、治疗及预后判断策略。其范畴包括精准预防(患病风险的预测及预防性干预)、精准诊断(疾病早期发现与诊断，分子分型)、精准治疗(分子靶向疗法、疗效的预测与监控、精准外科技术等)。
生物标志物的发现和应用是精准医学的重点研究方向。生物标志物按照不同用途可以分为：明确疾病状态或分型的诊断生物标志物、反应疾病预后的预后生物标志物、预测对于干预措施的应答情况的预测生物标志物、反映接受治疗后产生生物学应答的药效学生物标志物、用于监测从而避免或降低安全性风险的安全生物标志物。其中预测生物标志物可用作筛选更有可能从治疗中获益的人群，排除临床不获益患者。如PD-1/PD-L1、CTLA-4等免疫检查点抑制剂在多种实体瘤中已有广泛研究，并已作为非小细胞肺癌的一线治疗方案，但其整体客观缓解率依然只有20％左右。PD-L1的表达可以作为抗PD-1/PD-L1治疗反应的预测标志物，肿瘤突变负荷(TMB)也已被证明与黑色素瘤、肺腺癌、膀胱癌的免疫检查点抑制剂的疗效有关。
CD26是一种多功能Ⅱ型跨膜糖蛋白，也可以以溶解形式存在于血浆中。CD26常以同源二聚体形式存在，其单体含766个氨基酸，相对分子质量约110kDa。CD26氨基酸残基从内向外分为5个部分：胞内区(1～6)、跨膜区(7～28)、高度糖基化区(29～323)、富含半胱氨酸区(324～551)和C端催化结构域(552～766)，其分子三维结构与功能密切相关。CD26(DPP4)抑制剂在临床上用于Ⅱ型糖尿病的治疗已有数十年的历史。此外，CD26在多种肿瘤细胞表面的 表达量明显升高，例如，恶性间皮瘤，肾癌，前列腺癌，肺癌等，对于这类型CD26表达量较高的肿瘤，CD26是一种具有重要价值的作用靶点(CD26/DPP4-a potential biomarker and target for cancer therapy,Pharmacology&Therapeutics(2019),198:135-159)。
目前以人CD26为靶点的抗癌药物研究进展最快的是Y’S Therapeutics公司的单克隆抗体YS110，已完成了I/II期临床试验。但根据已有文献资料分析可知，YS110的临床前研究已存在体内外活性均较低的问题，如在文献“A humanized anti-CD26monoclonal antibody inhibits cell growth of malignant mesothelioma via retarded G2/M cell cycle transition,Cancer Cell Int(2016)16:35”中，在YS110的浓度为250ug/ml，作用48h以后，对肿瘤细胞株生长的抑制率为18.3％，IC50值远远高于250ug/ml，足见其体外的活性较低；又如在专利“抗CD26抗体及其使用方法(申请号：CN200680034937.4)”中，展示了YS110对多种CD26高表达细胞株的小鼠肿瘤模型的治疗，YS110仅能一定程度缩小肿瘤体积，缓解肿瘤生长，并不能完全抑制肿瘤生长实现肿瘤的治愈，可见其在动物模型中的活性仍然不理想。YS110已经完成的临床二期研究结果显示，YS110具有较好的耐受性，但疾病控制率未达到预期，这与YS110在临床前研究中所表现出来的低活性相对应。
在CD26抗体的研究过程中，发明人发现部分CD26+细胞对CD26抗体或其衍生物敏感，但也有较多CD26+细胞对CD26抗体并不敏感。结合YS110在临床前研究及临床研究中较低的体内外活性，发明人推测CD26抗体对CD26+细胞的作用具有选择性。筛选出能够有效预测对CD26抗体治疗有效性的生物标记物，是CD26抗体实现临床应用亟待解决的问题。
发明内容
本申请提供了用于筛查受试者接受CD26抗体或其衍生物治疗有效性的生物标志物CD45。
第一个方面，本申请提供了CD26抗体或其衍生物在制备治疗CD26表达且CD45不表达的肿瘤的药物中的应用。所述CD26表达CD45不表达的肿瘤包括实体肿瘤和血液瘤。
本申请还提供了CD26抗体或其衍生物在制备治疗CD26表达且CD45低表达的肿瘤的药物中的应用。所述CD26表达CD45低表达的肿瘤包括实体肿瘤和血液瘤。
本申请的“抗体”既可以是含有两条重链和两条轻链的全长抗体；又可以是抗原结合片段，即保留与抗原特异性结合能力的抗体片段，例如保留一个或多个CDR区的片段，包括但不限于Fab片段、FV片段、线性抗体、单链抗体、纳米抗体、双特异性抗体、多特异性抗体等。
本申请的“衍生物”指直接给予受试者的活性药物分子不是传统抗体(上述完整结构抗体或者抗体的抗原结合片段)，而是含有抗体的其他形式，包括但不限于靶向CD26的CAR-T等，或者是能在体内生成抗体的其他形式，包括但不限于含有抗CD26抗体编码基因的rAAV载体(腺相关病毒载体)等。
第二个方面，本申请提供了CD45抗原或抗CD45抗体在制备用于筛查受试者接受CD26抗体或其衍生物治疗有效性的试剂盒中应用。
上述试剂盒的使用方法如下：以所述试剂检测病灶CD45表达情况；CD45低表达时，判断该受试者接受CD26抗体或其衍生物治疗有效或有效的概率高，CD45高表达或较高表达时，判断该受试者接受CD26抗体或其衍生物治疗无效或无效的概率高。
或者，上述试剂盒的使用方法如下：以所述试剂检测病灶CD45表达情况；CD45不表达时，判断该受试者接受CD26抗体或其衍生物治疗有效或有效的概率高，CD45表达时，判断该受试者接受CD26抗体或其衍生物治疗无效或无效的概率高。
第三个方面，本申请提供了一种筛查受试者接受CD26抗体或其衍生物治疗有效性的方法：检测病灶CD45表达情况；CD45低表达时，判断该受试者接受CD26抗体或其衍生物治疗有效或有效的概率高；CD45高表达或较高表达时，判断该受试者接受CD26抗体或其衍生物治疗无效或无效的概率高。
优选地，本发明提供的筛查受试者接受CD26抗体或其衍生物治疗有效性的方法：检测病灶CD45表达情况；CD45不表达时，判断该受试者接受CD26抗 体或其衍生物治疗有效或有效的概率高；CD45表达时，判断该受试者接受CD26抗体或其衍生物治疗无效或无效的概率高。
CD45是一种跨膜蛋白酪氨酸磷酸酯酶(PTPase)，在血液细胞中广泛表达，由细胞外结构域、跨膜结构域、细胞内结构域构成。CD45存在多种亚型，不同亚型胞外结构域不同、胞内及跨膜结构域相同，各亚型在不同种类细胞亚群上有表达差异。CD45胞膜外区是约有391～552个氨基酸的片段，有11～15个N-糖基化位点及多个O-糖基化位点。CD45胞内区结构域高度保守，含有2个重复的蛋白酪氨酸磷酸酯酶(PTPase)结构域，其中一个具有PTPase活性，而另一个因催化活性所必须的关键氨基酸的变化而无显著PTPase活性，但后者可能对前者的PTP ase酶活性起调节作用。CD45通过其胞质区PTPase活性，调节Src家族蛋白激酶，在淋巴细胞发育和活化中发挥关键作用。CD45是否与CD26抗体发挥药效有关在本申请以前是未知的。
本申请提供了CD45筛查受试者接受CD26抗体或其衍生物治疗有效性的应用，能有效改善CD26抗体或其衍生物肿瘤免疫治疗的精准性和有效性，提高患者的临床获益；本申请发现了CD26作为肿瘤免疫治疗新靶点的治疗有效性和精准性的新机制。
附图说明
图1：CD4+T细胞的CD45、CD4、CD26表达流式检测图
图2：CD8+T细胞的CD45、CD8、CD26表达流式检测图
图3：抗体所介导的PBMC对细胞的杀伤效果图
图4：18G272、19G294在PC-3细胞NOD-SCID小鼠皮下移植模型中的动物肿瘤体积变化。
图5：18G272、19G294在PC-3细胞NOD-SCID小鼠皮下移植模型中的动物体重变化。
图6：18G272在NCI-H596细胞NOD/SCID小鼠皮下移植模型中的动物肿瘤体积变化。
图7：19G294在NCI-H226细胞NOD/SCID小鼠皮下移植模型中的动物肿瘤体积 变化。
图8：19G294在NCI-H226细胞NOD/SCID小鼠皮下移植模型中的动物体重变化。
图9：19G294在A498细胞NOD-SCID小鼠皮下移植模型中的动物肿瘤体积变化。
图10：18G272在OS-RC-2细胞NOD-SCID小鼠皮下移植模型中的动物肿瘤体积变化。
图11：19G294在OS-RC-2细胞NOD-SCID小鼠皮下移植模型中的动物肿瘤体积变化。
图12：AAV表达载体骨架示意图，包括质粒复制起点pUC ori，Amp抗性基因，腺相关病毒5’末端反向重复序列(ITR)，CMV启动子，β内含子增强序列，多克隆位点，polyA终止序列与3’末端反向重复序列(ITR)。
图13：AAV递送BiTE基因在荷有OSRC-2(肾癌)异种移植瘤NOD/SCID小鼠中的肿瘤体积变化。
图14：AAV递送BiTE基因在荷有OSRC-2(肾癌)异种移植瘤NOD/SCID小鼠中的小鼠体重变化。
图15：AAV递送BiTE基因在荷有OSRC-2(肾癌)异种移植瘤NOD/SCID小鼠中的小鼠生存期变化。
图16：CAR-T细胞采用流式细胞仪检测CAR阳性率。
图17：CD26靶向的CAR-T细胞对肿瘤细胞的杀伤。
具体实施方式
除非在本文中明确定义，否则本文使用的技术术语具有本领域普通技术人员通常理解的含义。
单数形式词语如“一个(a、an)”“所述(the)”包括它们对应的复数指示物。
术语“或”意指“和/或”，并可与“和/或”互换使用。
术语“CD26”又称二肽基肽酶(DPP4，dipeptidyl peptidase 4)，人CD26氨基酸序列能以登录号NP_001926.2在Genbank中找到，其cDNA序列能以登录号NM_001935.3在Genbank中找到。
术语“抗体”是指可以非共价地、可逆地且以特异性方式结合相应抗原的免疫球蛋白家族。例如，天然存在的IgG抗体是四聚体，其包含通过二硫键相互连接的至少两条重链和两条轻链。每条重链由重链可变区(VH)和重链恒定区组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL可以进一步细分为具有高变性的互补决定区(CDR，又称高变区)，以及更保守的框架区(FR)。每个VH和VL由三个CDR和四个FR组成，按照以下顺序从氨基末端到羧基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。HCDR1、HCDR2、HCDR3为三个重链互补决定区，LCDR1、LCDR2、LCDR3为三个轻链链互补决定区。重链可变区(VH)及轻链可变区(VL)负责抗原识别，尤其是其中的互补决定区(CDR)，通常对抗原的不同表位具有特异性。恒定区主要负责效应子功能。
术语“BiTE”是一种双特异性抗体，全称“双特异性T细胞接合器”，由两个具有抗原特异性的单链可变片断(single chain variable fragment，scFv)通过接头连接而成，其中一个scFv靶向识别肿瘤相关表面抗原，另一个scFv靶向识别T细胞表面CD3。单链可变片断(scFv)由抗体重链可变区和轻链可变区通过接头连接而成。
CD3是T细胞信号传导的组成部分。当BiTE分子同时与T细胞和肿瘤细胞结合时，T细胞被激活，促进CD8+T细胞直接分泌穿孔素和颗粒酶，CD4+T细胞分泌细胞因子进一步招募活化杀伤性T细胞，从而杀伤肿瘤细胞。
在本申请具体的实施例中“双特性抗体”如18G272，19G294均为靶向CD26的双特异性T细胞接合器(ZHBiTE)，序列分别如SEQ ID NO：1与SEQ ID NO：2所示。
ZB：ZHBody，BiTE分子融合Fc片段以氨基至羧基的顺序包含：BiTE-连接肽-CH2-CH3-肽接头-CH2-CH3。ZB09氨基酸序列如SEQ ID NO：4所示。可以按照常规方法制备ZB抗体，如抗体序列进行CHO细胞密码子优化，在基因上游添加AvrII酶切位点与kozak序列，下游添加终止密码子和BstZ17I酶切位点，全基因合成后，PCR扩增得到目的基因，通过酶切酶连反应连入pCHO1.0载体的AvrII与BstZ17I位点，形成表达载体。表达载体稳定转化CHO-S细胞，MTX与嘌呤霉素筛选高表达稳定细胞株。将高表达稳定细胞株接种于Dynamis培养基(A2617501,Thermo Fisher)，37℃，8％CO2，130rpm进行分批补料流加培养。 取上清培养液，离心收集上清，0.45μm收集滤膜过滤即得处理后培养液上清，进行层析纯化。得到目的抗体分子。
ZA：ZHBiAb，双特异性类IgG全长抗体，与天然抗体结构基本相同，由两条重链和两条轻链组成，每条轻链通过二硫键连接至一条重链；两个轻链-重链二聚体经由重链之间的二硫键连接，从而形成Y形分子。ZA16、ZA23其中一个Fab片段识别CD26，另一个Fab片段识别CD3。ZA16重链A氨基酸序列如SEQ ID NO：5所示，轻链A氨基酸序列如SEQ ID NO：6所示，重链B氨基酸序列如SEQ ID NO：7所示，轻链B氨基酸序列如SEQ ID NO：8所示。ZA23重链A氨基酸序列如SEQ ID NO：9所示，轻链A氨基酸序列如SEQ ID NO：10所示，重链B氨基酸序列如SEQ ID NO：11所示，轻链B氨基酸序列如SEQ ID NO：12所示。
CD26靶向CAR-T细胞：表达靶向CD26CAR分子的T细胞，本申请的CD26CAR分子结构包含CD8α信号肽、以VL-Linker-VH连接的抗CD26单链抗体、CD8α铰链、CD8α跨膜区、CD28共刺激域、CD3zeta胞内域。
含有抗CD26抗体编码基因的rAAV载体，是将抗CD26抗体编码基因导入到腺相关病毒(AAV)中，通过重组腺相关病毒(rAAV)载体将抗CD26抗体基因递送至体内，利用rAAV感染后的细胞持续合成抗CD26抗体。
实施例1 CD26抗体对PBMC细胞的作用
外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)主要由T淋巴细胞、B淋巴细胞、单核细胞、巨噬细胞、树突状细胞等免疫细胞组成，它们都表达CD26，为观察CD26抗体是否对正常PBMC细胞有杀伤作用及与CD45表达的关联性，开展如下实验：
一、细胞株CD26、CD45阳性率的检测
贴壁细胞用T75细胞培养瓶培养靶细胞，待细胞融合到80％以上时，胰蛋白酶消化细胞并收集，用MACS buffer清洗一次，血球计数板进行计数，分为5×105个细胞每份；用抗CD26单克隆抗体作为一抗，与靶细胞于室温共孵育约40min，孵育结束后，离心弃上清，细胞沉淀用MACS buffer重悬，再次离心弃上清，收集细胞沉淀；再用相应的抗体Alexa Fluor 488鼠抗人IgG1做为二抗，重悬细胞沉淀，于室温避光孵育约30min，孵育结束后，用PBS清洗两次，离心弃上清，收集细胞沉淀；用约200ul的MACS buffer将细胞沉淀重悬，在1h内 用流式细胞仪ACCURI C6进行检测并分析CD26阳性率，结果如表1所示。
用T75细胞培养瓶培养靶细胞，收集，用MACS buffer清洗一次，血球计数板进行计数，分为5×105个细胞每份；用抗CD45-FITC单克隆抗体(货号130-113-679，厂家Miltenyi，可识别所有CD45亚型)(1:1000)与靶细胞于2～8℃共孵育约10min，孵育结束后，用MACS buffer清洗两次，离心弃上清，收集细胞沉淀；用约200ul的MACS buffer将细胞沉淀重悬，在1h内用流式细胞仪ACCURI C6进行检测并分析CD45阳性率，结果如表1所示。
表1
二、CD26抗体介导PBMC对PBMC的毒性评价
1、实验方法
按下述方法分别制备人肾癌786-0细胞、OS-RC-2细胞模型。
786-0细胞模型
PBMC细胞采用荧光染料Calcein-AM标记上绿色荧光信号，786-0细胞以6×105cells/ml的细胞浓度，每孔50μl接种到U-型96孔细胞培养板中，在相应的样品反应孔中分别加入50μl终浓度为100ng/ml、10ng/ml、1ng/ml、0.1ng/ml、0.01ng/ml、0.001ng/ml的抗体，在空白对照孔中加入50μl培养基，在阳性对照孔中加入50μl终浓度为1％TritonX-100，再按E/T为10:1的比例加入50μl浓度为9×106cells/ml的PBMC细胞，将反应体系置37℃二氧化碳培养下孵育5h，反应结束后，离心取上清置于一块新的96孔板中，再次离心后，取80μl上清置于黑色96孔酶板中，用酶标仪在470nm激发光波长和515nm的发射波波长的条件下进行检测。
OS-RC-2细胞模型
PBMC细胞采用荧光染料Calcein-AM标记上绿色荧光信号，OS-RC-2细胞以6×105cells/ml的细胞浓度，每孔50μl接种到U-型96孔细胞培养板中，在相应的样品反应孔中分别加入50μl终浓度为100ng/ml、10ng/ml、1ng/ml、0.1ng/ml、0.01ng/ml、0.001ng/ml的抗体，在空白对照孔中加入50μl培养基，在阳性对照孔中加入50μl终浓度为1％TritonX-100，再按E/T为15:1的比例加入50μl浓度为 9×106cells/ml的PBMC细胞，将反应体系置37℃二氧化碳培养下孵育5h，反应结束后，离心取上清置于一块新的96孔板中，再次离心后，取80μl上清置于黑色96孔酶板中，用酶标仪在470nm激发光波长和515nm的发射波波长的条件下进行检测。
细胞裂解率用公式为：(Vsample-Vvehicle control)/(VTritonX-100-Vvehicle control)×100％。其中Vsample为药物处理组的荧光信号读数的平均值，Vvehicle control为空白对照组荧光信号读数的平均值，VTriton-100为阳性对照组荧光信号读数的平均值。将细胞裂解率和样品浓度值用GraphPad Prism 7.00软件计算各样品所介导的PBMC细胞对PBMC细胞的IC50值。
2、结果分析
在抗体18G272介导的PBMC对靶细胞786-0进行杀伤的过程中，不会介导PBMC对PBMC细胞发生杀伤。抗体18G272介导的PBMC(4#)对PBMC(4#)细胞的细胞毒效应IC50值约为99730000pg/ml；抗体18G272介导的PBMC(6#)对PBMC(6#)细胞无细胞毒效应。
在抗体18G272介导的PBMC对靶细胞OS-RC-2进行杀伤的过程中，不会介导PBMC对PBMC细胞发生杀伤。抗体18G272介导的PBMC(1#)对PBMC(1#)细胞无细胞毒效应；抗体18G272介导的PBMC(3#)对PBMC(3#)细胞无细胞毒效应。
在抗体19G294介导的PBMC对靶细胞786-0进行杀伤的过程中，不会介导PBMC对PBMC细胞发生杀伤。抗体19G294介导的PBMC(2#)对PBMC(2#)细胞的细胞毒效应IC50值约为1915313pg/ml；抗体19G294介导的PBMC(3#)对PBMC(3#)细胞的细胞毒效应IC50值约为1947224pg/ml。
在抗体19G294介导的PBMC对靶细胞OS-RC-2进行杀伤的过程中，不会介导PBMC对PBMC细胞发生杀伤。抗体19G294介导的PBMC(2#)对PBMC(2#)细胞的细胞毒效应IC50值约为47920315pg/ml；抗体19G294介导的PBMC(3#)对PBMC(3#)细胞的细胞毒效应IC50值约为1406115138pg/ml。
综上所述，PBMC表达CD26同时高表达CD45，靶向CD26的抗体不会介导PBMC细胞对PBMC细胞产生细胞毒效应。
二、CD26抗体介导PBMC对CD4+T细胞、CD8+T细胞的毒性评价
实验一：从PBMC中分离CD4+T、CD8+T细胞
1、实验方法
采用CD4+磁珠(货号130-045-101，Miltenyi)从PBMC细胞中分离CD4+T细胞，采用CD8磁珠(货号130-045-201，Miltenyi)从PBMC细胞中分离CD8+T细胞。
收集分离到的CD4+T细胞，用MACS buffer清洗一次，血球计数板进行计数，分为5×105个细胞每份；分别用抗CD4抗体(货号53-0048-42，e Bioscinece)、抗CD45-FITC单克隆抗体(货号130-113-679，厂家Miltenyi，可识别所有CD45亚型)按说明书稀释后，与靶细胞于2～8℃共孵育约10min，孵育结束后，用MACS buffer清洗两次，离心弃上清，收集细胞沉淀；用约200ul的MACS buffer将细胞沉淀重悬，在1h内用流式细胞仪ACCURI C6进行检测，结果如图1，表2所示。
表2
收集分离到的CD8+T细胞，用MACS buffer清洗一次，血球计数板进行计数，分为5×105个细胞每份；分别用抗CD8抗体(货号130-098-075，Miltenyi)、抗CD45-FITC单克隆抗体(货号130-113-679，厂家Miltenyi，可识别所有CD45亚型)按说明书稀释后，与靶细胞于2～8℃共孵育约10min，孵育结束后，用MACS buffer清洗两次，离心弃上清，收集细胞沉淀；用约200ul的MACS buffer将细胞沉淀重悬，在1h内用流式细胞仪ACCURI C6进行检测，结果如图2，表3所示。
表3
实验二：细胞毒效应检测
1、实验方法
CD4+T、CD8+T细胞采用荧光染料Calcein-AM标记上绿色荧光信号，以3×105cells/ml的细胞浓度，每孔50μl接种到U-型96孔细胞培养板中，在相应的样品反应孔中分别加入50μl终浓度为100ng/ml、10ng/ml、1ng/ml、0.1ng/ml、0.01ng/ml、0.001ng/ml的抗体，在空白对照孔中加入50μl培养基，在阳性对照孔中加入50μl终浓度为1％TritonX-100，再按E/T为15:1的比例加入50μl浓度为4.5×106cells/ml的PBMC细胞，将反应体系置37℃二氧化碳培养下孵育5h，反应结束后，离心取上清置于一块新的96孔板中，再次离心后，取80μl上清置于黑色96孔酶板中，用酶标仪在470nm激发光波长和515nm的发射波波长的条件下进行检测。
细胞裂解率用公式为：(Vsample-Vvehicle control)/(VTritonX-100-Vvehicle control)×100％。其中Vsample为药物处理组的荧光信号读数的平均值，Vvehicle control为空白对照组荧光信号读数的平均值，VTriton-100为阳性对照组荧光信号读数的平均值。将细胞裂解率和样品浓度值用GraphPad Prism 7.00软件计算各样品所介导的PBMC细胞对PBMC细胞的IC50值。
2、结果分析
实验结果如表4所示。
表4 CD26抗体所介导的PBMC对CD4+、CD8+T细胞的细胞毒效应
*NA表示未检测到抗体所介导的PBMC对细胞的细胞毒效应
从上述结果可知，CD4+T cell、CD8+T细胞高表达CD45，靶向CD26的抗体19G294不会介导PBMC细胞对CD4+、CD8+T细胞亚型产生细胞毒效应。
实施例2 CD26抗体对血液瘤的作用
为观察CD26抗体是否对血液瘤细胞有杀伤作用及与CD45表达的关联性，开展如下实验：
一、淋巴瘤细胞株CD26、CD45阳性率的检测
检测方法同实施例1，不同淋巴瘤细胞的CD26、CD45表达情况如表5所示。
表5
二、CD26抗体介导PBMC对淋巴瘤细胞的毒性评价
1、实验方法
按下述方法分别制备人淋巴瘤细胞Jurkat细胞、U937细胞、Z-138细胞、NAMALWA细胞、HL-60细胞、SNK-6细胞、MOLT-4细胞模型。
肿瘤细胞采用荧光染料Calcein-AM标记上绿色荧光信号，以6×105cells/ml的细胞浓度，每孔50μl接种到U-型96孔细胞培养板中，在相应的反应孔中分别加入50μl终浓度为100ng/ml、10ng/ml、1ng/ml、0.1ng/ml、0.01ng/ml、0.001ng/ml的双特异性抗体，在空白对照孔中加入50μl培养基，在阳性对照孔中加入50μl终浓度为1％TritonX-100为阳性对照，再按E/T为15:1的比例加入50μl浓度为9×106cells/ml的PBMC细胞，将反应体系置37℃二氧化碳培养下孵育5h，反应结束后，离心取上清置于一块新的96孔板中，再次离心后，取80μl上清置于黑色96孔板中，用酶标仪在470nm的激发光波长和515nm的发射波波长的条件下进行检测。
细胞裂解率用公式为：(Vsample-Vvehicle control)/(VTriton-100-Vvehicle control)×100％。其中Vsample为药物处理组的荧光信号读数的平均值，Vvehicle control为空白对照组荧光信号读数的平均值，VTriton-100为阳性对照组荧光信号读数的平均值。
将细胞裂解率和样品浓度值用GraphPad Prism 7.00软件计算样品所介导的PBMC对靶细胞的IC50值。
3、结果分析
不同结构类型的双特异性抗体ZA23、ZA16、ZB09、19G294，等均无法介导PBMC细胞对CD45+CD26+的肿瘤细胞株Jurkat、U937、Z-138、NAMALWA、HL-60、SNK-6、MOLT-4等产生细胞毒效应，结果如表6所示，代表性附图如图3所示。
表6不同结构类型的双特异性抗体对Jurkat等细胞胞毒效应的IC50值

*NA表示未检测到抗体所介导的PBMC对细胞的细胞毒效应
综上所述，图中荧光信号没有随着抗体浓度的增加而改变(与抗体浓度无剂量依赖关系)，说明抗体没有介导PBMC细胞对相应的细胞产生细胞毒效应。多数淋巴瘤细胞表达CD26同时高表达CD45，靶向CD26的抗体不会介导PBMC细胞对高表达CD45淋巴瘤细胞产生细胞毒效应。
实施例3 CD26抗体对实体瘤的作用
为观察CD26抗体是否对实体瘤细胞有杀伤作用及与CD45表达的关联性，开展如下实验：
一、实体瘤细胞株CD26、CD45阳性率的检测
检测方法同实施例1，不同实体瘤细胞的CD26、CD45表达情况表7所示。
表7


注：“-”表示未检测；“不表达”表示使用该方法未检测出有表达，可以认为是不表达
或低表达，并非严格意义上的不表达，可能与方法检测限有关。
二、CD26抗体介导PBMC对实体瘤细胞的毒性评价
1、实验方法
实验方法同实施例2，制备人肾癌细胞OS-RC-2细胞、786-0细胞、人肺鳞癌NCI-H226细胞、人前列腺癌PC-3细胞模型。
2、结果分析
在相同的实验条件下，双特异性抗体ZA23、ZA16、ZB09、19G294等对介导PBMC细胞对CD26+CD45-肿瘤细胞OS-RC-2产生杀伤活性，见表8。双特异性抗体19G294、18G272等均可介导PBMC细胞对多种CD26+CD45-的肿瘤细胞产生强烈的细胞毒效应，见表9。
表8不同双特异性抗体介导的PBMC对OS-RC-2的细胞毒效应的IC50值(pM)
表9不同双特异性抗体介导的PBMC对不同靶细胞细胞毒效应的IC50值(pM)
综上所述，在体外实验中，不同类型的CD26抗体对多种CD45不表达或低表达的实体瘤细胞株均有杀伤作用。
为进一步观察CD26抗体对实体瘤的体内药效，开展如下三～五项体内实验。
三、双特异性抗体18G272、19G294在荷有PC-3异种移植瘤NOD/SCID小鼠 中的药效
方法：选取体重18～22g，大约5～7周龄的NOD-SCID小鼠，将5×106cells/0.1ml PC-3细胞悬液与1×107cells/0.1ml PBMC细胞悬液按照1:1(体积比)的比例充分混合后接种于NOD-SCID小鼠皮下，每只接种0.2ml。按照体重将动物随机分成3组，分别是Model组(PBS)、19G294组(30μg/只/次)、18G272组(30μg/只/次)，每组6只动物。Model组和各治疗组静脉注射给药，接种当天开始给药，连续给药五日。停药两天后，进行第二疗程的给药。首次给药时间为小鼠皮下接种细胞1h后，即第一疗程为D1、D2、D3、D4、D5；第二疗程为D8、D9、D10、D11、D12。给药频率为1天/次。
肿瘤体积：每周检测2～3次，采用游标卡尺分别量取肿瘤长径与短径，肿瘤体积(mm3)＝长径*短径2/2。相对肿瘤抑制率：TGI(％)＝(1-T/C)×100％。公认地T表示给药组某一时间点的相对肿瘤体积(当次测量的肿瘤体积与分组时肿瘤体积比值)，C表示模型组某一时间点的相对肿瘤体积(当次测量的肿瘤体积与分组时肿瘤体积比值)。而本申请实验接种当天根据体重分组，计算TGI时T、C分别表示给药组、模型组当次实际测量的肿瘤体积。
瘤重：末次检测结束后动物实施安乐死，剥离肿瘤块，生理盐水冲洗并用滤纸吸干水分，称量瘤块重量，并拍照。相对肿瘤抑制率TGI(％)＝(1-TTW/CTW)×100％，TTW表示治疗组实验终结时平均瘤重，CTW表示模型组实验终结时平均瘤重。
结论：在30μg/只的剂量下，18G272可以抑制PC-3人前列腺癌模型小鼠的肿瘤增长，19G294几乎可以完全抑制PC-3人前列腺癌模型小鼠的肿瘤增长，结果如图4、表6所示。
供试品19G294、18G272在30μg/只的剂量下，动物体重呈上升趋势，与模型组动物体重相当，治疗期间耐受良好，结果如图5、表10所示。
表10

注：*：P＜0.05，vs Model组；**：P＜0.01，vs Model组
四、双特异性抗体18G272在荷有NCI-H596(肺癌)异种移植瘤NOD/SCID小鼠中药效
方法：选取体重18～22g，大约5～7周龄的NOD/SCID小鼠，将8×106cells/0.1ml NCI-H596细胞悬液与1.6×107cells/0.1ml PBMC细胞悬液按照1:1(体积比)的比例充分混合后接种于NOD/SCID小鼠皮下，每只接种0.2ml。按照体重将动物随机分成2组，分别是Model组(PBS，6只/组)、18G272组(30μg/只/次，6只/组)。
给药途径，给药周期同上。
一般临床观察、体重、肿瘤体积检测同上。
结论：供试品18G272在30μg/只的剂量下，可显著抑制人肺腺鳞癌模型小鼠的肿瘤增长；供试品18G272在30μg/只的剂量下，动物体重呈上升趋势，治疗期间耐受良好。结果如图6、表11所示。
表11 NCI-H596人肺鳞癌细胞小鼠皮下移植模型中的肿瘤生长、体重统计

注：*：P＜0.05，vs Model组。
五、双特异性抗体19G294在荷有NCI-H226(间皮瘤)异种移植瘤NOD/SCID小鼠中药效
方法：选取体重18～22g，大约5～7周龄的NOD/SCID小鼠，将5×106cells/0.1ml NCI-H226细胞悬液与1.5×107cells/0.1ml PBMC细胞悬液按照1:1(体积比)的比例充分混合后接种于NOD/SCID小鼠皮下，每只接种0.2ml。按照体重将动物随机分成2组，分别是Model组(PBS，5只/组)、19G294组(60μg/只/次，5只/组)。给药途径，给药周期同上。
一般临床观察、体重、肿瘤体积检测同上。
结论：供试品19G294在60μg/只的剂量下，可显著抑制人肺腺鳞癌(间皮瘤)模型小鼠的肿瘤增长，如图7所示；供试品19G294在60μg/只的剂量下，动物体重呈上升趋势，治疗期间耐受良好。结果如图8、表12所示。
表12 NCI-H226人肺鳞癌(间皮瘤)细胞小鼠皮下移植模型中的肿瘤生长、体重统计

注：*：P＜0.05，vs Model组。a：表示TGI根据细胞接种后第57天瘤重计算。
六、双特异性抗体19G294在荷有A498异种移植瘤NOD/SCID小鼠中药效
方法：选取体重18～22g，大约5～7周龄的NOD-SCID小鼠，将1×107cells/0.1ml A498细胞悬液与1×107cells/0.1ml PBMC细胞悬液按照1:1(体积比)的比例充分混合后接种于NOD-SCID小鼠皮下，每只接种0.2ml。按照体重将动物随机分成2组，分别是Model组(PBS)、19G294组(30μg/只/次)，每组5只动物。给药途径，给药周期同上。
结论：19G294在30μg/只的剂量下，可完全抑制人肾癌模型小鼠的肿瘤增长，结果如图9、表13所示；供试品19G294在30μg/只的剂量下，动物没有出现体重下降，治疗期间耐受良好。
表13

注：*：P＜0.05，vs Model组；**：P＜0.01，vs Model组。a：表示TGI根据细胞接种
后第61天瘤重计算。
七、双特异性抗体18G272、19G294等在荷有OS-RC-2异种移植瘤NOD/SCID小鼠中药效
实验一：
方法：选取体重18～22g，大约5～7周龄的NOD-SCID小鼠，3×106cells/0.1ml OS-RC-2细胞悬液与6×106cells/0.1ml PBMC细胞悬液按照1:1(体积比)的比例充分混合后接种于NOD-SCID小鼠皮下，每只接种0.2ml。按照体重将动物随机分成2组，分别是Model组(PBS)、18G272组(30μg/只/次)；每组6只动物。给药途径，给药周期同上。
结论：18G272在30μg/只的剂量下，可显著抑制人肾癌模型小鼠的肿瘤增长，结果如图10、表14所示。OS-RC-2人肾癌模型在试验期间会导致动物死亡，在本试验条件下，18G272可降低动物死亡率，可显著提高OS-RC-2人肾癌模型小鼠的中位生存期(MST)，动物没有出现体重下降，治疗期间耐受良好，结果如14所示。
表14

注：*：P＜0.05，vs Model组；**：P＜0.01，vs Model组；D15后各组动物因肿瘤生长
开始死亡或安乐。a：表示TGI根据细胞接种后第29天瘤重计算。
实验二：
方法：选取体重18～22g，大约5～7周龄的NOD-SCID小鼠，3×106cells/0.1ml OS-RC-2细胞悬液与6×106cells/0.1ml PBMC细胞悬液按照1:1(体积比)的比例充分混合后接种于NOD-SCID小鼠皮下，每只接种0.2ml。按照体重将动物随机分成2组，分别是Model组(PBS)、19G294组(30μg/只/次)；每组5只动物。给药途径，给药周期同上。
结论：19G294在30μg/只的剂量下，可显著抑制人肾癌模型小鼠的肿瘤增长，结果如图11、表15所示。OS-RC-2人肾癌模型在试验期间会导致动物死亡，在本试验条件下，19G294可降低动物死亡率、延长动物生存期，动物没有出现体重下降，治疗期间耐受良好，结果如表15所示。
表15

注：*：P＜0.05，vs Model组；**：P＜0.01，vs Model组；D23后动物开始死亡或安乐
从三～七的体内实验结果可知，多种CD26抗体在CD45不表达或低表达的多种不同实体瘤细胞的动物模型中都显示出较好的治疗效果。
实施例4含有抗CD26抗体编码基因的rAAV载体在荷有肿瘤小鼠中的药效
以上实施例观察了CD26抗体对不同种类细胞的体内外杀伤作用，为进一步观察靶向CD26的基因疗法对肿瘤细胞的作用，开展如下实验：
一、AAV表达载体的构建
AAV表达载体骨架示意图如图12所示。
pAAV-GFP表达载体构建：
GFP为绿色荧光蛋白，GFP基因序列(SEQ ID NO：13)扩增后构建入pAAV-CMV载体的“β内含子增强序列”及“polyA终止序列”之间的多克隆位点MCS(XbaI与BamHI酶切位点之间)形成pAAV-GFP表达载体，作为对照。
pAAV-BiTE表达载体构建：
将信号肽基因(SEQ ID NO：14)与BiTE基因序列(SEQ ID NO:15)融合并合成扩增后构建入pAAV-CMV载体的β内含子增强序列及polyA终止序列之间的多克隆位点MCS(BamHI与EcoRI酶切位点之间)，形成AAV表达载体：pAAV-21R23(含CD26-CD3BiTE)。
二、重组AAV病毒的生产和纯化
293T细胞培养：293T细胞复苏后在含10％FBS，1％Glutamax的DMEM培养基中贴壁培养，传代2-3次后细胞状态恢复可用于包装。150mm细胞培养皿使用15ml 0.1％明胶室温包被30min。293T细胞胰酶消化后重悬、计数，吸弃培养皿中包被液，每个150mm皿接种1.21.8×10e7个293T细胞，补293T培养基至30ml，37℃，5％CO2培养过夜。
转染：第二天上午，293T细胞生长至70-80％汇合，更换新鲜293T培养基， 每皿30ml，放回培养箱。准备转染混合物，将构建好的2种AAV表达载体质粒分别与pRC6、pHelper质粒按质量比1：1：1加入无血清DMEM培养基，每个15cm皿总DNA量30μg，再轻柔加入Lipo8000脂质体(碧云天生物技术货号：C0533)，轻柔混匀，室温静置30min。每皿加入1.5ml转染混合物，轻柔混匀，放回培养箱。转染24h后，更换为30ml AAV收获培养基(含2％FBS，1％Glutamax，1％1M HEPES的DMEM培养基)，放回培养箱。
rAAV收集纯化：感染72h后，加入培养液体积的1/80的0.5M EDTA(pH8.0)到含293T细胞的培养皿中,吹打使细胞悬浮，收集到50ml无菌离心管中。于4℃2000g离心10分钟，弃上清，完全去除上清液后收集细胞沉淀，即为含有AAV颗粒的宿主293T细胞。后续步骤按照Purification Kit Maxi试剂盒(TAKARA货号：6666)说明书进行AAV的纯化与浓缩，分装后-80℃保存。
三、重组AAV递送BiTE基因在荷有OS-RC-2(肾癌)异种移植瘤NOD/SCID小鼠中的药效
方法：选取体重18～22g，大约5～7周龄的NOD-SCID小鼠，4×107cells/ml OS-RC-2细胞悬液与6×107cells/ml PBMC细胞悬液按照1:1(体积比)的比例充分混合，GFP、21R23AAV纯化样品稀释至1×1011vg/ml。将OS-RC-2+PBMC细胞悬液0.1ml分别与溶媒PBS或给药组AAV样品0.1ml混匀后接种于NOD-SCID小鼠皮下，每只接种0.2ml。分别是Model组、A组(GFP组)、B组(21R23组)、每组6只动物。建模第5天开始测量小鼠肿瘤体积、体重，观察小鼠生存状态。
结论：本试验通过NOD-SCID小鼠皮下注射OS-RC-2和PBMC细胞混合液，建立人肾癌小鼠异种移植模型。在该模型的基础上，对每只小鼠给予1次10e10vg表达BiTE双特异抗体的重组AAV进行治疗。从建模第8天开始，模型组与GFP-A组肿瘤开始明显增大，给予AAV药物治疗B组肿瘤体积出现显著抑制，第12天B组肿瘤完全消退，结果如图13、表16所示。
表16
此外，OS-RC-2人肾癌模型在试验期间会导致动物死亡，而给予AAV治疗组的动物没有出现体重下降，治疗期间耐受良好，如图14、15所示。
综上所述，重组AAV递送的BiTE双特异性抗体仅通过1次给药，即完成对CD26表达同时CD45不表达或低表达的肾癌细胞的的完全抑制，具有显著的治疗效果。
实施例5 CD26靶向CAR-T细胞对CD26-CD45-、CD26+CD45-、CD26+CD45+细胞的细胞毒性评价
为进一步观察靶向CD26的CAR-T细胞免疫疗法对肿瘤细胞的作用及与CD45表达的关联性，开展如下实验：
一、CAR-T细胞构建
重组CAR基因包含BamHI酶切位点、Kozak序列、CD8α信号肽、以VL-Linker-VH连接的抗CD26单链抗体、CD8α铰链、CD8α跨膜区、CD28共刺激域、CD3zeta胞内域、终止密码子、SalI酶切位点，将重组CAR基因全序列合成(SEQ ID NO：3)后，通过BamHI与SalI双酶切位点连入慢病毒质粒载体pWPT-GFP中替换GFP基因，形成慢病毒表达载体pWPT-44529。
将构建的PWPT-44529质粒进行慢病毒包装和纯化，进一步使用重组CAR慢病毒感染健康供体T细胞，使用IL2与激活剂刺激扩增CAR-T细胞，收获扩增后的CAR-T细胞采用流式细胞仪检测CAR阳性率，由图16可知，慢病毒感染T细胞的阳性率达到40.7％。
二、CAR-T细胞药效评价
在96孔板中将转染后的效应细胞：CAR-T细胞(44529)、未转染T细胞(NTD)分别与不同靶细胞进行共培养，靶细胞的选择包括：CD26+CD45-的肾癌细胞A498与769-P；CD26-的淋巴瘤细胞Raji与肾癌细胞G401；CD26+CD45+的淋巴瘤细胞U937与Jurkat。共培养24h后取96孔板中的共培养上清，使用DOJINDO公司LDH检测试剂盒(CK12)检测上清中靶细胞的LDH释放量来计算不同效 靶比情况下CAR-T细胞和NTD细胞的杀伤率，结果如图17。
由检测结果可见CD26靶向的CAR-T细胞对CD26阴性靶细胞Raji与G401无特异性杀伤作用，对CD26+CD45-细胞(A498、769-P)，CD26CAR-T体现出高于对照T细胞明显的杀伤效果，证明了CD26CAR-T的靶点特异性杀伤。但是对于CD26+CD45+双阳性细胞(U937、Jurkat)，CD26CAR-T的杀伤效果被明显抑制，未检测到特异性杀伤。
综上所述，CD26抗体、其他与CD26抗体有关的基因和细胞治疗方法(如靶向CD26的AAV基因治疗、靶向CD26的CAR-T细胞治疗)均显示出对CD45不表达或低表达的肿瘤细胞有显著的体内外杀伤活性；而对CD45高表达的肿瘤细胞无显著杀伤。
基于上述研究结果本申请提供了CD45筛查受试者接受CD26抗体或其衍生物治疗有效性的应用，能有效改善CD26抗体或其衍生物肿瘤免疫治疗的精准性和有效性，提高患者的临床获益；发现了CD26作为肿瘤免疫治疗新靶点的治疗有效性和精准性的新机制。

Claims (10)

  1. CD26抗体或其衍生物在制备治疗CD26表达且CD45不表达、或者CD26表达且CD45低表达的肿瘤的药物中的应用，所述肿瘤是实体肿瘤或血液瘤。
  2. 权利要求1所述的应用，所述肿瘤是实体肿瘤。
  3. 如权利要求1或2任一项所述应用，所述CD26抗体为是特异性结合CD26的全长抗体、Fab片段、FV片段、线性抗体、单链抗体、纳米抗体、双特异性抗体或多特异性抗体。
  4. 如权利要求1或2任一项所述应用，所述CD26抗体衍生物是靶向CD26的Car-T。
  5. 如权利要求1或2任一项所述应用，所述CD26抗体衍生物是含有抗CD26抗体编码基因的rAAV载体。
  6. CD45抗原或抗CD45抗体在制备用于筛查受试者接受CD26抗体或其衍生物治疗有效性的试剂中应用。
  7. 如权利要求6所述的应用，所述试剂的使用方法如下：以所述试剂检测病灶CD45表达情况；CD45低表达时，判断该受试者接受CD26抗体或其衍生物治疗有效或有效的概率高；CD45高表达时，判断该受试者接受CD26抗体或其衍生物治疗无效或无效的概率高。
  8. 如权利要求7所述的应用，所述试剂的使用方法如下：以所述试剂检测病灶CD45表达情况；CD45不表达时，判断该受试者接受CD26抗体或其衍生物治疗有效或有效的概率高；CD45表达时，判断该受试者接受CD26抗体或其衍生物治疗无效或无效的概率高。
  9. 一种筛查受试者接受CD26抗体或其衍生物治疗有效性的方法，检测病灶CD45表达情况；CD45低表达时，判断该受试者接受CD26抗体或其衍生物治疗有效或有效的概率高；CD45高表达时，判断该受试者接受CD26抗体或其衍生物治疗无效或无效的概率高。
  10. 一种筛查受试者接受CD26抗体或其衍生物治疗有效性的方法，检测病灶CD45表达情况；CD45不表达时，判断该受试者接受CD26抗体或其衍生物治疗有效或有效的概率高；CD45表达时，判断该受试者接受CD26抗体或其衍生物治疗无效或无效的概率高。
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