CN110954697A - Method for detecting anti-PD-1 antibody expression positive immune effector cells and application thereof - Google Patents

Method for detecting anti-PD-1 antibody expression positive immune effector cells and application thereof Download PDF

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CN110954697A
CN110954697A CN201811133922.4A CN201811133922A CN110954697A CN 110954697 A CN110954697 A CN 110954697A CN 201811133922 A CN201811133922 A CN 201811133922A CN 110954697 A CN110954697 A CN 110954697A
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antibody
cells
detection reagent
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金华君
唐熙
刘祥箴
何江川
黄晨
钱其军
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Shanghai Cell Therapy Research Institute
Shanghai Cell Therapy Group Co Ltd
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Shanghai Cell Therapy Research Institute
Shanghai Cell Therapy Group Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4633—Antibodies or T cell engagers
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4636—Immune checkpoint inhibitors
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152

Abstract

The invention provides a detection reagent for detecting anti-PD-1 antibody expression positive immune effector cells. Specifically, the detection reagent comprises: a marker that specifically binds to an anti-PD-1 antibody or is conjugated to an anti-PD-1 antibody, the anti-PD-1 antibody being secreted by the immune effector cell. The detection reagent and the method have the advantages of high sensitivity, good specificity and more accurate detection result.

Description

Method for detecting anti-PD-1 antibody expression positive immune effector cells and application thereof
Technical Field
The invention belongs to the fields of biotechnology and medicine, and particularly relates to a method for detecting positive CAR-T cells expressed by an anti-PD-1 antibody and application thereof.
Background
Chimeric Antigen Receptors (CARs) are a class of hybrid antigen receptors, one part being antibodies and the other part being TCRs, comprising an extracellular antigen-binding region and an intracellular signaling domain. By using single-chain antibodies (scFv) derived from tumor-specific antibodies as the extracellular antigen-binding domain of the CAR, T cells expressing the CAR (CAR-T cells) are endowed with entirely new antigen targeting. The CAR gene targeting the tumor associated antigen is transduced into lymphocytes by retroviral or lentiviral vectors, transposon or transposase systems or direct mRNA electrotransfer and expresses the fused CAR protein on the cell surface, enabling the CAR-T lymphocytes formed to recognize specific tumor associated antigens in a non-MHC-restricted manner, thereby increasing their ability to recognize and kill tumors.
CAR-T cells, while achieving significant efficacy in the treatment of hematological tumors, have encountered difficulties in the treatment of solid tumors, one of the major reasons being the activation of the PD-1/PD-L1 inhibitory immune checkpoint pathway in tumor cells. PD-1 is an immunosuppressive receptor, a type i transmembrane protein belonging to a member of the CD28 family, and is widely expressed on the surface of activated T cells, B cells, monocytes, and dendritic cells. PD-1 has 30% homology with CTLA-4 in structure, two tyrosine residues exist in an intracellular region, and are respectively involved in an immunoreceptor tyrosine-based inhibition motif (ITIM) forming an N terminal and an immunoreceptor tyrosine-based switch motif (ITSM) forming a C terminal; the extracellular domain is composed of an IgV-like domain, contains multiple glycosylation sites and is heavily glycosylated, and the domain can be combined with a ligand so as to play a role in inhibiting T cell activation. PD-L1 is also known as CD274, and a member belonging to the B7 family is designated B7 homolog 1 (B7-H1). PD-L1 is widely expressed on activated T cells, B cells, macrophages, dendritic cells and tumor cells. The combination of PD-1 and PD-L1 promotes tyrosine in ITSM domain of PD-1 to generate phosphorylation, further causes dephosphorylation of downstream protein kinases Syk and PI3K, inhibits activation of downstream channels such as AKT, ERK and the like, finally inhibits transcription and translation of genes and cytokines required by T cell activation, and plays a role in negatively regulating T cell activity. In malignant tumors, PD-1 expression is up-regulated on the surfaces of tumor cells, tumor interstitial cells and immune cells. In addition, abnormal secretion of cytokines in the tumor microenvironment, such as excessive secretion of interferon g, can induce over-expression of PD-L1 on the surface of tumor cells and other cells, and cells over-expressing PD-L1 can escape immune monitoring of T cells through a PD-1/PD-L1 pathway or other unknown mechanisms, can also induce apoptosis of T cells, or lead to immune exhaustion of T cells. Thus, functional inactivation of CAR-T cells after entry into tumor tissue is an important obstacle to CAR-T cell treatment of solid tumors. The PD-1 inhibitor, such as a PD-1 antibody, is combined with the CAR-T cell to reduce the inhibition effect of tumor cells on the T cell and obviously improve the tumor killing effect of the CAR-T cell, so that the method becomes an important direction of CAR-T treatment.
The PD-1 antibody is one of the PD-1 inhibitors which is most frequently used and is most widely used. One of the methods for combining the PD-1 antibody with CAR-T cells is to construct CAR-T cells expressing secreted anti-PD-1 antibody, i.e. to integrate and express secreted anti-PD-1 single-chain antibody (scFv) in addition to the integration of CAR gene on the genome of T cells ("targeting mesothelin chimeric antigen receptor modified T cells expressing PD-1 antibody for treating biliary tract malignancy preliminary exploration" doctor university of tusche university of celebration paper 2017.5).
Compared with a method for combining a PD-1 monoclonal antibody drug, the CAR-T cell for self-expressing secretory PD-1scFv has the advantages that 1) the anti-PD-1 antibody drug is high in price, and the treatment cost can be remarkably reduced; 2) because the CAR-T cell expresses the secretory anti-PD-1 antibody, the PD-1 antibody has higher concentration in local tumor tissues and is closer to the CAR-T cell in physical distance, so that the immunosuppression effect of tumor cells on the CAR-T cell can be more effectively relieved, and the killing of the CAR-T cell on tumors is promoted; 3) at present, PD-1 monoclonal antibody medicines are not on the market in China, only 3 enterprises (BMS, Merck and Daidabiology) are provided for marketing application to China, even if PD-1 monoclonal antibodies of the 3 enterprises are on the market in China in the future, the foreseeable medicine types of anti-PD-1 antibodies which can be selected recently are relatively limited, and the self-expressed secretory anti-PD-1 antibody is generally a single-chain antibody, so that the structure is simpler and more choices can be provided. When this strategy is employed from CAR-T cells expressing a secretory anti-PD-1 antibody, the positive rate of expression of the secretory anti-PD-1 antibody in the CAR-T cells becomes an important indicator. Expression of anti-PD-1 scFv in CAR-T Cells can be expressed by fusion of CAR with anti-PD-1 scFv (Enhanced Cancer immunothergy by clinical antibody Receptitor-Modified T Cells Engineered to Secrete Checkpoint inhibitors Li S, Siriwon N, Zhang X, Yang S, Jin T, He F, KimYJ, Mac J, Lu Z, Wang S, Han X, Wang P Clin Cancer Res.2017 Nov 15; 23(22)) or by separate expression. The fusion expression has the advantages that the anti-PD-1 scFv is fused with the CAR gene in frame for expression, the anti-PD-1 scFv is also positively expressed in the CAR-positive T cells without further detection, but the defects are that the length of the CAR gene expression frame is increased, the expression of the CAR gene is burdened, and the overlong length of the expression frame can increase the burden of virus packaging when the CAR-T cells are prepared by a lentivirus packaging method, so that the virus packaging efficiency is reduced. Expression of the CAR separately from the anti-PD-1 scFv overcomes the above-mentioned drawbacks, but the expression-positivity of the anti-PD-1 scFv needs to be separately tested because it is expressed separately.
Therefore, there is an urgent need in the art to develop a test method for detecting the positive rate of expression of anti-PD-1 scFv in CAR-T cells.
Disclosure of Invention
The invention aims to provide a method for detecting CAR-T cells positive to the expression of an anti-PD-1 antibody, which has good specificity and low false positive rate.
In a first aspect of the present invention, there is provided a detection reagent for detecting an immune effector cell positive for expression of an anti-PD-1 antibody, the detection reagent comprising: a marker that specifically binds to an anti-PD-1 antibody or is conjugated to an anti-PD-1 antibody, the anti-PD-1 antibody being secreted by the immune effector cell.
In another preferred embodiment, the immune effector cell is selected from a CAR-T cell, a CAR-NK cell, a CAR-TIL cell, a CAR-CIK cell, a CAR-macrophage, and a TCR-T cell, or a combination thereof.
In another preferred embodiment, the immune effector cell comprises a CAR-T cell.
In another preferred embodiment, the anti-PD-1 antibody is a fusion protein.
In another preferred embodiment, the anti-PD-1 antibody has a structure according to formula I:
A-B-C (I)
in the formula (I), the compound is shown in the specification,
a is a single chain antibody (scFv) against PD-1;
b is an optional linking element;
c is an Fc fragment;
and, "-" is a linker peptide or peptide bond.
In another preferred embodiment, the scFv has the structure shown in formula A1 or A2:
VL-VH(A1) (ii) a Or
VH-VL(A2)
Wherein, VLIs the light chain variable region of an anti-PD-1 antibody; vHIs the heavy chain variable region of an anti-PD-1 antibody; "-" is a linker peptide (or flexible linker) or peptide bond.
In another preferred embodiment, V isLAnd VHConnected by a flexible joint.
In another preferred embodiment, the flexible joint is 1-5 (preferably, 2-4, more preferably, 3-4) continuous (G)4S.
In another preferred embodiment, VLHas the amino acid sequence shown in the 21 st to 131 nd positions of SEQ ID No. 4, and VHThe amino acid sequence of (1) is shown in 147 th and 266 th parts of SEQ ID NO. 4.
In another preferred embodiment, the length of the connecting element is 1 to 50nt, preferably 1 to 30 nt.
In another preferred embodiment, the Fc fragment is selected from the group consisting of: IgG4Fc, IgG1Fc, IgG2Fc, or a combination thereof.
In another preferred embodiment, the amino acid sequence of the single-chain antibody against PD-1 is shown in SEQ ID No. 4.
In another preferred embodiment, the nucleotide sequence of the single-chain antibody for encoding anti-PD-1 is shown in SEQ ID No. 3.
In another preferred embodiment, the label that specifically binds to the anti-PD-1 antibody or is conjugated to the anti-PD-1 antibody is the PD-1 antigen, and the PD-1 antigen has the cargo number H82E 4.
The commercial product here under the designation H82E4 is biotin-labeled PD-1 antigen from the company acrobiosystems
In another preferred embodiment, the detection reagent is an anti-PD-1 antibody, which is purchased from Kinsley and has a cat # A01853-40.
In another preferred embodiment, the positive immune effector cells comprise CAR immune cells and CAR non-immune cells.
In another preferred embodiment, the positive immune effector cell is selected from the group consisting of: t cells, NK cells, CIK cells, NKT cells, or a combination thereof.
In another preferred embodiment, the positive immune effector cells comprise: HEK-293T cells, Jurkat cells, or a combination thereof.
In another preferred embodiment, the positive immune effector cell specifically expresses an anti-mesothelin CAR.
In another preferred embodiment, the mesothelin-resistant CAR has the structure shown in formula II:
L-scFv-H-TM-C-CD3ζ-Z-P (II)
in the formula (I), the compound is shown in the specification,
each "-" is independently a linker peptide or a peptide bond;
l is a null or signal peptide sequence;
scFv is the single chain variable region sequence of targeting mesothelin;
h is a null or hinge region;
TM is a transmembrane domain;
c is a costimulatory signal molecule;
CD3 ζ is the cytoplasmic signaling sequence derived from CD3 ζ;
z is an optional self-cleaving protein coding sequence;
p is the coding sequence of a fusion protein comprising an anti-PD-1 antibody.
In another preferred embodiment, the self-cleaving protein comprises T2A.
In another preferred embodiment, the fusion protein has a structure according to formula I:
A-B-C (I)
in the formula (I), the compound is shown in the specification,
a is a single chain antibody (scFv) against PD-1;
b is an optional linking element;
c is an Fc fragment;
and, "-" is a linker peptide or peptide bond.
In another preferred embodiment, the scFv has the structure shown in formula A1 or A2:
VL-VH(A1) (ii) a Or
VH-VL(A2)
Wherein, VLIs the light chain variable region of an anti-PD-1 antibody; vHIs the heavy chain variable region of an anti-PD-1 antibody; "-" is a linker peptide (or flexible linker) or peptide bond.
In another preferred embodiment, V isLAnd VHConnected by a flexible joint.
In another preferred embodiment, the flexible joint is 1-5 (preferably, 2-4, more preferably, 3-4) continuous (G)4S.
In another preferred embodiment, VLThe amino acid sequence of (a) is as shown in SEQ ID No.:4 from bits 21 to 131, and VHThe amino acid sequence of (1) is shown in the 147 th and 266 th positions of SEQ ID NO. 4.
In another preferred embodiment, the scFv is a single chain antibody variable region fragment of murine, human, chimeric of human and murine, or fully humanized.
In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CSF2RB, CD4, CD137, IL-2, IFNr, or a combination thereof.
In another preferred embodiment, H is the hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, CD80, CD86, or a combination thereof.
In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.
In another preferred embodiment, C is a costimulatory signal molecule for a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
In another preferred embodiment, the C is a costimulatory signal molecule derived from CD28 and/or 4-1BB (CD 137).
In a second aspect of the present invention, there is provided a kit for detecting an immune effector cell positive for expression of an anti-PD-1 antibody, the kit comprising:
(T1) a first container, and a detection reagent in the first container, the detection reagent being a label that specifically binds to an anti-PD-1 antibody or is conjugated to an anti-PD-1 antibody, the anti-PD-1 antibody being secreted by CAR-T cells; and
(t0) description.
In another preferred embodiment, the detection reagent is selected from the group consisting of: a PD-1 antigen, an antibody against a PD-1 antibody, or a combination thereof.
In another preferred embodiment, the detection reagent is an antibody against the PD-1 antibody.
In another preferred embodiment, the anti-PD-1 antibody is purchased from Kinsery, having a cat # A01853-40.
In another preferred embodiment, the detection reagent is a biotin-labeled PD-1 antigen with a cargo number of H82E4 from the company acrobiosystems.
In another preferred embodiment, the detection reagent is detectably labeled.
In another preferred embodiment, the detectable label comprises fluorescence.
In another preferred embodiment, the test kit further comprises one or more additional test reagents selected from the group consisting of:
(t2) a second container, and a sealant located within the second container;
(t3) a third container, and a positive control (such as a PD-1CAR scFv recombinant protein) located within the third container; and
(t4) a fourth container, and a negative control reagent located in the fourth container.
In another preferred embodiment, the additional detection reagents are located in the same or different containers.
In another preferred embodiment, the instructions describe a method for detecting immune effector cells positive for anti-PD-1 antibody expression.
In a third aspect of the invention, there is provided a use of the detection reagent of the first aspect of the invention for preparing a reagent or a kit for detecting anti-PD-1 antibody expression positive immune effector cells.
In another preferred embodiment, the positive immune effector cells comprise CAR immune cells and CAR non-immune cells.
In another preferred embodiment, the positive immune effector cell is selected from the group consisting of: t cells, NK cells, CIK cells, NKT cells, or a combination thereof.
In another preferred embodiment, the positive immune effector cells comprise: HEK-293T cells, Jurkat cells, or a combination thereof.
In a fourth aspect of the present invention, there is provided a method for detecting an immune effector cell positive for expression of an anti-PD-1 antibody, comprising the steps of:
(I) providing a detection reagent according to the first aspect of the invention;
(II) detecting the immune effector cell group to be detected by using the detection reagent so as to obtain a qualitative or quantitative detection result of the anti-PD-1 antibody expression positive immune effector cells.
In another preferred embodiment, the method is an in vitro detection method.
In another preferred embodiment, the method is a flow cytometry detection method.
In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.
In another preferred example, the method is a quality control method.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Figure 1 shows a schematic of the structure of a CAR targeting mesothelin domain III and an anti-PD-1 antibody.
FIG. 2 shows the results of detection of expression of PD-1 antigen of surface inhibitory phenotype by flow cytometry using biotin-labeled anti-PD-1 antibody. FIG. 2A shows the detection results of Mock-T cells; FIG. 2B shows the results of detection of Meso-CAR-T-antipiPD 1 cells.
FIG. 3 shows the results of comparing the positive rates of Meso-CAR-T-anti PD1 detected by flow cytometry for biotin-labeled anti-mesothelin antibody, biotin-labeled anti-PD-1 antibody, and PE-labeled goat anti-human IgG4Fc antibody. Figure 3A shows the proportion of mesothelin CAR positive cells measured with mesothelin domain III antigen; FIG. 3B shows the proportion of PD-1 antibody-positively expressing cells measured with an anti-PD-1 antibody.
Figure 4 shows the results of flow cytometry detection of positive CAR-T cells with anti-PD-1 antibody, IgG4Fc antibody, and mesothelin domain III antigen. FIG. 4A is the results of detection of PD-1 secretion positive Meso3CAR-T cells with antibodies against PD-1 antibody; FIG. 4B is the result of detection of PD-1 secretion-positive Meso3CAR-T cells with IgG4Fc antibody; figure 4C is the results of detecting mesothelin domain IIICAR-expressing positive cells with mesothelin domain III antigen.
FIG. 5 shows the results of detection of Meso3CAR-T cells that were secretion positive for anti-PD-1 antibodies using PD-1 antigen flow cytometry. FIG. 5A shows the results of detecting Mock-T cells with PD-1 antigen; FIG. 5B shows the results of different amounts of PD-1 antigen to detect Meso3CAR-T cells secreting anti-PD-1 antibodies.
Detailed Description
The present inventors have made extensive and intensive studies and, as a result of extensive screening, have unexpectedly developed a detection reagent having high sensitivity for detecting the positive rate of secretory anti-PD-1 scFv expressed in cells, good specificity and low false positive rate for the first time. The test result shows that the detection specificity of the detection reagent is far higher than that of other detection reagents, and in addition, the detection reagent has higher affinity for anti-PD-1 positive immune effector cells. The present invention has been completed based on this finding.
Term(s) for
As used herein, the term "CAR" refers to a chimeric antigen receptor, including an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding member (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens.
As used herein, "single chain variable fragment (ScFv)" refers to a single chain polypeptide derived from an antibody that retains the ability to bind antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA techniques, and in which the Fv regions of immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via a spacer sequence. Various methods of engineering ScFv are known to those skilled in the art.
Detection application
The antibodies of the invention are useful in detection applications, for example, for detecting a sample, thereby providing diagnostic information.
In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus, a biopsy as used in the present invention may comprise a tissue sample prepared, for example, by endoscopic methods or by needle or needle biopsy of an organ.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
Reagent kit
The invention also provides a kit for detecting the anti-PD-1 antibody expression positive immune effector cells, which comprises the following components in percentage by weight:
(T1) a first container, and a detection reagent in the first container, the detection reagent being a label that specifically binds to an anti-PD-1 antibody or is conjugated to an anti-PD-1 antibody, the anti-PD-1 antibody being secreted by CAR-T cells; and
(t0) description.
In another preferred embodiment, the detection reagent is selected from the group consisting of: a PD-1 antigen, an antibody against a PD-1 antibody, or a combination thereof.
In another preferred embodiment, the detection reagent is an antibody against the PD-1 antibody.
In another preferred example, the anti-PD-1 antibody is an anti-PD-1 antibody from Kinsley having a cat H82E 4.
In another preferred embodiment, the detection reagent is detectably labeled.
In another preferred embodiment, the detectable label comprises fluorescence.
In another preferred embodiment, the test kit further comprises one or more additional test reagents selected from the group consisting of:
(t2) a second container, and a sealant located within the second container;
(t3) a third container, and a positive control (such as a PD-1CAR scFv recombinant protein) located within the third container; and
(t4) a fourth container, and a negative control reagent located in the fourth container.
In another preferred embodiment, the additional detection reagents are located in the same or different containers.
In another preferred embodiment, the instructions describe a method for detecting immune effector cells positive for anti-PD-1 antibody expression.
Detection method
The invention also provides a method for detecting positive immune effector cells based on the antibody. Because the antibodies of the invention have high affinity and high specificity for the antigen binding region of the matching or corresponding CAR, the polyclonal antibodies of the invention can be used to detect positive immune effector cells with high efficiency, sensitivity and accuracy.
Representative examples of positive immune effector cells that can be detected by the methods of the invention include (but are not limited to): t cells, NK cells, NKT cells, CIK cells, and the like.
The detection method can be used for scientific research, research and development of cell medicines, quality control of the cell medicines, clinical treatment monitoring of the cell medicines, accompanying diagnosis of clinical patients and the like.
The main advantages of the invention include:
(1) the kit developed based on the antibody has high detection sensitivity and good specificity.
(2) Compared with the detection of anti-IgG 4Fc fragment antibody or PD-1 antigen, the detection of the anti-PD-1 scFv expression positive cells by using the antibody has low false positive rate and more accurate detection result.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight. The reagents or instruments used in the present invention are not indicated by manufacturers, and are all conventional products available on the market.
General procedure
The sources of antibodies or antigens used in the embodiments of the invention are as follows:
PD-1 antigen was purchased from Acrobiosytems under the cat # H82E 4;
antibodies to PD-1 antibodies were purchased from kasugar, cat #: a01853-40;
PE-labeled anti-IgG 4Fc antibodies were purchased from Abcam, cat No.: ab 99825;
streptomycin-PE was purchased from BD under cat # 554061;
the expichho expression system kit was purchased from ThermoFisher, cat #: a29133
MabSelect SureTMLx purchased from GE Healthcare, cat No.: 17547403
anti-CD 28 antibody: purchased from Abcam, cat # s: ab88034
The mesothelin domain II I antigen is prepared as follows:
artificially synthesizing a sequence shown in SEQ ID NO. 5, connecting the sequence to a pCDNA3.4 vector multiple cloning site, and constructing an expression vector for over-expressing the mesothelin domain III antigen and IgG4Fc fragment fusion protein. According to ExpicHOTMThe specification of the expression system, after the above fusion protein is over-expressed by using ExpichOTM expression system, the expression product is purified by using MabSelect affinity chromatography resin of GE Healthcare according to the operation steps of the specification, which are as follows:
1. cell recovery and culture
An ExpiCHO-S cell was removed from the liquid nitrogen tank, programmed to recover in a 125mL Erlenmeyer flask, 30mL of pre-heated ExpiCHO expression medium was added, and the cells were placed on a orbital shaker (37 ℃ incubator with 8% CO)2Under humidified air conditions) and the shaker speed was set at 125 rpm. After three days, samples were taken and counted, and when the cell density reached 4X 106-6×106At individual viable cells/mL, cell passages were performed.
2. Cell transfection and protein Collection
Transfection systems were prepared according to the following table
Volume of culture flask 125mL
Required amount of cells 1.5×108
Volume of transfected culture 25mL
OptiPRO SFM① 1mL
Expifeactine CHO reagent 80μL
OptiPRO SFM② 920μL
ExpicHO enhancer 150μL
ExpicHO adjuvant (Standard)③ 6mL
ExpicHO adjuvant (high titer) 6mL
ExpicHO adjuvant (maximum titer)④ Day 1 and day 5, 4mL
Final culture volume ～35mL
Plasmid DNA was divided into heavy chain and light chain (1: 1); the total plasmid DNA was 0.8ug/ml transfection culture volume, 25ml was added to adjust cell density to 6X 106Preparing mixture of ExpifeactamineCHO/plasmid DNA at a viable cell/mL ratio, incubating the mixture for 1-5 minutes at room temperature, and adding the mixture to the cell suspensionThe cells were cultured on an orbital shaker (37 ℃ incubator containing 8% CO2 in humidified air). 18-22 hours after transfection, 50. mu.L of cell suspension was aspirated for counting and the Expifeactina CHO boosters and ExpicHO adjuvants were added proportionally. 8-10 days after transfection, the culture is finished, the cell suspension transfected this time is clarified by centrifugation at 4000rpm for 30min, and then deep filtration is carried out.
3. Protein purification
MabSelect Sure Lx affinity chromatography
3.1 preparation: the MabSelect Sure Lx column was loaded on the corresponding AKTA purifier, confirming that the column packing was: mabselect Sure Lx, confirming the type of the chromatographic column and the volume of the filler; the AKTA purifier was turned on, the UNICORN software was turned on and the instrument connected.
3.2 cleaning balance: after the system pipeline is washed by purified water, a 20mM PB equilibrium chromatography column is used, the equilibrium flow rate of an xk16X20 column is not more than 6ml/min, and the equilibrium flow rate of an xk 50X 20 column is not more than 30 ml/min; the volume was equilibrated to 2-3CV until the UV and conductance baselines leveled off.
3.3 ultraviolet zero calibration: the ultraviolet absorption wavelength was confirmed to be 280nm and ultraviolet zero calibration was performed by pressing Auto zero bond.
3.4 loading: sample name, number, volume, PH were confirmed and loaded. Sample loading flow rate: the xk16X20 column is not more than 6ml/min, and the xk 50X 20 column is not more than 30 ml/min; and calculating the sample loading volume according to the expression amount and the column volume, ensuring that the filler loading capacity does not exceed 6mg/ml, and recording the actual sample loading volume.
3.5, leaching: and (3) leaching by using 20mM PB, wherein the leaching flow rate xk16x20 column is not more than 6ml/min, the leaching flow rate xk50 x20 column is not more than 30ml/min, the leaching volume is 3-5CV until the ultraviolet baseline and the conductance baseline are leveled, and the ultraviolet absorption value is less than 10 mAu.
3.6 elution: elution of a sample with 25mM citric acid at pH 3.0, elution flow rate xk16x20 column no greater than 6ml/min, xk50 x20 column no greater than 30ml/min, collection of the UV absorption > 10mAu peak, and recording the collected sample volume.
3.7, cleaning and storing the chromatographic column: washing 2CV with purified water, washing 2CV with 0.1M NaOH, washing 2CV with 20% ethanol, sealing the chromatographic column, and storing in refrigerator at 2-8 deg.C.
3.8 Low pH incubation Virus kill: the sample collected by affinity chromatography was allowed to stand at room temperature for 1-1.5h, and the pH was adjusted to 5.0 with 1M Tris buffer pH 8.
3.9 sterilizing and filtering; sterilizing and filtering with 0.22 μm, and selecting needle filter, 500ml filter bottle (harvesting) or 1L filter bottle (harvesting).
3.10 sample preservation: labeling: the product is numbered-M, is stored in a refrigerator at 2-8 ℃, and needs to be frozen in the refrigerator at-20 ℃ or-40 ℃ if the storage time exceeds one week.
Preparation method of anti-PD-1 single-chain antibody (PD-1 scFv):
the sequence shown in SEQ ID NO. 3 is artificially synthesized and is connected to the multiple cloning sites of the pCDNA3.4 vector to construct an expression vector for over-expressing the fusion protein of the PD-1 single-chain antibody and the IgG4Fc fragment. The rest is the same as the preparation method of the mesothelin structural domain III antigen.
Biotin labeling of mesothelin domain III antigen, PD-1 antigen, anti-PD-1 antibody and IgG4Fc antibody was specifically assigned to Kinsley, and labeling was performed according to a method conventional in the art.
Example 1.
Construction of recombinant expression vector expressing mesothelin-targeted CAR and recombinant expression vector expressing anti-PD-1 single-chain antibody
The nucleotide sequence of the Meso3CAR gene (containing a CD8 signal peptide, an anti-mesothelin single-chain antibody, an IgG4CH2CH3 hinge region, a CD8 transmembrane region, a CD28 intracellular domain and a tyrosine activation motif of CD3 zeta, the nucleotide sequence of which is shown in SEQ ID NO:1 and the encoded amino acid sequence of which is shown in SEQ ID NO: 2) was delegated to be synthesized by Shanghai by Czeri, and was inserted between EcoRI and SalI cleavage sites of the pNB328 vector (the structure and sequence of pNB328 are shown in CN 201510638974.7, the entire contents of which are incorporated herein by reference), and the constructed recombinant plasmid was named as pNB328-Meso3-CAR, and the structural schematic diagram is shown in FIG. 1.
A nucleotide sequence (comprising VL, a joint, VH, an IgG4CH2CH3 hinge region and an IgG4Fc region) of an anti-PD-1 scFv gene is synthesized by Shanghai Czeri, the nucleotide sequence is shown as SEQ ID NO:3, and the coded amino acid sequence is shown as SEQ ID NO: 4), the nucleotide sequence is loaded between EcoRI enzyme cutting sites and SalI enzyme cutting sites of a pS328 vector (compared with pNB328, the pS328 lacks a PB transposon sequence, and other elements are the same as the pNB328 vector), and a plasmid pS328-anti PD-1 is constructed, and the structural schematic diagram is shown as figure 1.
Example 2.
Preparation of mesothelin-targeted CAR-T cells and mesothelin-targeted CAR-T cells self-expressing secreted PD-1 antibodies
Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Ficoll separation. Culturing PBMC for 2-4h in adherent manner, wherein nonadherent suspension cells are initial T cells, collecting the suspension cells in a 15ml centrifuge tube, centrifuging for 3min at 1200rmp, discarding the supernatant, adding physiological saline, centrifuging for 3min at 1200rmp, discarding the physiological saline, and repeating the steps; 2 1.5ml centrifuge tubes were added to each tube at 5X 106Centrifuging cells with numbers a and b of 1200rmp for 3min, discarding supernatant, taking an electrotransfer kit (purchased from Lonza company), adding an electrotransfer reagent into tubes a and b in proportion to obtain 100 mu L, adding the constructed recombinant plasmid pNB328-meso3CAR 4 mu g into tube a, and adding the recombinant plasmids pNB328-meso3CAR and pS328-anti PD-1 into tube b to obtain 4 mu g respectively; transferring the mixed solution into an electric rotating cup, putting the electric rotating cup into an electric rotating instrument, selecting a required program, and carrying out electric shock; transferring the cell suspension to six-well plate (AIM-V culture solution containing 2% FBS) containing culture solution with micropipette, mixing, standing at 37 deg.C and 5% CO2Culturing in incubator, adding stimulating factor IL-2 to final concentration of 500IU/mL after six hours, adding mesothelin III antigen and anti-CD 28 antibody to final concentration of 0.5 μ g/mL, 37 deg.C, and 5% CO2Culturing for 3-4 days, observing the growth condition of the T cells, and obtaining the mesothelin-targeted CAR-T cell Meso3CAR-T and the mesothelin-targeted CAR-T cell Meso3CAR-T-antiPD1 which self-expresses the secretory PD1 antibody.
In addition, 5X 10 of the suspension was added to another centrifuge tube6The individual cells, number c, 1200rmp were centrifuged for 3min, the supernatant was discarded, and an electrotransfer kit (from Lonza) was prepared by adding 100 μ L of the electrotransfer reagent in proportion, and adding 4 μ g each of pNB 328-empty and pS 328-empty cells, and a control T cell, i.e., Mock-T cell, was constructed as described above.
Example 3.
Detection of Meso3CAR-T-AntiPD1 cells expressing the inhibitory phenotypic antigen PD-1 on their surface
1. Dissolving a PD-1 antibody marked by biotin in PBS to prepare working solution with the concentration of 1 mg/mL;
2. taking Mock-T and Meso3CAR-T-anti iPD1 cells at 1X 106Centrifuging at 1000rpm for 3min, removing upper layer culture medium, adding 2 μ L biotin-labeled anti-PD-1 antibody working solution into 2 cells, respectively, and treating at 37 deg.C with 5% CO2Incubating for 1 h;
3. the incubated cells in step 1 were washed three times with cold PBS, resuspended in 100. mu.L physiological saline, added with 1. mu.L streptomycin-PE and incubated at 4 ℃ for 30 minutes. After three washes with physiological saline, the cells were examined for their fluorescence intensity by flow cytometry, and the expression of PD-1 antigen on the surface of each cell was analyzed.
The results are shown in FIG. 2A (Mock-T) and FIG. 2B (Meso3CAR-T-anti PD 1). Compared with 0.62% of Mock-T cells, the positive rate of PD-1 antigen expression of the Meso3CAR-T-anti PD1 cells is obviously improved to 25.81%, which shows that compared with Mock-T cells, the proportion of activated cells in the Meso3CAR-T-anti PD1 cells is obviously increased.
Example 4.
Detection of mesothelin-positive Meso3CAR-T and anti-PD-1 scFv-positive Meso3 CAR-T-antipid 1 cells
1. The biotin-labeled anti-PD-1 antibody and the biotin-labeled mesothelin III antigen were dissolved in PBS to prepare a working solution having a concentration of 1 mg/mL.
2. 2 parts of Mock-T, Meso3CAR-T and Meso3CAR-T-anti iPD1 cells were taken, and 1X 10 cells were taken for each cell in each group6And (2) centrifuging at 1000rpm for 3min, removing the upper culture medium, adding 2 mu L of biotin-labeled mesothelin domain III antigen working solution obtained in the step (1) into one portion of Mock-T, Meso3CAR-T and Meso3 CAR-T-antiiPD 1 cells, adding 2 mu L of biotin-labeled anti-PD-1 antibody working solution into the other portion of Mock-T, Meso3CAR-T and Meso3 CAR-T-antiiPD 1 cells, and carrying out centrifugation at 37 ℃ and 5% CO for 3min2Incubating for 1 h;
3. two sets of CAR-T cells after incubation in step 2 were washed three times with cold PBS, resuspended in 100 μ L physiological saline, added 1 μ L streptomycin-PE, and incubated for 30min at 4 ℃. After three washes with physiological saline, the cells were examined for fluorescence intensity using a flow cytometer, and the positive rate of each CAR-T cell was analyzed.
The results are shown in FIG. 3. FIG. 3A shows the proportion of mesothelin CAR positive cells, Meso3CAR-T-antiPD1 cells, measured with mesothelin domain III antigen was significantly higher than the mesothelin CAR positive proportion of control Mock-T cells; FIG. 3B shows that the proportion of PD-1 antibody-positive expressing cells measured with anti-PD-1 antibody, the proportion of anti-PD-1 antibody-positive cells in Meso3CAR-T-antipD1 cells was significantly higher than the anti-PD-1 antibody-positive proportion of Mock-T cells to Meso3CAR-T cells.
Example 5.
Detection of specificity of anti-PD-1 scFv-positive Meso3CAR-T-anti PD1 cell positivity by different antibodies
The biotin-labeled anti-PD-1 antibody and the mesothelin domain III antigen were dissolved in PBS to prepare a working solution at a concentration of 1 mg/mL. Taking 3 parts of Mock-T cells and Meso3CAR-T-anti PD1 cells, each part is 1 multiplied by 106And the cells are divided into an anti-PD 1 antibody group, an Ig4Fc antibody group and a mesothelin domain III antigen group, each group comprises 1 part of Mock-T cells and 1 part of Meso3CAR-T-anti PD1 cells, centrifugation is carried out at 1000rpm for 3min to remove an upper culture medium, 2 mu L of biotin-labeled anti-PD-1 antibody, 2 mu L of PE-labeled anti-IgG 4Fc antibody and 2 mu L of biotin-labeled mesothelin domain III antigen are respectively added into each cell of the above 3 groups, and the temperature is 37 ℃, and the CO content is 5 percent2After incubation for 1h and washing the cells three times with cold PBS, the anti-IgG 4Fc antibody panel was directly examined for fluorescence intensity using flow cytometry. The anti-PD 1 antibody group and the mesothelin domain III antigen group were resuspended in cells again with 100. mu.L of physiological saline, 1. mu.L of streptomycin-PE antibody was added, incubated at 4 ℃ for 30 minutes, and the fluorescence intensity of the cells was detected by flow cytometry after washing three times with physiological saline.
The results are shown in FIG. 4. Fig. 4A shows the antibody detection group for the anti-PD 1 antibody, fig. 4B shows the IgG4Fc antibody detection group, and fig. 4C shows the mesothelin domain III antigen detection group. In FIG. 4A, the ratio of Meso3CAR-T-anti PD1 cells to PD-1scFv expression positive cells was 31.56%, which is lower than the 39.85% positive rate of Meso3CAR-T-anti PD1 cells to IgG4Fc cells in the IgG4Fc antibody detection group shown in FIG. 4B. Figure 4C shows that the proportion of mesothelin CAR-positive cells was about 39.85%. Meanwhile, the positive rate of 0.75% of Meso3CAR-T control cells in fig. 4A is also much lower than that of 9.15% of Meso3CAR-T control cells in fig. 4B, which indicates that the anti-PD-1 antibody detecting CAR-T cells expressing positive anti-PD-1 antibody has better specificity and can effectively reduce the false positive rate compared with the anti-IgG 4Fc antibody.
Comparative example 1.
Detection of specificity of anti-PD-1 scFv positive Meso3CAR-T-anti iPD1 cell positivity by different doses of PD-1 antigen
The biotin-labeled PD-1 antigen was dissolved in PBS to prepare a working solution having a concentration of 1 mg/mL. Taking 1 part of Mock-T cells and 1 multiplied by 1063 aliquots of Meso3CAR-T-AntiPD1 cells, each at 1X 106Centrifuging at 1000rpm for 3min, removing upper layer culture medium, adding 2 μ L biotin-labeled PD-1 antigen to Mock-T cells, adding 1 μ L, 2 μ L, and 5 μ L biotin-labeled PD-1 antigen to Meso3CAR-T-antipD1 cells, respectively, at 37 deg.C and 5% CO2After 1h incubation, cells were washed three times with cold PBS, and each group was resuspended in 100. mu.L of physiological saline, 1. mu.L of the streptomycin-PE antibody was added, incubated at 4 ℃ for 30 minutes, and the fluorescence intensity of the cells was measured by flow cytometry after three times of physiological saline washes.
The results are shown in FIG. 5. FIG. 5A shows the results of detecting Mock-T cells with PD-1 antigen; FIG. 5B shows the results of different amounts of PD-1 antigen to detect Meso3CAR-T cells secreting anti-PD-1 antibodies. According to the results of fig. 5, the CAR positive rate of Meso3CAR-T-anti PD1 cells measured by using different doses of PD-1 antigen is higher than that of Mock-T group and increases with increasing dose, but the ratio is still very low compared with the antibody group using anti-PD 1 antibody and the anti-IgG 4Fc antibody group, which indicates that the antibody of anti-PD-1 antibody selected by the invention has higher affinity with PD-1 antibody, dominates in competition with PD-1 antigen, and can identify PD-1 antibody-expressing positive cells more accurately.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shanghai cell therapy group Co., Ltd
Shanghai cell therapy research institute
<120> method for detecting anti-PD-1 antibody expression positive immune effector cells and application thereof
<130>P2018-0979
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>2049
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<213> Artificial Sequence (Artificial Sequence)
<400>1
gccaccatgg aagccccagc tcagcttctc ttcctcctgc tactctggct cccagatacc 60
accggagagg tgcagctggt ggagtccggg ggaggcctgg tccagcctgg gggatccctg 120
agactctcct gcgcagcctc tggattcgac ctcggtttct acttttacgc ctgttgggtc 180
cgccaggctc cagggaaggg cctggagtgg gtctcatgca tttatactgc tggtagtggt 240
agcacgtact acgcgagctg ggcgaaaggc cgattcacca tctccagaga caattcgaag 300
aacacgctgt atctgcaaat gaacagtctg agagccgagg acacggccgt gtattactgt 360
gcgagatcta ctgctaatac tagaagtact tattatctta acttgtgggg ccaaggcacc 420
ctggtcaccg tctcctcagg cggaggcgga tcaggtggtg gcggatctgg aggtggcgga 480
agcgacatcc agatgaccca gtctccatcc tccctgtctg catctgtggg agacagagtc 540
accatcactt gccaggccag tcagaggatt agtagttact tatcctggta tcagcagaaa 600
ccagggaaag ttcccaagct cctgatctat ggtgcatcca ctctggcatc tggggtcccc 660
tcgcggttca gtggcagtgg atctgggaca gatttcactc tcaccatcag cagcctgcag 720
cctgaagatg ttgccactta ctactgtcag agttatgctt attttgatag taataattgg 780
catgctttcg gcggagggac caaggtggag atcaaagagt ccaaatatgg tcccccatgc 840
ccaccatgcc cagcacctcc cgtggccgga ccatcagtct tcctgttccc cccaaaaccc 900
aaggacactc tcatgatctc ccggacccct gaggtcacgt gcgtggtggt ggacgtgagc 960
caggaagacc ccgaggtcca gttcaactgg tacgtggatg gcgtggaggt gcataatgcc 1020
aagacaaagc cgcgggagga gcagttccag agcacgtacc gtgtggtcag cgtcctcacc 1080
gtcctgcacc aggactggct gaacggcaag gagtacaagt gcaaggtctc caacaaaggc 1140
ctcccgtcct ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agagccacag 1200
gtgtacaccc tgcccccatc ccaggaggag atgaccaaga accaggtcag cctgacctgc 1260
ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1320
gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1380
agcaggctaa ccgtggacaa gagcaggtgg caggagggga atgtcttctc atgctccgtg 1440
atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tctgggtaaa 1500
cccttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 1560
gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 1620
atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 1680
ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 1740
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1800
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1860
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1920
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1980
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 2040
cgctgataa 2049
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Ala Thr Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Asp Thr Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly
20 25 30
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
35 40 45
Phe Asp Leu Gly Phe Tyr Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro
50 55 60
Gly Lys Gly Leu Glu Trp Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly
65 70 75 80
Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg
85 90 95
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
100 105 110
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg
115120 125
Ser Thr Tyr Tyr Leu Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val
130 135 140
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
165 170 175
Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser
180 185 190
Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu
195 200 205
Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser
210 215 220
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
225 230 235 240
Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp
245 250 255
Ser Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
260 265 270
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val
275 280285
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
290 295 300
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
305 310 315 320
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
325 330 335
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr
340 345 350
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
355 360 365
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
370 375 380
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
385 390 395 400
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
405 410 415
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
420 425 430
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
435 440445
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
450 455 460
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
465 470 475 480
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
485 490 495
Ser Leu Gly Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu
500 505 510
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
515 520 525
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
530 535 540
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
545 550 555 560
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser
565 570 575
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
580 585 590
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
595 600 605
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
610 615 620
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
625 630 635 640
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
645 650 655
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
660 665 670
Leu His Met Gln Ala Leu Pro Pro Arg
675 680
<210>3
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60
gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 120
ctctcctgca gggccagcaa aggtgtcagt acatctggct atagttattt gcactggtat 180
caacagaaac ctggccaggc tcccaggctc ctcatctatc ttgcatccta cctagaatct 240
ggcgtcccag ccaggttcag tggtagtggg tctgggacag acttcactct caccatcagc 300
agcctagagc ctgaagattt tgcagtttat tactgtcagc acagcaggga ccttccgctc 360
acgttcggcg gagggaccaa agtggagatc aaaggtggag gcggttcagg cggaggtggc 420
agcggcggtg gcgggtcgca ggtgcagctg gtgcagtccg gcgtggaggt gaagaagcct 480
ggcgcctccg tcaaggtgtc ctgtaaggcc tccggctaca ccttcaccaa ctactacatg 540
tactgggtgc ggcaggcccc aggccaggga ctggagtgga tgggcggcat caacccttcc 600
aacggcggca ccaacttcaa cgagaagttc aagaaccggg tgaccctgac caccgactcc 660
tccaccacaa ccgcctacat ggaactgaag tccctgcagt tcgacgacac cgccgtgtac 720
tactgcgcca ggcgggacta ccggttcgac atgggcttcg actactgggg ccagggcacc 780
accgtgaccg tgtcctccga gtccaaatat ggtcccccat gcccaccatg cccagcacct 840
gagttcgagg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 900
atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 960
gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 1020
gaggagcagt tccagagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080
tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1140
gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1200
ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260
taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320
accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1380
gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1440
cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatga 1488
<210>4
<211>495
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>4
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
20 25 30
Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly
35 40 45
Val Ser Thr Ser Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro
50 55 60
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser
65 70 75 80
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
100 105 110
Gln His Ser Arg Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
115 120 125
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro
145 150 155 160
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
165 170 175
Asn Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
180 185 190
Trp Met Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu
195 200 205
Lys Phe Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr
210 215 220
Ala Tyr Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr
225 230 235 240
Tyr Cys Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp
245 250 255
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro
260 265 270
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val
275 280 285
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
290 295 300
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
305 310 315 320
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
325 330 335
Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser
340 345 350
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
355 360 365
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
370 375 380
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
385 390 395 400
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
405 410 415
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
420 425 430
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
435 440 445
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
450 455 460
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
465 470 475 480
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
485 490 495
<210>5
<211>753
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60
aacgggtccg aatacttcgt gaagatccag tccttcctgg gtggggcccc cacggaggat 120
ttgaaggcgc tcagtcagca gaatgtgagc atggacttgg ccacgttcat gaagctgcgg 180
acggatgcgg tgctgccgtt gactgtggct gaggtgcaga aacttctggg accccacgtg 240
gagggcctga aggcggagga gcggcaccgc ccggtgcggg actggatcct acggcagcgg 300
caggacgacc tggacacgct ggggctgggg ctacagggcg gcatccccaa cggctacctg 360
gtcctagacc tcagcatgca agaggccctc tcggagtcca aatatggtcc cccatgccca 420
ccatgcccag ggcagccccg agagccacag gtgtacaccc tgcccccatc ccaggaggag 480
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc 540
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 600
ctggactccg acggctcctt cttcctctac agcaggctaa ccgtggacaa gagcaggtgg 660
caggagggga atgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 720
cagaagagcc tctccctgtc tctgggtaaa tga 753

Claims (10)

1. A detection reagent for detecting an anti-PD-1 antibody-expressing positive immune effector cell, wherein the detection reagent comprises: a marker that specifically binds to an anti-PD-1 antibody or is conjugated to an anti-PD-1 antibody, the anti-PD-1 antibody being secreted by the immune effector cell.
2. The detection reagent of claim 1, wherein the anti-PD-1 antibody has the structure of formula I:
A-B-C (I)
in the formula (I), the compound is shown in the specification,
a is a single chain antibody (scFv) against PD-1;
b is an optional linking element;
c is an Fc fragment;
and, "-" is a linker peptide or peptide bond.
3. The detection reagent of claim 2, wherein the scFv has the structure of formula a1 or a 2:
VL-VH(A1) (ii) a Or
VH-VL(A2)
Wherein, VLIs the light chain variable region of an anti-PD-1 antibody; vHIs the heavy chain variable region of an anti-PD-1 antibody; "-" is a linker peptide (or flexible linker) or peptide bond.
4. Such asThe detection reagent according to claim 3, wherein V isLHas the amino acid sequence shown in the 21 st to 131 nd positions of SEQ ID No. 4, and VHThe amino acid sequence of (1) is shown in 147 th and 266 th parts of SEQ ID NO. 4.
5. The detection reagent of claim 2, wherein the amino acid sequence of the anti-PD-1 single-chain antibody is shown in SEQ ID No. 4.
6. The detection reagent of claim 2, wherein the nucleotide sequence encoding the single-chain antibody against PD-1 is as shown in SEQ ID No. 3.
7. The detection reagent of claim 1, wherein the label that specifically binds to the anti-PD-1 antibody or is conjugated to the anti-PD-1 antibody is an anti-PD-1 antibody, which is purchased from kasei corporation and has a cat No. a 01853-40.
8. A kit for detecting an anti-PD-1 antibody-expressing positive immune effector cell, wherein the kit comprises:
(T1) a first container, and a detection reagent in the first container, the detection reagent being the detection reagent of any one of claims 1-7, the anti-PD-1 antibody being secreted by CAR-T cells; and
(t0) description.
9. Use of the detection reagent of any one of claims 1-7 for the preparation of a reagent or kit for detecting anti-PD-1 antibody expression positive immune effector cells.
10. A method of detecting an anti-PD-1 antibody-expressing positive immune effector cell, comprising the steps of:
(I) providing a detection reagent of any one of claims 1-7;
(II) detecting the immune effector cell group to be detected by using the detection reagent so as to obtain a qualitative or quantitative detection result of the anti-PD-1 antibody expression positive immune effector cells.
CN201811133922.4A 2018-09-27 2018-09-27 Method for detecting anti-PD-1 antibody expression positive immune effector cells and application thereof Pending CN110954697A (en)

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