CN114395532A

CN114395532A - Cell strain for detecting activity of antibody-immune agonist coupled drug and detection method

Info

Publication number: CN114395532A
Application number: CN202210280605.5A
Authority: CN
Inventors: 徐涵文; 马赛; 秦刚
Original assignee: Genequantum Healthcare Suzhou Co Ltd
Current assignee: Genequantum Healthcare Suzhou Co Ltd
Priority date: 2022-03-22
Filing date: 2022-03-22
Publication date: 2022-04-26
Anticipated expiration: 2042-03-22
Also published as: CN114395532B

Abstract

The invention provides a cell strain for detecting the activity of an antibody-immune agonist coupled drug and a detection method, wherein the cell strain expresses an antigen or a fragment thereof capable of binding an antibody in the antibody-immune agonist coupled drug, a receptor capable of binding an immune agonist in the antibody-immune agonist coupled drug and an immune signal path reporter gene. The cell strain can be used for activity detection of immune agonist conjugate drugs with different target antigens and different activity levels. The detection method can overcome the defects of a co-culture mode of PBMC and antigen positive cells, has the advantages of simple operation, low cost, higher sensitivity, larger detection dynamic range and the like, and the activity result of the antibody-immune agonist coupled drug obtained by the detection method is closer to the in vivo efficacy experiment verification result.

Description

Cell strain for detecting activity of antibody-immune agonist coupled drug and detection method

Technical Field

The invention relates to a method for detecting the activity and the drug effect of an antibody-immune agonist coupled drug, in particular to a cell strain for detecting the activity of the antibody-immune agonist coupled drug and a detection method.

Background

Tumor immunotherapy is a very promising cancer treatment method, and is also a hot direction in drug development. The principle of tumor immunotherapy is to eliminate tumors and prevent tumor recurrence by enhancing host immune function. Currently, a widely used tumor immunotherapy in clinical practice uses checkpoint inhibitors (CPI) represented by monoclonal antibodies targeting PD-1 and PD-L1, which show good efficacy on multiple tumor species, but many cancer patients still do not respond to checkpoint inhibitors or have new progression of the disease after treatment. One of the reasons is that many solid tumors are internally immunosuppressed, have low availability of neoantigens, and have low T cell infiltration, making them ineffective for immunotherapy. These tumors are often rich in tumor-associated myeloid cells (myeloid cells), providing an immunosuppressive environment.

TLR7/8 (Toll-like receptor 7/8), expressed in these myeloid cells. TLR7/8 agonists bind to TLR7/8 receptors and activate downstream signaling, activating IFN Regulatory Factor (IRF) and NF- κ B signaling pathways, leading to the production of type I interferons and other proinflammatory cytokines. TLR7/8 agonists are potent activators of innate immunity and have been demonstrated to have potent antiviral and antitumor activity in a number of animal and human studies. Agonists can activate T/B cell-mediated humoral immune responses by enhancing phagocytosis and antigen presentation by macrophages and dendritic cells, thereby producing a long-lasting and systemic anti-tumor effect on tumor antigens. However, such small molecule agonists are poor in tolerance, and generally need to be administered by intratumoral administration (intracancer) or epidermal smearing (topical) mode, but cannot be administered systemically, which greatly increases the difficulty of further clinical application and popularization.

The use of antibody-immune agonist conjugate drugs (i.e., covalent attachment of an immune agonist to a tumor-targeting antibody) in order to specifically deliver an immune agonist into the tumor microenvironment, triggering a local immune activation cascade, is a promising approach to overcome current drug resistance mechanisms and immune agonist side effects of cancer immunotherapy. The products currently in clinical trials are NJH395 from Nowa, BDC-1001 from Bolt Biotherapeutics, Inc. (Bolt Biotherapeutics) and SBT6050 from Hiwolback Therapeutics, both of which target HER2, and small molecule agonists of TLR 7/8. The mechanism of action of these products includes several aspects: first, the antibody moiety inhibits HER2 receptor dimerization and HER2 ectodomain shedding, promotes receptor internalization and/or degradation, inhibits downstream signaling pathways, and stimulates antibody-dependent cellular cytotoxicity (ADCC) killing of tumor cells overexpressing HER 2. Second, antigen presenting cells (e.g., macrophages and dendritic cells) directly phagocytose and clear tumor cells via antibody-dependent cellular phagocytosis (ADCP). Release of the immune agonist within the antigen presenting cell results in further activation of the antigen presenting cell and enhances the antigen presenting effect. Finally, activated antigen presenting cells can enhance tumor antigen-specific T cell activation, resulting in cytotoxic T cell-mediated immune responses against tumor neoantigens.

The activity detection of the antibody-immune agonist conjugate drug is an important ring for target selection, compound screening, drug effect evaluation and drug quality control. The main effector cell of the TLR7/8 agonist is an immune cell, and an antibody targets a tumor cell. The antibody is specifically bound to a target antigen, the antibody is brought into an effector cell together with an immune agonist, and the release of the immune agonist and the activation of a downstream signal path are important steps for the antibody-immune agonist conjugate drug to exert the drug effect. The existing cell strain or method for detecting the activity of the small molecule agonist by expressing TLR7 or TLR8 and a downstream NF-kB-reporter gene cannot be used for detecting the activity of the antibody-immune agonist conjugate. The activity of the small-molecule immune agonist in the reporter cell is not only related to the receptor agonistic activity, but also related to the membrane permeability of the small-molecule immune agonist, so that the activity detection result of the small-molecule immune agonist in the existing reporter cell cannot be used as the activity detection result of the antibody-immune agonist conjugate.

The existing antibody-immune agonist conjugate activity detection system generally uses a co-culture mode of immune cells and tumor cells to detect downstream cytokines. The most commonly used method at present is to co-culture Peripheral Blood Mononuclear Cells (PBMCs) with antigen-positive tumor cells of interest and then detect downstream cytokines. The defects of the method comprise:

1. the detection method of the PBMC and antigen positive cell co-culture system is complex, relates to multiple steps of blood collection, PBMC separation, cell culture, plate laying, ELISA and the like, and has high material and manual cost;

2. the PBMC activity of different blood donors is greatly different, and the activity difference has obvious influence on the experimental result;

3. for some agonists with weaker activity, the co-culture system of PBMC and antigen positive cells is not sensitive enough, and the change of cytokines can not be detected frequently;

4. for different targets and agonists, different inflammatory cytokines need to be screened to select a suitable detection index, and the method cannot be universally used among multiple targets and agonists.

Therefore, it is necessary to develop a stable and highly sensitive cell line for detecting the activity of the antibody-immunoactivator conjugate, and to develop a method and a kit for detecting the activity of the antibody-immunoactivator conjugate with simple operation, low test cost, accuracy and high efficiency.

Disclosure of Invention

The invention aims to provide a cell strain for detecting the activity of an antibody-immune agonist coupled drug, and a method and a kit for detecting the activity of the antibody-immune agonist coupled drug, which have the advantages of good stability, high sensitivity, simple operation and low cost, are developed by using the cell strain.

In order to achieve the purpose, the invention adopts the technical scheme that:

a cell line for detecting the activity of an antibody-immunoactivator conjugate drug, said cell line expressing an antigen or a fragment thereof capable of binding to an antibody of said antibody-immunoactivator conjugate drug, a receptor capable of binding to an immunoactivator of said antibody-immunoactivator conjugate drug, and an immune signaling pathway reporter gene.

In some embodiments, the antigen is a target protein overexpressed by tumor cells.

In some embodiments, the antigen is a target protein HER2, CD47, Trop2, CEACAM5 (CEA Cell addition Molecule 5), CD22, sirpa (signal regulatory protein a), PD-L2, or PD-L1.

In some embodiments, the antibody is a monoclonal antibody (e.g., a monoclonal antibody, a diabody, an scFv fragment) or an antibody fusion protein.

In some embodiments, the antibody is an immune checkpoint inhibitor.

In some embodiments, the immune agonist is a Toll-like receptor agonist or a STING (Stimulator of Interferon gene stimulating protein) agonist.

In some embodiments, the immune signaling pathway reporter is an NF- κ B signaling pathway reporter or an IRF (interferon regulatory factor) signaling pathway reporter.

According to some preferred embodiments, the Toll-like receptor is TLR7 and/or TLR8 and the immune agonist is TLR7 and/or TLR8

、

、

、

、

Or

。

In some embodiments, the STING (Stimulator of Interferon gene stimulating proteins) agonist is 2',3' -cGAMP.

According to some preferred embodiments, the cell line is HEK293 cells.

According to some preferred embodiments, the reporter gene sequence in the immune signaling pathway reporter gene is a luciferase gene sequence or an alkaline phosphatase gene sequence.

For example: the HER2 antigen, TLR7 and NF-kappa B-alkaline phosphatase reporter gene are expressed in HEK293 cells and are used for detecting the activity of an antibody-TLR 7 immune agonist coupling drug targeting HER 2.

Another example is: PD-L1 antigen, TLR8 and NF-kB-luciferase genes are expressed in HEK293 cells and are used for detecting the activity of an antibody-TLR 8 immune agonist coupling drug targeting PD-L1.

According to some preferred embodiments, the antigen or fragment thereof is expressed from a lentivirus infected cell strain of the antigen or fragment thereof, and the lentivirus expression plasmid of the antigen or fragment thereof comprises a CMV promoter, a gene coding region sequence of the antigen or fragment thereof located downstream of the CMV promoter, a first selection gene for selecting positive cell strains, and a nucleic acid sequence encoding a 2A linker peptide for linking the gene coding region sequence of the antigen or fragment thereof and the first selection gene. Preferably, the 2A linker peptide is a P2A linker peptide or a T2A linker peptide. In some embodiments, the 2A linker peptide is a P2A linker peptide.

According to some preferred embodiments, the immune agonist receptor is expressed from an immune agonist receptor lentivirus infected cell line, and the immune agonist receptor lentivirus expression plasmid comprises a CMV promoter, a gene coding region sequence of the immune agonist receptor located downstream of the CMV promoter, a second selection gene for selecting positive cell lines, and a nucleic acid sequence for linking the gene coding region sequence of the immune agonist receptor and the nucleic acid sequence of the second selection gene encoding a 2A linker peptide. Preferably, the 2A linker peptide is a P2A linker peptide or a T2A linker peptide. In some embodiments, the 2A linker peptide is a P2A linker peptide.

Specifically, the first screening gene and the second screening gene are different so as to carry out positive screening on each lentivirus infected cell strain.

Further, the first screening gene and the second screening gene are independently a fluorescent protein gene or a resistance gene.

According to some embodiments, the first selection gene and the second selection gene are fluorescent protein genes encoding different fluorescent proteins, or resistance genes corresponding to different antibiotics, or one is a fluorescent protein gene and the other is a resistance gene.

According to some embodiments, the resistance gene is a puromycin resistance gene, blasticidin s (blasticidin s) resistance gene or bleomycin (Zeocin) resistance gene.

According to some embodiments, the fluorescent protein gene is a red fluorescent protein gene, a green fluorescent protein gene, or a blue fluorescent protein gene. According to some embodiments, the immune signaling pathway reporter gene is infected into the cell line by a NF- κ B signaling pathway reporter lentivirus or an IRF signaling pathway reporter lentivirus.

According to some preferred embodiments, the lentiviral expression plasmid for the NF- κ B signaling pathway reporter comprises a CMV promoter, a third selection gene located downstream of the CMV promoter, an NF- κ B response element, an IFN- β promoter, and a reporter sequence.

According to some preferred embodiments, the IRF signaling pathway reporter lentiviral expression plasmid comprises a CMV promoter, a third selection gene located downstream of the CMV promoter, an ISRE response element, a TATA promoter, and a reporter sequence.

Specifically, the third screening gene is a fluorescent protein gene or a resistance gene, and the third screening gene, the first screening gene and the second screening gene are different from each other in pairs so as to carry out positive screening on cell strains infected by various lentiviruses.

In some embodiments, the first screenable gene, the second screenable gene, and the third screenable gene are fluorescent protein genes encoding different fluorescent proteins.

In other embodiments, the first selection gene, the second selection gene and the third selection gene are resistance genes corresponding to different antibiotics, such as puromycin resistance gene, blasticidin s (blasticidin s) resistance gene and bleomycin (Zeocin) resistance gene. In some embodiments, the first selection gene is a puromycin resistance gene, the second selection gene is blasticidin s (bleticidin s) resistance gene, and the third selection gene is a bleomycin (Zeocin) resistance gene.

In other embodiments, one of the first selection gene, the second selection gene, and the third selection gene is a resistance gene, and the other two are fluorescent protein genes encoding different fluorescent proteins. For example, the first selection gene is a resistance gene, and the second selection gene and the third selection gene are fluorescent protein genes encoding different fluorescent proteins. In some embodiments, the first selection gene is a puromycin resistance gene, the second selection gene is a green fluorescent protein gene encoding a green fluorescent protein, and the third selection gene is a red fluorescent protein gene encoding a red fluorescent protein.

The invention also provides a construction method of the cell strain for detecting the activity of the antibody-immune agonist coupled drug, which comprises the following steps:

firstly, constructing an antigen or a fragment lentivirus containing the gene coding region sequence of the antigen or the fragment thereof;

secondly, infecting a reporter gene cell strain with the antigen or the fragment of the antigen in the first step, wherein the reporter gene cell strain simultaneously expresses the receptor of the immune agonist and the immune signal path reporter gene;

and thirdly, screening positive cell strains by adopting a fluorescent label screening and/or resistance screening mode to obtain the cell strains for detecting the activity of the antibody-immune agonist coupled drug.

Preferably, the construction method specifically comprises the following steps:

1) inserting the gene coding region sequence of the antigen or the fragment thereof at the downstream of the CMV promoter, and connecting the nucleic acid sequence of the gene coding region sequence with a first screening gene by using a nucleic acid sequence coding a 2A connecting peptide (such as a P2A connecting peptide) to construct an antigen or fragment thereof slow virus expression plasmid;

2) carrying out lentivirus packaging on the antigen or the fragment lentivirus expression plasmid of the antigen in the step 1) to obtain lentivirus;

3) infecting a reporter gene cell strain by adopting the lentivirus obtained in the step 2);

4) and screening positive cell strains by using antibiotics and/or fluorescence corresponding to the first screening gene to obtain the cell strains for detecting the activity of the antibody-immune agonist coupled drug.

According to a specific and preferred embodiment, said first selection gene is a puromycin resistance gene and said antibiotic is puromycin.

According to some preferred embodiments, the reporter cell line is constructed by:

(1) embedding the gene coding region sequence of the receptor of the immune agonist at the downstream of the CMV promoter, and connecting a nucleic acid sequence coding a 2A connecting peptide (such as T2A connecting peptide) with a second screening gene to construct a receptor lentivirus expression plasmid of the immune agonist;

(2) carrying out lentivirus packaging on the receptor lentivirus expression plasmid of the immune agonist in the step (1) to obtain lentivirus;

(3) respectively adopting the lentivirus of the step (2) and the lentivirus of the immune signal path reporter gene to infect cells;

(4) and screening the positive cell strain by adopting a fluorescent label screening and/or resistance screening mode to obtain a reporter gene cell strain which simultaneously expresses a receptor of the immune agonist and an immune signal path reporter gene.

Specifically, the second selection gene is different from the first selection gene.

According to some embodiments, the lentiviral vector is obtained by packaging an expression plasmid of the lentiviral vector, wherein the expression plasmid of the lentiviral vector is an expression plasmid of NF-kB signal pathway reporter lentiviral vector or an expression plasmid of IRF signal pathway reporter lentiviral vector.

Specifically, the construction method of the NF-kB signal path reporter gene lentiviral expression plasmid comprises the following steps: and a third screening gene, an NF-kB response element, an IFN-beta promoter and a reporter gene sequence are sequentially embedded in the downstream of the CMV promoter.

Specifically, the construction method of the IRF signal pathway reporter gene lentiviral expression plasmid comprises the following steps: and a third screening gene, an ISRE response element, a TATA promoter and a reporter gene sequence are sequentially embedded in the downstream of the CMV promoter.

Specifically, the first screening gene, the second screening gene and the third screening gene are different from each other in pairs so as to carry out positive screening on cell strains infected by various lentiviruses. In some embodiments, the first selection gene is a puromycin resistance gene, the second selection gene is a green fluorescent protein gene encoding a green fluorescent protein, and the third selection gene is a red fluorescent protein gene encoding a red fluorescent protein.

Specifically, the reporter gene sequence is a luciferase gene sequence or an alkaline phosphatase gene sequence.

For example: embedding a gene coding region sequence of a receptor of the immune agonist at the downstream of the CMV promoter, and connecting a nucleic acid sequence coding a T2A connecting peptide with a third screening gene to construct a receptor lentivirus expression plasmid of the immune agonist; sequentially embedding a red fluorescent protein label, an NF-kB reaction element, an IFN-beta promoter and a reporter gene sequence at the downstream of the CMV promoter to construct the NF-kB signal pathway reporter gene lentiviral expression gene; or, sequentially embedding a red fluorescent protein label, an ISRE reaction element, a TATA promoter and a reporter gene sequence at the downstream of the CMV promoter to construct the IRF signal pathway reporter gene lentiviral expression plasmid.

In some embodiments, the reporter cell line is a TLR7 or TLR8 agonist reporter cell line currently used for detection of small molecule immune agonists, such as HEK-hTLR7 (cat # hkb-hTLR 7) and HEK-hTLR8 (cat # hkb-hTLR 8) from Avoweo root (Invivogen).

The invention also provides a method for detecting the activity of the antibody-immune agonist conjugate drug,

setting up the experimental group and the control group: the experimental group is that the cell strain for detecting the activity of the antibody-immune agonist coupled drug is cultured in a culture medium containing the antibody-immune agonist coupled drug and a chromogenic substrate (substrate of alkaline phosphatase) corresponding to the immune signal path reporter gene, and the control group is that the cell strain for detecting the activity of the antibody-immune agonist coupled drug is cultured in a culture medium containing only the chromogenic substrate corresponding to the immune signal path reporter gene;

and respectively detecting the light absorption values of the experimental group and the control group at 600-650 nm, and judging the activity of the antibody-immune agonist coupled drug according to the light absorption values of the experimental group and the control group.

Preferably, 3-10 detection concentrations of the antibody-immune agonist coupled drug are set, the multiple of the light absorption value of the experimental group relative to the light absorption value of the control group and the EC50 value are calculated, and the activity of the antibody-immune agonist coupled drug in the test range is determined.

Preferably, the detection concentration range of the antibody-immune agonist coupling drug is 0.001 nM-60 nM; more preferably 0.01nM to 30 nM; e.g., 0.001nM, 0.005nM, 0.008nM, 0.01nM, 0.02nM, 0.05nM, 0.09nM, 0.1nM, 0.6nM, 1nM, 2nM, 3nM, 5nM, 8nM, 10nM, 14nM, 17nM, 20nM, 25nM, 30nM, 35nM, 40nM, 43nM, 50nM, 51nM, 60 nM.

According to some specific and preferred embodiments, said detection method comprises in particular the following steps:

a) taking the cell strain with the cell fusion degree of 70-80% and good growth state for detecting the activity of the antibody-immune agonist coupled drug, and incubating for 2-3min by using PBS;

b) adjusting the cell density to 1X 10 using a medium containing a chromogenic substrate corresponding to said immune signaling pathway reporter gene⁵~6×10⁵Cells/ml are plated in 50-150 muL/hole, an antibody-immune agonist coupling drug with the concentration of 0.01 nM-30 nM is added into an experimental group, and a culture medium which is the same in volume as the experimental group and only contains a chromogenic substrate corresponding to the immune signal pathway reporter gene is added into a control group;

c) and respectively placing the experimental group and the control group in an incubator for culturing for 20-30 h, detecting the light absorption value at 600-650 nm, calculating the multiple of the light absorption value of the experimental group relative to the light absorption value of the control group and EC50 (half maximum effect concentration), and determining the activity of the antibody-immune agonist coupled drug in the test range.

Wherein the concentration refers to the concentration of the antibody-immune agonist conjugate drug in the culture medium.

In addition, the invention also provides a kit, which comprises the cell strain for detecting the activity of the antibody-immune agonist coupled drug.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

the detection method can overcome the defects of complicated detection method, large activity difference of PBMC of donors, low sensitivity, poor universality and the like in a PBMC and antigen positive cell co-culture mode, has the advantages of simple operation, low cost, higher sensitivity, larger detection dynamic range and the like, can be used for the activity detection of the coupling drugs of the immune agonists with different target antigens and different activity levels, and has an activity detection result closer to the in vivo efficacy experiment verification result.

Drawings

FIG. 1 is a strategy and map of plasmid construction in an example of the present invention;

FIG. 2 is a graph of the results of the activation effect of different immune agonists on HEK-hTLR7 cells;

FIG. 3 is a graph of the results of the activation effect of different immune agonists on HEK-hTLR8 cells;

FIG. 4 is a HER2-GFP lentiviral expression plasmid map;

FIG. 5 is a graph of the results of cell detection after flow sorting;

FIG. 6 is a graph showing the results of a pressure screening FACS assay;

FIG. 7 is a graph showing the results of affinity assay of HEK-hTLR7-HER2-GFP and HEK-hTLR8-HER2-GFP with HER2 antibody, in which HCC1954 (HER 2+ + +) indicates the expression of HER 2;

FIG. 8 is a graph showing the results of activity assays of antibody mAb control and HER2 AIAC1 in two reporter cells HEK-hTLR7 and HEK-hTLR7-HER2-GFP, respectively;

FIG. 9 is a graph showing the results of activity assays of antibody mab controls and different antibody-immune agonist conjugates in two reporter cells, HEK-hTLR7-HER2-GFP and HEK-hTLR8-HER 2-GFP;

FIG. 10 is a graph showing the results of the detection of HER2 AIAC1 and its mab control for TNF-. alpha.release activation in a PBMC (from donor 1 or donor 2) co-culture assay with antigen positive cells of interest;

FIG. 11 is a graph showing the results of the detection of HER2 AIAC1, HER2 AIAC6 and its mab control for TNF- α release activation in a PBMC (from donor 3) co-culture assay with antigen positive cells of interest;

FIG. 12 is a graph showing the results of activity assays of antibody-immunoactivator conjugates of different targets in a PBMC and tumor cell co-culture system, wherein A shows the secretion of TNF- α, B shows the secretion of IP-10, and C shows the secretion of MCP-1;

FIG. 13 is a graph showing the results of activity assays of antibody mab control, HER2 AIAC1 and HER2 AIAC6 in HEK-hTLR7-HER2-GFP reporter cells;

FIG. 14 is a graph showing the results of the drug efficacy test of HER2 AIAC4 and HER2 AIAC6 in HER2 positive HCC1954 CDX model;

FIG. 15 is a graph showing the results of drug efficacy testing of HER2 AIAC1 and HER2 AIAC6 in HER2 positive NCI-N87 CDX model;

fig. 16 is a graph showing the results of drug efficacy test of HER2 AIAC1 and HER2 AIAC6 in HER2 positive gastric cancer PDX model.

Detailed Description

All of the features disclosed in the specification of the invention, or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where mutually exclusive features or steps are claimed. The invention will now be further described with reference to specific examples, but the invention should not be limited to these examples, but may be substituted by other equivalent or similarly purposed alternative features unless specifically stated. Unless expressly stated otherwise, each feature is only an example of a generic series of equivalent or similar features. Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The conventional co-culture mode of PBMC and antigen positive cells for detecting the activity of the antibody-immune agonist coupled drug has the problems of complicated operation, large activity difference of PBMCs from different sources, low sensitivity, incapability of accurately detecting and evaluating the drug activity of some immune agonists with low coupling activity, high detection cost and the like. The cell strain simultaneously expresses an antigen or a fragment thereof capable of binding to an antibody in an antibody-immune agonist coupled drug, a receptor capable of binding to an immune agonist in the antibody-immune agonist coupled drug, and an immune signal path reporter gene.

According to some embodiments, the cell line for detecting the activity of the antibody-immunoactivator conjugate is constructed as follows:

first, plasmid construction

As shown in fig. 1, 4 lentiviral expression plasmids were constructed.

1. Lentivirus expression plasmids (also referred to simply as target gene expression plasmids) for antigens or fragments thereof: the sequence encoding the target gene (e.g., cell surface receptor such as HER 2) is inserted downstream of the CMV promoter and linked to a first selection gene (e.g., puromycin resistance gene) with a nucleic acid sequence encoding a P2A linker peptide.

2. An immune agonist receptor lentiviral expression plasmid (e.g., an expression plasmid for TLR7 or TLR 8): the gene coding region sequence for TLR7 or TLR8 was inserted downstream of the CMV promoter and linked to a second selection gene (e.g., a green fluorescent protein (EGFP) tag) with a nucleic acid sequence encoding a T2A linker peptide. In some embodiments, the second selection gene may also be a different resistance gene than the first selection gene.

3. NF-kB signal path reporter lentivirus expression plasmid (also called NF-kB reporter plasmid for short): and a third screening gene (such as a red fluorescent protein (mChery) label), an NF-kB reaction element, an IFN-beta promoter and a reporter gene sequence are sequentially embedded at the downstream of the CMV promoter, and the reporter gene sequence can be a luciferase gene sequence or an alkaline phosphatase gene sequence. In some embodiments, the third selection gene may also be replaced with a resistance gene that is different from both the first selection gene and the second selection gene.

4. IRF signaling pathway reporter lentiviral expression plasmid (also abbreviated IRF reporter plasmid): detecting the activation of signal transduction pathways induced by interferon regulatory factor 7 (IRF 7), IRF3, etc., and sequentially embedding a third screening gene (such as a red fluorescent protein (mCherry) label), an ISRE response element, a TATA promoter (minor TATA promoter) and a reporter gene sequence at the downstream of the CMV promoter, wherein the reporter gene sequence can be a luciferase gene sequence or an alkaline phosphatase gene sequence. In some embodiments, the third selection gene may also be replaced with a resistance gene that is different from both the first selection gene and the second selection gene.

Wherein the resistance genes (i.e. the first resistance gene, the second resistance gene and the third resistance gene) of

plasmids

1, 2 and 3 or 4 were used for the screening of different resistances.

The preparation method of the trastuzumab of the invention mainly comprises the following steps: transferring the nucleic acid sequences of the heavy chain and the light chain into an expression cell, and culturing and purifying to obtain the product.

Secondly, constructing cell strains:

the genome of the reporter cell line includes a gene of interest (a nucleic acid sequence encoding a tumor antigen or fragment thereof), a nucleic acid sequence encoding TLR7 or TLR8, and a reporter gene. For example: HER2, TLR7 and NF-kB-alkaline phosphatase are expressed in HEK293 cells, and the activity of an antibody-TLR 7 immune agonist targeting HER2 is detected; another example is: PD-L1, TLR8 and NF-kB-luciferase are expressed in HEK293 cells and are used for detecting the activity of an antibody-TLR 8 immune agonist targeting PD-L1.

Cell line selection:

the method comprises the following steps: TLR7 agonist or TLR8 agonist reporter gene cell lines (e.g., HEK-hTLR7 (the HEK-Blue hTLR7 cell line with the product number of hkb-hTLR7 from Avova root (InvivoGen)) and HEK-hTLR8 (the HEK-Blue hTLR8 cell line with the product number of hkb-hTLR8 from Avova root (InvivoGen)) for detection of small molecule agonists are commercially available, and TLR7 or TLR8, NF- κ B reporter gene or IRF reporter gene is expressed in such cell lines. The target gene can be further infected directly on the basis of the reporter gene cell strain.

The second method comprises the following steps: toll-like receptors (TLR 7 or TLR 8) and reporter genes (NF-kB reporter genes or IRF reporter genes) are sequentially infected in HEK293 cells, cell strains expressing the Toll-like receptors and the reporter genes simultaneously are screened, and then target genes are further infected.

And (3) respectively carrying out lentivirus packaging, concentration and titer test on the lentivirus expression plasmids constructed in the step one to prepare virus stock solution for infecting host cells.

Specific operation of viral infection:

1) inoculating cells: taking HEK293 cells with 70% -80% fusion degree and good growth state, digesting, counting and inoculating about 1.5 × 10⁶Culturing the cells in a T25 bottle containing 6mL of DMEM complete medium, and placing the cells in a cell culture box for overnight culture;

2) viral infection: preparing a mixture of a DMEM complete medium and polybrene (ploybrene), wherein the final concentration of polybrene is 10 mug/mL; before infection, taking out the virus stock solution from a refrigerator, placing the virus stock solution on ice to melt the virus stock solution, sucking out original cell culture medium, adding 4mL of a mixture of DMEM complete culture medium and polybrene, and then adding 25 muL of the virus stock solution, wherein the MOI (multiflicity of infection) =5 of the virus stock solution;

3) after 24 hours of infection, the virus-containing medium was discarded, and fresh DMEM complete medium was added thereto, and the culture was continued at 37 ℃.

Screening positive cell strains:

for those containing fluorescent tags, the positive selection procedure was as follows:

(1) after 48h of infection, the expression of the fluorescent protein can be observed by a fluorescent microscope, and the fluorescent protein is expanded and cultured to a T75 culture bottle;

(2) flow type separation: cell count, filtration on 70 μm filter, 1mL of PBS resuspension (cell density 1X 10 or less)⁷Cells/ml), the department with small fluorescence intensity span and the most dense cells among the positive cells is collected, and the fluorescence intensity span is approximately in the positive region of 50% -60% of the positive cell population;

(3) culturing cells, and performing secondary sorting or pressurized screening to ensure the purity of positive cells;

(4) and (3) continuing cell amplification culture, freezing and storing, wherein the fluorescence positive rate is more than 99.5% by FACS detection before freezing.

For those containing resistance genes, the positive selection procedure was as follows:

(1) after infection for 72h, carrying out amplification culture, and adding corresponding antibiotics; preferably, according to the resistance gene, 8 mug/mL Puromycin (Puromycin) screening, or 10 mug/mL blasticidin S (Blasticidin S), or 100 mug/mL bleomycin (Zeocin) can be selected for screening;

(2) subculturing for 14-21 days, wherein the culture medium contains corresponding antibiotics;

(3) and (4) freezing and storing (the target protein is subjected to fluorescent staining before freezing and FACS detection, and the positive rate is more than 98%).

In the invention, the detection method of the activity of the antibody-immune agonist coupled drug comprises the following steps:

setting up the experimental group and the control group: the experimental group is that the cell strain for detecting the activity of the antibody-immune agonist coupled drug is cultured in a culture medium containing the antibody-immune agonist coupled drug and a chromogenic substrate (a substrate of alkaline phosphatase), and the control group is that the cell strain for detecting the activity of the antibody-immune agonist coupled drug is cultured in an equal volume of the culture medium containing only the chromogenic substrate; and respectively detecting the light absorption values of the experimental group and the control group at 600-650 nm, and judging the activity of the antibody-immune agonist coupled drug according to the light absorption values of the experimental group and the control group.

For better comparison of the absorbance difference between the experimental group and the control group, the activity of the antibody-immunoactivator conjugate drug in the test range can be determined by calculating the multiple of the absorbance of the experimental group relative to the absorbance of the control group and the EC50 value.

Compared with the prior art, the cell strain and the detection method have the following advantages:

1. cells stably expressing the target gene can be kept stable for a long time, and the expression level of the cells is close to that of the cells, so that the stability among experimental batches can be ensured;

2. by simultaneously expressing TLR7/8 and a target antigen gene on a cell, the binding, internalization and release of an antibody and activation of a TLR7/8 downstream pathway can be realized on the same reporter cell;

3. usually, after the PBMC is co-cultured with the target antigen positive cells, one or more downstream cytokines are detected to indicate the activation level of the TLR 7/8; however, the downstream of a key transcription factor NF-kB or IRF7 in a TLR7/8 signaling pathway can transcribe a plurality of interferon or inflammatory cytokines, and a selected marker (the interferon or the inflammatory cytokine) is difficult to be universally used for detecting the activity of different antibody-immune agonist coupling medicaments; the way of reporter gene can better respond the activation condition of the channel;

4. the cell line detection result is comparable to the PBMC co-culture detection result, such as similar EC50, the strength of an agonist can be distinguished, and the like, but the cell line detection is more sensitive and has a larger detection dynamic range.

The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry, for example, the cell culture conditions, culture media and the like which are not noted are conventional conditions and conventional culture media. The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.

Example 1

Activity detection of TLR7/8 immune agonist in HEK-hTLR7 and HEK-hTLR8 cell lines

Reporter gene cell lines for TLR7 or TLR8 agonists for small molecule detection are commercially available or can be constructed according to the experimental methods mentioned above, and in this example, reporter gene cell lines for TLR7 or TLR8 agonists (HEK-hTLR 7 and HEK-hTLR 8) are purchased from the company InvivoGen.

1. And respectively taking HEK-hTLR7 and HEK-hTLR8 cells with the cell fusion degree of 70% -80% and good growth state, incubating for 2-3min by PBS, beating the cells, blowing the cells by a pipette, and staining and counting by trypan blue.

2. The cells were resuspended in HEK medium (HEK-Blue medium for detecting alkaline phosphatase activity, hb-det2 from Avova root (Invivogen)) to adjust the cell density to 4X 10⁵Cells/ml, plated at 100 μ L/well, and immune agonists were added to the experimental groups (see Table 1 for immune agonists used in this example). The highest detection concentration of the immune agonist in the experimental group is 10nM, the gradient dilution is carried out by 3 times, the detection concentration is 8 in total, and the immune agonist is diluted by a HEK culture medium. The control group was an equal volume of HEK medium without any added immune agonist.

3. Cells and an immune agonist are mixed uniformly and then placed in a cell culture box to be cultured for 24h, then the absorbance at 630nm is detected, and the multiple relation of the absorbance of an experimental group and a control group is calculated (the experimental result on the HEK-hTLR7 cells is shown in figure 2, the experimental result on the HEK-hTLR8 cells is shown in figure 3) and the EC50 value (the result is shown in figure 2).

As can be seen from figure 2, compounds other than compound 3 were detectable for activity on HEK-hTLR7, indicating that only compound 3 of these compounds was not a TLR7 activator and that compounds other than compound 3 were TLR7 activators. Furthermore, the activation potency of different compounds is different, for example compound 4 is less active on TLR7, while other TLR7 agonists are slightly more active but also differentiated, indicating that HEK-hTLR7 has a high degree of differentiation of the high and low activity of different activators.

As can be seen from fig. 3 and table 2, compound 1 and compound 3 detected higher activity on HEK-hTLR8, compound 6, compound 1 and compound 3 were TLR8 activators, and compound 2 also showed some activity.

Thus, the compound 1 is a TLR7/8 double agonist, the compound 5 is a TLR7 agonist, the compound 3 is a TLR8 agonist, the compound 2 is a TLR7 agonist and has certain TLR8 agonist activity, the compound 4 is a TLR7 agonist, and the compound 6 is a TLR7/8 double agonist.

It can also be seen from Table 2 that different agonists are activated to different degrees on HEK-hTLR7 and/or HEK-hTLR8 cells. Table 2 results show that as TLR7 agonist, the activity of compound 2 > the activity of compound 6 > the activity of compound 1 > the activity of compound 5, as TLR8 agonist, the activity of compound 3> the activity of compound 1 > the activity of compound 6.

As shown in FIG. 2, FIG. 3 and Table 2, different agonists activate Toll-like receptors in HEK-hTLR7 and/or HEK-hTLR8 cells, and are suitable for use in constructing the cell line models of the present invention.

Example 2

Construction of HEK-hTLR7-HER2-GFP and HEK-hTLR8-HER2-GFP stably transfected cell lines

1. HER2-GFP construction plasmid, lentivirus packaging concentration and titer detection are served by Jinzhi Biotechnology, Inc. The HER2-GFP lentiviral expression plasmid map is shown in FIG. 4.

2. Inoculating cells: respectively taking an HEK-hTLR7 cell strain and an HEK-hTLR8 cell strain with 70-80 percent of fusion degree and good growth state, digesting, counting and inoculating about 1.5 multiplied by 10⁶The individual cells were cultured in T25 flasks and placed in a cell incubator at 37 ℃ overnight.

3. Preparing a mixture of a DMEM complete culture medium and polybrene, wherein the final concentration of the polybrene is 10 mug/mL; before infection, the virus stock solution was removed from the refrigerator and placed on ice to melt the virus stock solution, the cell culture medium was aspirated, a mixture of DMEM complete medium and polybrene was added, and the virus stock solution was added to the cells, the moi (multiple of infection) =5 of the virus stock solution.

4. After 24 hours of infection, the GFP expression efficiency was observed by a fluorescence microscope. The virus-containing culture medium was aspirated, and fresh DMEM complete culture medium was added thereto to continue the culture at 37 ℃.

5. Observing the state of the cells, and carrying out amplification culture: t25 transferred to T75 flask.

6. Flow type separation: cell count, filtration on 70 μm filter, 1mL PBS resuspension (cell density less than 1X 10)⁷Cells/ml, the sector where the fluorescence intensity span among positive cells is small and the cells are most dense, is collected at approximately 50% -60% of the positive cell population.

7. Cell culture and FACS detection. If the negative cells are more than 2 percent, secondary sorting or pressure screening is needed.

8. Flow results show that the GFP positive rates of infected HEK-hTLR7 and HEK-hTLR8 cells, namely HEK-hTLR7-HER2-GFP and HEK-hTLR8-HER2-GFP, are respectively 93.7% and 92.3%, as shown in FIG. 5, two peaks are respectively shown, and puromycin screening is required in later culture.

9. After the puromycin is added for culture, the GFP detection result is shown in figure 6, the GFP positive rates of HEK-hTLR7-HER2-GFP and HEK-hTLR8-HER2-GFP are respectively 99.7% and 99.6%. After the puromycin screening, the fluorescence intensity of the positive cells is more concentrated, and the discrimination is better.

Example 3

Affinity assay of HEK-hTLR7-HER2-GFP and HEK-hTLR8-HER2-GFP stably transfected cell line and HER2 antibody

1. And respectively taking HEK-hTLR7-HER2-GFP, HEK-hTLR8-HER2-GFP, HEK-hTLR7, HEK-hTLR8 and HCC1954 cells with cell fusion degree of 70% -80% and good growth state, and carrying out pancreatin digestion and counting. HCC1954 cells served as positive control cells. HEK-hTLR7 and HEK-hTLR8 cells served as negative control cells.

2. At a rate of 1X 10 per hole⁵And (4) paving the cells, wherein the number of the cells is 50 muL/hole.

3. Trastuzumab (monoclonal antibody targeting HER2, also known as HER2 mAb) was added at a concentration setting of 50 μ g/mL (333.3 μ M), 30 μ g/mL (200 nM), 40nM, 8nM, 1.6nM, 0.32nM, 0.064nM, 50 μ L/well.

Incubating at 4.4 ℃ in the dark for 1h, and washing twice with PBS; APC-labeled anti-human IgG antibody was added, incubated at 4 ℃ for 30min, washed twice with PBS, and subjected to FACS detection.

The results are shown in fig. 7 and table 3: the HEK-hTLR7-HER2-GFP and HEK-hTLR8-HER2-GFP respectively have higher affinity to HER2 antibodies, which indicates that the HEK-hTLR7-HER2-GFP and the HEK-hTLR8-HER2-GFP can stably express the antigen HER 2.

Example 4

Activity test of different antibody-immune agonist conjugate drugs (AIAC for short) on HEK-hTLR7-HER2-GFP or HEK-hTLR8-HER2-GFP

1. Response of trastuzumab-compound 1 conjugate drug (labeled HER2 AIAC 1) on HEK-hTLR7 and HEK-hTLR7-HER2-GFP cell lines, wherein the conjugation method of trastuzumab to compound 1 is described in WO 2015165413.

(1) Respectively taking HEK-hTLR7 and HEK-hTLR7-HER2-GFP cells with cell fusion degree of about 70% -80% and good growth state, incubating the cells for 2-3min by PBS, beating the cells, blowing the cells by a pipette, and staining and counting by trypan blue.

(2) The HEK medium (Invivogen) is usedHEK-Blue medium of hb-det 2) resuspended cells, which medium contains a chromogenic substrate for alkaline phosphatase, adjusted to a cell density of 4X 10⁵Cells/ml, 100 μ L/well plated, experimental groups added HER2 AIAC1 or trastuzumab. The highest concentration detected was 30nM, 3-fold gradient dilution, for a total of 8 concentrations detected. HER2 AIAC1 was diluted with HEK medium. The control group was added an equal volume of HEK medium containing only the chromogenic substrate for alkaline phosphatase as the experimental group.

(3) The cells and HER2 AIAC1 or trastuzumab are mixed uniformly and then placed in a cell culture box to be cultured for 24h, light absorption at 630nm is detected, the light absorption value is in direct proportion to the activity of alkaline phosphatase, and the multiple relation with a control group is calculated.

The results are shown in FIG. 8, in which trastuzumab control has no activation in both HEK-hTLR7 and HEK-hTLR7-HER2-GFP reporter cells; HER2 AIAC1 coupled drug has no activation effect in a reporter cell strain without the expression of the target antigen HER2 (left figure 8), but activates the expression of NF-kB downstream gene in HER 2-expressed reporter cell HEK-hTLR7-HER2-GFP (right figure 8).

2. The method of the embodiment is adopted to detect the activity of different HER2 antibody-TLR 7/8 agonist conjugate drugs on HEK-hTLR7-HER2-GFP and HEK-hTLR8-HER2-GFP stable cell strains respectively, and the result is shown in figure 9.

As can be seen from the left panel of FIG. 9, HER2 AIAC5 and HER2 AIAC2 have activating effect on HEK-hTLR7-HER2-GFP cell line, while HER2 AIAC3 has no activating effect on HEK-hTLR7-HER2-GFP cell line because Compound 3 is only an agonist of TLR 8; compound 4 is not highly active on its own, so HER2 AIAC4 does not show activation of HEK-hTLR7-HER2-GFP cell line in the tested concentration range.

As can be seen from the right panel of FIG. 9, since Compound 3 is an agonist of TLR8, HER2 AIAC3 has strong activating effect on HEK-hTLR8-HER2-GFP cell strain; compound 5, Compound 4 and Compound 2 are agonists of TLR7, and therefore have no activating effect on HER2 AIAC5, HER2 AIAC4 and HER2 AIAC2 on HEK-hTLR8-HER2-GFP cell lines.

In conclusion, the different AIACs have consistency with the conclusion obtained by detecting the small molecule agonist alone on the cell line established in the application, so that the accuracy and the sensitivity of the cell strain model design are further verified.

The different AIACs were prepared as follows:

HER2 AIAC2 (trastuzumab-compound 2 conjugate drug): see patent CN110678183A example 1;

HER2 AIAC3 (trastuzumab-compound 3 conjugate drug): see connector (linker) -compound 2.14 in patent US10239862B 2;

HER2-AIAC4 (trastuzumab-compound 4 conjugate drug): see patent WO2020190725a1, example 17;

HER2 AIAC5 (trastuzumab-compound 5 conjugate drug): see patents WO 2015165413;

HER2 AIAC6 (trastuzumab-compound 6 conjugate drug): see patent WO2015165413 for a preparation method.

Comparative example 1

Detection of HER2 AIAC1 Activity in PBMC (from donor 1 or donor 2) in Co-culture with tumor cells

1. PBMC acquisition

(1) 40mL of fresh peripheral human whole blood was collected using an EDTA anticoagulant tube and stored on ice or at 4 ℃.

(2) And (4) reversing and mixing for several times, averagely transferring the whole blood into a 50mL centrifuge tube, adding PBS according to the volume ratio of 1:1 for dilution, and mixing uniformly.

(3) Taking SepMate^TM50mL of the lymphocyte separation medium was added, and diluted whole blood was slowly added to the cells adherent thereto.

(4) Centrifuging for 10min at 1200g, setting the acceleration of the centrifugation rotating speed to 9 and the deceleration to 0, and keeping the temperature at room temperature.

(5) After centrifugation, the liquid surface was divided into 4 layers, which were red blood cell and granulocyte layers, a separation liquid layer, PBMC, and plasma in this order. The supernatant above the 25mL level in the separator tube was discarded and the remaining liquid was quickly poured into a 50mL centrifuge tube.

(6) PBS wash 2 times, 500g centrifuge for 8 min.

(7) Trypan blue staining count, adjusting cell density to1.25×10⁶Cells/ml, 100 μ L/well were seeded in 96-well plates.

2. Co-culture of PBMC with HER2 high expressing cells

(1) Taking NCI-N87 cells with good growth state, digesting, staining trypan blue, counting, adjusting cell density to 2.5 × 10⁵Cells/ml were added at 100 μ L/well to 96-well plates containing PBMCs.

(2) HER2 AIAC1 or trastuzumab was added. The highest concentration detected was 10nM, 3-fold gradient dilution, for a total of 7 concentrations detected.

(3) Put at 37 ℃ with 5% CO₂After incubation in the incubator for 18h, centrifugation was carried out at 2000rpm for 3min, 200. mu.L of the supernatant was pipetted into a new 96-well plate and TNF-. alpha.secretion was detected using the Elisa method, the results are shown in FIG. 10.

As shown in FIG. 10, the secretion of TNF- α by HER2 AIAC1 was more than 2 times that of HER2 mAb (trastuzumab), and was related to PBMC of different origins, which released more TNF- α than donor 1 after HER2 AIAC1 treatment. It can be seen that PBMCs from different donors had a significant effect on the experimental results.

Comparative example 2

Detection of HER2 AIAC1 and HER2 AIAC6 Activity in PBMC (from donor 3) co-culture with tumor cells

The procedure was essentially the same as in comparative example 1, except that PBMC was replaced with PBMC from donor 3, and the results are shown in FIG. 11.

As shown in figure 11, the activity of the agonist in HER2 AIAC6 was lower than the agonist in HER2 AIAC 1. In the detection system, compared with the monoclonal antibody, the secretion amount of TNF-alpha after HER2 AIAC6 treatment is basically consistent with that of trastuzumab, and the activity of HER2 AIAC6 cannot be evaluated in a PBMC and tumor cell co-culture system.

Comparative example 3

Comparing the detection effect of AIAC activity of different targets in the PBMC and tumor cell co-culture system

The method is basically the same as comparative example 1, and is distinguished by increasing the co-culture system of PBMC and human T lymphoblast cell line (Jurkat cells, expressing CD 47), and replacing the drug to be tested with SIRP alpha-Fc-compound 1 conjugate drug targeting target CD47 (CD 47 AIAC1, see WO 2015165413), SIRP alpha-Fc-compound 2 conjugate drug (CD 47 AIAC2, see CN 110678183A) SIRP alpha-Fc with the amino acid sequence: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISAITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Seq ID number 1). The results are shown in FIG. 12.

As shown in fig. 12 a and B: under the same target, in a PBMC and tumor cell co-culture system, the same AIAC shows different activity responses on different cytokines by taking the secretion amount of the cytokines as an activity judgment basis in the same experiment. As shown in fig. 12C: the cytokines activated were also different for different targets using the same small molecule agonist conjugated AIACs. Therefore, the PBMC and tumor cell co-culture system may not accurately respond to the activation of the pathway if only a single or several fixed cytokine indexes are adopted, which is not favorable for large-scale general screening.

Example 5

Verification of Activity on HEK-hTLR7-HER2-GFP cell line

In order to solve the problems that the TNF-alpha secretion amount after HER2 AIAC6 (AIAC 6 for short) treatment is basically consistent with that of trastuzumab, and HER2 AIAC6 activity cannot be evaluated in a co-culture system of PBMC and tumor cells, the HEK-hTLR7-HER2-GFP cell line established by the application is used for evaluating the HER2 AIAC6 activity, and the specific experimental operation refers to example 4.

The results are shown in fig. 13 and table 4, the obvious activity of HER2 AIAC6 can be detected by using HEK-hTLR7-HER2-GFP reporter cells, and the activity is weaker than that of HER2 AIAC1, which indicates that the detection method of the invention can be used for activity test of coupling drugs of immune agonist with weaker activity, and has higher sensitivity and larger detection dynamic range compared with the method of co-culturing PBMCs and tumor cells.

Example 6

In vivo experimental verification

A mouse HER2 tumor model is established to detect the in vivo drug effects of HER2 AIAC1, HER2 AIAC4 and HER2 AIAC 6.

As shown in figure 14, the objective of this experiment was to evaluate the efficacy of HER2 AIAC4, HER2 AIAC6 and their mAb controls (HER 2 mAb) in vivo in a female severe combined immunodeficiency mouse model (SCID Beige mouse model) of human breast cancer HCC1954 (HER 2 positive) subcutaneous xenografts. Will be 5X 10⁶Individual HCC1954 tumor cells were placed in 0.2 mL of a mixture of PBS and Matrigel (Matrigel) (PBS: Matrigel = 1: 1) and subcutaneously inoculated on the right back of each mouse. The mean volume reached 140 mm 10 days after tumor inoculation³The grouped administration is started. All drugs were given at 5mg/kg dose with 6 dosing frequency for 5 days. The tumor volume of the tumor-bearing mice in the vehicle control group reaches 2,181 mm 28 days after the start of the administration³. The HER2 mAb had a tumor volume of 1,977 mm at a 5mg/kg dose compared to vehicle control³No drug effect; HER2 AIAC4 has a tumor volume of 1,697 mm at a dose of 5mg/kg³While HER2 AIAC6 showed significant tumor growth inhibition, all tumors were cleared (0 mm)³). In this experiment, the test substance HER2 AIAC4 had no antitumor activity (consistent with the conclusion that HER2 AIAC4 had no activating activity shown in fig. 9 of example 4), whereas HER2 AIAC6 had antitumor activity (consistent with the conclusion that HER2 AIAC6 had activating activity shown in fig. 13 of example 5), indicating that the results of the in vitro drug activity test performed on the cell lines of the present invention were consistent with the in vivo efficacy results. In fig. 14, IV indicates tail vein injection, Q5d × 6 indicates administration once every 5 days, for a total of 6, and n =6 indicates that each group contains 6 mice.

As shown in FIG. 15, the objective of this experiment was to evaluate the efficacy of HER2 AIAC1 and HER2 AIAC6 in human gastric carcinoma NCI-N87 (HER 2 positive) subcutaneous xenografts in a female SCID Beige mouse model. Will be 1 × 10⁷NCI-N87 tumor cell setThe right dorsal aspect of each mouse was inoculated subcutaneously in 0.2 mL of a mixture of PBS and matrigel (PBS: matrigel = 1: 1). The mean volume reached 175 mm 6 days after tumor inoculation³The grouped administration is started. The tumor volume of the tumor-bearing mice of the vehicle control group reaches 2,057 mm 49 days after the beginning of the administration³. Compared with the vehicle control group, the test substance HER2 AIAC1 shows obvious tumor growth inhibition effect by single administration under the dosage of 5mg/kg, and the tumor volume is 3 mm³. The test object HER2 AIAC6 also shows obvious tumor growth inhibition effect under the conditions of 5mg/kg dose and 4 times of administration frequency of once-administered-once-5 days, and the tumor volume is 1 mm³. In this experiment, HER2 AIAC6 showed good antitumor activity, similar to the single administration of HER2 AIAC1 at 4 doses. In fig. 15 IV represents tail vein injection, Q5d × 4 represents dosing once every 5 days, 4 times in total, and n =6 represents that each group contains 6 mice.

As shown in FIG. 16, the objective of this experiment was to evaluate the efficacy of HER2 AIAC1, HER2 AIAC6 and their monoclonal antibody controls (HER 2 mAb) in vivo in a female BALB/c nude mouse model of subcutaneous xenograft tumors in human gastric carcinoma ST-02-0103 PDX. The volume is about 30 mm³The gastric cancer tumor tissue was subcutaneously inoculated on the right back of each mouse. The 34-scale average volume after tumor inoculation reaches 137 mm³The grouped administration is started. The tumor volume of the tumor-bearing mice in the vehicle control group reaches 1,629 mm 35 days after the beginning of the administration³. HER2 mAb has a tumor volume of 1,779 mm at a 5mg/kg dose³No drug effect. Compared with the vehicle control group, the test object HER2 AIAC1 shows obvious tumor growth inhibition effect by single administration at 5mg/kg dose, and the tumor volume is 189 mm³. Meanwhile, the test object HER2 AIAC6 also shows a certain effect of inhibiting tumor growth under the conditions of 5mg/kg dose and 4 times of administration frequency of once administration for 5 days, and the tumor volume is 865 mm³. HER2 AIAC6 had some antitumor effect but the potency was weaker than HER2 AIAC1, and the in vivo potency results were similar to those of example 5. In fig. 16 IV represents tail vein injection, Q5d × 4 represents administration once every 5 days, 4 times in total, and n =5 represents that each group contains 5 mice.

In summary (fig. 14-16), HER2 AIAC4 has no anti-tumor activity, HER2 AIAC1 and HER2 AIAC6 have significant anti-tumor activity in vivo efficacy experiments, in vivo efficacy test results are consistent with in vitro activity test results performed by using the cell strain of the present invention, and in vitro test results of a PBMC and tumor cell co-culture system show that HER2 AIAC6 has no activity and is inconsistent with in vivo efficacy test results, so that in vitro drug activity test results performed by using the method of the present invention are closer to in vivo efficacy test results, which indicates that the reliability of the test method of the present invention is higher.

The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.

Sequence listing

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Claims

1. A cell line for detecting the activity of an antibody-immunoactivator conjugate drug, wherein said cell line expresses an antigen or a fragment thereof capable of binding to an antibody of said antibody-immunoactivator conjugate drug, a receptor capable of binding to an immunoactivator of said antibody-immunoactivator conjugate drug, and an immune signaling pathway reporter gene.

2. The cell line of claim 1, wherein the antigen is a target protein HER2, CD47, Trop2, CEACAM5, CD22, sirpa, PD-L2 or PD-L1, and the antibody is an anti-HER 2 antibody, an anti-CD 47 antibody, an anti-Trop 2 antibody, an anti-CEACAM 5 antibody, an anti-CD 22 antibody, an anti-PD-L2 antibody or an anti-PD-L1 antibody.

3. The cell line for detecting the activity of an antibody-immunoactivator conjugate drug as claimed in claim 1, wherein said immunoactivator is a Toll-like receptor agonist or an interferon gene-stimulating protein agonist, and said immune signaling pathway reporter is an NF- κ B signaling pathway reporter or an IRF signaling pathway reporter.

4. The cell line for detecting the activity of an antibody-immunoactivator conjugate according to claim 3, wherein said Toll-like receptor is TLR7 and/or TLR8, and said immunoactivator is a peptide of the same type as that of said antibody-immunoactivator conjugate

、

、

、

、

Or

。

5. The cell line for detecting the activity of an antibody-immunoactivator conjugate drug as claimed in claim 1, wherein the cell line is HEK293 cells, and/or the reporter gene sequence in the immune signaling pathway reporter gene is a luciferase gene sequence or an alkaline phosphatase gene sequence.

6. The cell line of claim 1, wherein the antigen or fragment thereof is expressed from a lentivirus infected cell line, and the lentivirus expression plasmid comprises a CMV promoter, a gene coding region sequence of the antigen or fragment thereof located downstream of the CMV promoter, a first selection gene for selecting positive cell lines, and a nucleic acid sequence for linking the gene coding region sequence of the antigen or fragment thereof to a 2A linker peptide of the first selection gene;

and/or the presence of a gas in the gas,

the receptor of the immune stimulant is obtained by expressing after the receptor lentivirus of the immune stimulant infects cell strains, the receptor lentivirus expression plasmid of the immune stimulant comprises a CMV promoter, a gene coding region sequence of the receptor of the immune stimulant positioned at the downstream of the CMV promoter, a second screening gene for screening positive cell strains, a gene coding region sequence connecting the receptor of the immune stimulant and a nucleic acid sequence of a coding 2A connecting peptide of the second screening gene,

and/or the presence of a gas in the gas,

the immune signal channel reporter gene is infected by NF-kB signal channel reporter gene lentivirus or IRF signal channel reporter gene lentivirus to enter a cell strain,

the NF-kB signal path reporter gene lentivirus expression plasmid comprises a CMV promoter, a third screening gene positioned at the downstream of the CMV promoter, an NF-kB reaction element, an IFN-beta promoter and a reporter gene sequence,

the IRF signal path reporter gene lentiviral expression plasmid comprises a CMV promoter, a third screening gene positioned at the downstream of the CMV promoter, an ISRE reaction element, a TATA promoter and a reporter gene sequence,

the first screening gene, the second screening gene and the third screening gene are different from each other so as to carry out positive screening on cell strains infected by each lentivirus.

7. The cell line for detecting the activity of an antibody-immunoactivator conjugate according to claim 6, wherein said first selection gene, said second selection gene and said third selection gene are independently a fluorescent protein gene or a resistance gene.

8. A method for constructing a cell line for detecting the activity of an antibody-immunoactivator conjugate according to any one of claims 1 to 7, wherein said method comprises:

firstly, constructing an antigen or a segment lentivirus expression plasmid containing the gene coding region sequence of the antigen or the segment thereof;

and thirdly, screening positive cell strains by adopting a fluorescent protein label screening and/or resistance screening mode to obtain the cell strains for detecting the activity of the antibody-immune agonist coupled drug.

9. The construction method according to claim 8, characterized in that the construction method specifically comprises the steps of:

1) embedding the gene coding region sequence of the antigen or the fragment thereof at the downstream of the CMV promoter, and connecting the nucleic acid sequence of the 2A connecting peptide with the first screening gene to construct an antigen or a fragment thereof lentiviral expression plasmid;

4) and (3) adopting a fluorescent protein label screening mode or using an antibiotic corresponding to the first screening gene to carry out resistance screening on the positive cell strain, thus obtaining the cell strain for detecting the activity of the antibody-immune agonist coupled drug.

10. The method of claim 9, wherein the reporter gene cell line is constructed by:

(1) embedding the gene coding region sequence of the immune agonist receptor at the downstream of the CMV promoter, and connecting the nucleic acid sequence of the 2A connecting peptide with a second screening gene to construct an immune agonist receptor lentivirus expression plasmid;

(3) respectively adopting the lentivirus of the step (2) and an immune signal path reporter gene lentivirus to infect cells;

(4) screening positive cell strains by adopting a fluorescent protein label screening or resistance screening mode to obtain a reporter gene cell strain which can simultaneously express a receptor capable of combining with the immune stimulant in the antibody-immune stimulant coupled drug and an immune signal channel reporter gene,

wherein, the first screening gene and the second screening gene are different so as to carry out positive screening on each cell strain after the lentivirus infection.

11. The method of claim 10, wherein the immune signaling pathway reporter lentivirus is obtained by packaging an immune signaling pathway reporter lentivirus expression plasmid with lentivirus,

the lentiviral expression plasmid of the immune signal pathway reporter gene is a NF-kB signal pathway reporter gene lentiviral expression plasmid or an IRF signal pathway reporter gene lentiviral expression plasmid.

12. The method of claim 11, wherein a third selection gene, an NF- κ B response element, an IFN- β promoter and a reporter sequence are sequentially inserted downstream of the CMV promoter to construct said NF- κ B signal pathway reporter lentiviral expression plasmid;

or the like, or, alternatively,

embedding a third screening gene, an ISRE reaction element, a TATA promoter and a reporter gene sequence in sequence at the downstream of the CMV promoter to construct the IRF signal pathway reporter gene lentiviral expression plasmid,

wherein, the first screening gene, the second screening gene and the third screening gene are different from each other in pairs so as to carry out positive screening on cell strains infected by each lentivirus,

the reporter gene sequence is a luciferase gene sequence or an alkaline phosphatase gene sequence.

13. A method for detecting the activity of antibody-immune agonist coupled drug is characterized in that,

setting up the experimental group and the control group: the experimental group is that the cell strain for detecting the activity of the antibody-immune agonist coupled drug of any one of claims 1 to 7 is cultured in a culture medium containing the antibody-immune agonist coupled drug and a chromogenic substrate corresponding to the immune signal path reporter gene, and the control group is that the cell strain for detecting the activity of the antibody-immune agonist coupled drug of any one of claims 1 to 7 is cultured in a culture medium containing only the chromogenic substrate corresponding to the immune signal path reporter gene;

14. The method for detecting the activity of the antibody-immunoactivator conjugate drug according to claim 13, wherein 3 to 10 antibody-immunoactivator conjugate drug detection concentrations are set, and the fold of the absorbance of the experimental group relative to the absorbance of the control group and the EC50 value are calculated to determine the activity of the antibody-immunoactivator conjugate drug in the test range.

15. The method for detecting the activity of an antibody-immunoactivator conjugate according to claim 13, wherein said method comprises the steps of:

b) adjusting the cell density to 1X 10 using a medium containing a chromogenic substrate corresponding to said immune signaling pathway reporter gene⁵~6×10⁵Cells/ml are plated in 50-150 muL/hole, the antibody-immune agonist coupling drug with the concentration of 0.01 nM-30 nM is added into an experimental group, and a culture medium which is the same in volume as the experimental group and only contains a chromogenic substrate corresponding to the immune signal pathway reporter gene is added into a control group;

c) and respectively culturing the experimental group and the control group in an incubator for 20-30 h, detecting the light absorption value at 600-650 nm, calculating the multiple of the light absorption value of the experimental group relative to the light absorption value of the control group and the EC50 value, and determining the activity of the antibody-immune agonist coupled drug in the test range.

16. A kit for detecting the activity of an antibody-immune agonist coupled drug, which is characterized by comprising the cell strain for detecting the activity of the antibody-immune agonist coupled drug, according to any one of claims 1 to 7.