Rat anti-mouse CD137 antibody or functional fragment thereof, tool antibody and application thereofTechnical Field
The invention relates to the technical field of biology, in particular to a rat anti-mouse CD137 antibody or a functional fragment thereof, a tool antibody and application thereof.
Background
Immune checkpoint therapy represented by PD-1 antibodies truly opens a new era of tumor immunotherapy, and some domestic PD-1 antibody drugs are already in medical insurance at present, so that more patients benefit. However, the PD-1 antibody has generally low effective rate, average 12%, and less than 20% in non-small cell lung cancer, and faces the actual problem of improving the curative effect. Scientists find that novel targets for immunomodulation other than PD-1 are important directions for deepening immunotherapy research. Besides negative immune checkpoint molecules such as PD-1, CTLA-4 and LAG3, other types of activated immune checkpoint molecules such as CD40, OX40, CD137 (4-1 BB) targets and the like are also used for T cell tumor regulation therapy research, are important research directions of patient groups benefiting from the expansion of tumor immunotherapy efficacy, and are of great interest internationally.
CD137 is one of the most interesting molecules, CD137 is one of TNFRSF members, a family involved in immune cell activation, expressed antigen-induced, and although CD137 is widely expressed, it is mainly expressed in T cells, biased toward cd8+ T cells and NK cells, which are the core cells of tumor immunity. The preclinical model has demonstrated that CD137 signaling modulates antitumor activity is mediated primarily by cd8+ T, NK can compensate for cd8+ T dysfunction, and thus both populations of cells eradicate mouse tumors together by functional complementation. In addition, CD137 signaling modified CAR-T cells are more potent and have been admitted to the U.S. food and drug administration (Food and Drug Administration, FDA) and drug administration for the treatment of B-cell leukemia and lymphoma. Accumulated studies indicate that CD137 signaling prolongs cd8+ T survival and formation of memory T cells.
Early foreign two CD137 antibodies were tested clinically Urelumab (BMS-663513,by Bristol-Myers Squibb) showing a clear in vivo immune activation effect, whereas excessive immune stimulation was observed, showing nonspecific immune activation, especially on liver and hematopoietic tissues, resulting in a degree of liver injury characterization, low dose application possibly preventing clinical antitumor efficacy, and Utomilumab (PF-05082566,by Pfizer) with well systemic toxicity controlled antibodies showing a weaker in vivo antitumor activity. Both of these early developed antibodies, despite finding certain drawbacks, have remained in clinical trials with strategies that reduce the amount and enhance the efficacy of the combined PD-1/PD-L1 antibodies. At present, 16 CD137 antibodies are globally researched, in recent years, china starts to stand on research and development of a safer and more effective new generation CD137 antibody, and 3 enterprises in China participate in research and development of a new CD137 target drug, namely anti-CD 137 monoclonal antibody, wherein 1 is in a phase I clinical stage, 2 is in a preclinical research stage, and still is in an early clinical stage.
Because of the limitation of human experiments, preclinical experiments, particularly preclinical in vivo experiments in mice, are important platforms for developing new generation of anti-tumor antibodies with low side effects and high efficiency. At present, the international rat CD137 antibodies are limited to a group of antibodies produced in the early international stage, no production report of the anti-mouse CD137 of the rat exists in China, and the whole anti-mouse CD137 antibodies have few resources and limit development and research. Different CD137 antibodies have different characteristics, including binding epitopes, affinity, class, which in turn determine their different immunomodulatory activities. In order to further advance the development of CD137 antibody medicines and exert stronger anti-tumor effect, simultaneously limit the related adverse reaction of the CD137 antibody, the preparation of the rat anti-mouse CD37 antibody with diversity and different characteristics is urgently needed, screening development and test evaluation basis are provided for the medicine experiments carried out in mice, and anti-tumor activity research is deepened, so that the research on the disease occurrence mechanism and the potential mechanism can be deeply conducted, the research and development of the CD137 antibody medicines and the clinical test progress can be really promoted, the time cost of preclinical test is reduced, the clinical experiment risk is also reduced, and the research and development of the new generation anti-human CD137 antibody are accelerated.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a rat anti-mouse CD137 antibody or a functional fragment thereof, a tool antibody and application thereof, thereby providing screening development and test evaluation basis for a drug experiment carried out in a mouse body, deepening anti-tumor activity research, being capable of deeply discussing a disease occurrence mechanism and a potential mechanism, being capable of really promoting research and development of CD137 antibody drugs and clinical test progress, reducing time cost of preclinical test, reducing clinical test risk and accelerating research and development of a new generation of anti-human CD137 antibody.
Noun definition
The term "binding protein" refers broadly to all proteins/protein fragments comprising CDR regions, in particular antibodies or antibody functional fragments. "antibody functional fragments" include antigen compound binding fragments of these antibodies, including Fab, fab ', F (ab') 2, fd, fv, scFv, bispecific antibodies, and antibody minimum recognition units, as well as single chain derivatives of these antibodies and fragments. The type of antibody may be selected from IgG1, igG2, igG3, igG4, igA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".
The term "antibody" is used herein in its broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments so long as they exhibit the desired biological activity, such as specifically binding to an antigen or fragment thereof. An "antibody fragment" includes a portion of a full length antibody, preferably an antigen binding or variable region thereof. Examples of antibody fragments include Fab, fab ', F (ab') 2, fd, fv, complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.
Typically, the variable regions VH/VL of the heavy and light chains of an antibody are obtained by ligating the CDRs numbered from the FR in a combination arrangement of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
The invention is realized in the following way:
In a first aspect, the invention provides a rat anti-mouse CD137 antibody or functional fragment thereof, the antibody or functional fragment thereof comprising the complementarity determining regions:
CDR-VH1:SDGVH;
CDR-VH2:IIYYDGDTDYDSAIKS;
CDR-VH3:IDFRY;
CDR-VL1:RASSSLSYMY;
CDR-VL2:ETSKLSS;
CDR-VL3:QQWNSTPLT。
The rat anti-mouse antibody has the advantages of difficult availability and high technical requirements. The inventors screened to identify rat anti-mouse CD137 antibodies or functional fragments thereof with high affinity and strong specificity for CD137 molecules. The binding epitope of the antibody and the mouse CD137 molecule is clear. Can be used for developing CD137 antibody drug research, thereby exerting stronger anti-tumor effect and simultaneously limiting the adverse reaction related to the CD137 antibody.
The identification shows that the antibody has a certain blocking capacity to CD137 molecule natural ligand, and has definite synergistic T cell activating capacity and antitumor activity, especially in mouse tumor. This information provides a novel tool for studying the role of specific CD137 antibodies in mouse tumor models, as well as the role and novel mechanisms of combination therapy of CD137 antibodies.
In a preferred embodiment of the present invention, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, which are shown in sequence SEQ ID NO. 1-4, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, which are shown in sequence SEQ ID NO. 5-8.
The sequences of SEQ ID NOS 1-8 are shown in the following table:
In a preferred embodiment of the invention, the antibody further comprises a constant region.
In an alternative embodiment, the constant region is selected from the group consisting of the constant region of any one of IgM, igD, igG, igA and IgE.
In an alternative embodiment, the constant region is derived from a mouse, the sequence of the light chain constant region of the constant region is shown in SEQ ID NO.9, and the sequence of the heavy chain constant region of the constant region is shown in SEQ ID NO. 10;
In an alternative embodiment, the functional fragment is selected from any one of the group consisting of F (ab ') 2, fab', fab, fv, bispecific antibody and scFv of an antibody.
The functional fragment of the above antibody generally has the same binding specificity as the antibody from which it is derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the antibodies described above may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In a second aspect, the invention also provides the use of a rat anti-mouse CD137 antibody or a functional fragment thereof in any one of the following:
(a) Preparing a product for detecting the CD137 level of the mice;
(b) Preparing an activation product for activating immune cells;
(c) Screening a mouse transplanted tumor curative effect model;
(d) Screening a mouse primary tumor curative effect model;
(e) Screening CD137 antibody medicines and combined therapeutic medicines;
(f) Preparing a CD137 ligand blocker.
Experiments prove that the rat anti-mouse CD137 antibody provided by the invention has good T cell activating effect and shows good in-vivo tumor inhibiting effect, so that the rat anti-mouse CD137 antibody provided by the invention has important significance and application potential for developing and researching CD137 monoclonal antibodies and double-targeting antibodies, and provides a brand-new tool for researching the action mechanism of a specific CD137 antibody in a mouse tumor curative effect model and the combined treatment action and new mechanism of the CD137 antibody.
The curative effect model is any tumor model of a mouse which is sensitive or insensitive to the CD137 antibody treatment, and comprises a naturally occurring tumor model, an induced tumor model and a transplanted tumor model.
The immune cells are immune cells expressing CD137, and are selected from lymphocyte, dendritic cell, macrophage, granulocyte, mast cell or natural killer cell.
The lymphocyte is T lymphocyte or B lymphocyte.
Such products include, but are not limited to, reagents, kits, chips or microwells. For example, an enzyme-labeled rat anti-mouse secondary antibody complex for detecting mouse CD137 is prepared.
In an alternative embodiment, the tumor is a mouse melanoma, a mouse breast cancer, a mouse lung cancer, a mouse colon cancer, a mouse mastadenoma, a mouse kidney cancer, a mouse lymphoma, a mouse lymphoid tumor, a mouse mast cell tumor, a mouse liver cancer, a mouse pituitary tumor, a mouse myeloma, a mouse brain neuroma, a mouse testicular stromal cell tumor, a mouse stomach cancer, an RM-1-mouse prostate cancer cell, or a mouse pancreatic cancer.
For example, the tumor is CT26 Colon cancer, MC 38-mouse intestinal cancer, B16-mouse melanoma, LLC-mouse lung cancer, CMT-93 mouse Colon cancer, colon 26-mouse Colon cancer, RENCA-mouse kidney cancer, C127-mouse mastadenoma, 4T 1-mouse breast cancer, NF 639-mouse breast cancer, YAC-1-mouse lymphoma, P388D 1-mouse lymphoid tumor, P815-mouse mast cell tumor, bpRc 1-mouse liver cancer, GT 1-1-mouse pituitary tumor, H22-mouse liver cancer, P3/ag-mouse myeloma, neuro-2 a-mouse brain neuroma, MLTC-1-mouse testicular interstitial cell tumor, MFC-mouse stomach cancer, RM-1-mouse prostate cancer cells, atT-20-mouse tumor, or LTPA-mouse pancreatic cancer.
Generally, the model of the therapeutic effect of the tumor in the mouse-transplanted tumor in (c) is an animal model in which a mouse immortalized tumor cell line or a mouse primary cancer tissue or cell is transplanted into a mouse and grown into a tumor. For example, the mouse breast cancer transplantation tumor model is an animal model in which a mouse immortalized breast tumor cell line or a mouse primary breast cancer tissue or cell is transplanted to a mouse to grow into a tumor, and the mouse breast cancer transplantation tumor model is selected from a luminal A breast cancer, a luminal B breast cancer, a HER-2 over-expression breast cancer or a basal-like breast cancer.
The mouse transplanted tumor curative effect model is suitable for drug screening, drug effect evaluation or clinical prediction. In particular for the study of immunomodulatory therapeutic drugs including the CD137 antibodies of the invention (or in combination with the present antibodies and other drugs or therapies).
The mouse primary tumor curative effect model of the application (d) can be selected from a mouse breast cancer primary tumor model, wherein the mouse breast cancer primary tumor model is selected from an induced breast cancer model, an induced breast cancer model or a genetically engineered mouse breast cancer model, and the induced breast cancer model is applied to experimental animals through gastric lavage, local smearing or intravenous injection and other ways by adopting an inducer such as methyl nitrosourea (N-methyl-N-nitrosourea, MNU) or DMBA and the like through a chemical induction way.
The genetically engineered mouse breast cancer model is selected from a transgenic model or a gene knockout model. The transgenic model adopts specific promoters such as mammary gland, colorectal and the like to express oncogenes in a targeted manner. Gene knockout models, for example, knockout of the tumor suppressor gene in mice, creates tumor models, for example, knockout of the tumor suppressor gene p53, mimicking naturally occurring tumor models.
The mouse primary tumor efficacy model can be used for researching the internal mechanism of tumor generation or metastasis.
In application (e), the combination therapeutic agent for mice comprises a rat anti-mouse CD137 antibody and at least one agent selected from the group consisting of a chemotherapeutic agent, a small molecule targeting agent, an anti-tumor immunomodulating agent, an oncolytic virus and a physiotherapy.
The chemotherapeutic agent is selected from platinum, docetaxel, paclitaxel (paclitaxel), vinorelbine, vinca alkaloids, 5-fluorouracil related agents, hilder, gemcitabine, anthracyclines or irinotecan.
The small molecule targeting drug is selected from at least one of an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), an anaplastic lymphoma kinase tyrosine kinase inhibitor (ALK-TKI), an echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene (EML 4-ALK) inhibitor, a KIF5B-RET fusion gene inhibitor, a BCL-2 inhibitor, an HDAC inhibitor, a Vascular Endothelial Growth Factor (VEGF) receptor Tyrosine Kinase Inhibitor (TKI), an Osteopontin (OPN) inhibitor, a Hedgehog (Hh) signaling pathway inhibitor, a WNT/beta-catenin signaling pathway inhibitor, a PI3K/AKT/mTOR signaling pathway inhibitor, a Ras/Raf/MAF signaling pathway inhibitor, a Notch signaling pathway inhibitor, and a transforming growth factor beta (TGF-beta) inhibitor.
The anti-tumor immunomodulatory drug is selected from at least one of anti-LAG 3 antibody, anti-Tim-3 antibody, tigit antibody, anti-CD 20 antibody, anti-CD 25 antibody, anti-OX 40 antibody, anti-CTLA-4 antibody, anti-PD-1 antibody, anti-BTLA antibody, anti-GITR antibody, anti-PD-L1 antibody, LAG3 inhibitor, CTLA-4 inhibitor, tim3 inhibitor, BTLA inhibitor, TIGIT inhibitor, CD38 inhibitor, CD47 inhibitor, IDO inhibitor, ang2 inhibitor, EGFR inhibitor, VISTA inhibitor, CSF1R inhibitor, CCR2 inhibitor, CXCR4 inhibitor, CXCR2 inhibitor, CCR4 inhibitor, CXCL12 inhibitor, IL-6R inhibitor, and IL-10 inhibitor.
The above platinum group includes, but is not limited to, antitumor platinum complexes, such as those selected from nedaplatin, carboplatin, cisplatin or oxaliplatin.
The above physiotherapy is selected from radiotherapy, thermotherapy, electrotherapy, microwave, ultrasound, radio frequency, acupuncture or cryotherapy.
The oncolytic virus is selected from the group consisting of an alphavirus, measles virus, vesicular stomatitis virus, human enterocytopathic orphan virus, adenovirus, herpes simplex virus, vaccinia virus, reovirus, and coxsackievirus, wherein the alphavirus is at least one selected from the group consisting of M1 virus and katavirus.
In application (f), the antibody provided by the invention has partial blocking capability on CD137 ligand at high concentration, so that the antibody can be used for preparing corresponding blocking agents.
In addition, the inventor finds that the antibody provided by the invention can cooperate with the anti-mouse CD3 antibody to activate the mouse T lymphocyte, has stronger synergistic activation capability than other anti-CD 137 antibodies, and has strongest proliferation of CD8+ T cell under the action of the anti-CD 137 antibody and the anti-CD 3 antibody.
In a third aspect, the invention also provides a tool antibody comprising the above-described rat anti-mouse CD137 antibody or a functional fragment thereof, which may be further engineered, such as potentially producing single chain antibodies and diabodies of various structures. The tool antibody or the further engineering fragment thereof can be used for the research of a mouse immune curative effect model, including, but not limited to, screening of mouse immune curative effects, screening development and test evaluation basis provided by a drug experiment carried out in a mouse, research of anti-tumor activity and mechanism, and the like.
In a fourth aspect, the invention also provides a reagent or kit for mouse CD137 immune detection, which contains the rat anti-mouse CD137 antibody or a functional fragment thereof, or contains the tool antibody.
The rat anti-mouse CD137 antibody provided by the invention has high binding affinity and specificity to CD137 molecules, and can be used for detecting mouse CD137 molecules.
The reagent or kit further comprises a label labeled with a detectable label.
A detectable label refers to a substance of a type having properties such as luminescence, color development, radioactivity, etc., that can be directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the detectable label includes, but is not limited to, fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In a fifth aspect, the invention also provides a nucleic acid encoding a rat anti-mouse CD137 antibody or a functional fragment thereof, comprising a nucleic acid encoding a heavy chain as shown in SEQ ID NO.11 and a nucleic acid encoding a light chain as shown in SEQ ID NO. 12.
In a sixth aspect, the invention also provides a vector comprising a nucleic acid encoding the above-described rat anti-mouse CD137 antibody or a functional fragment thereof.
In a seventh aspect, the invention also provides a host cell comprising the vector described above.
The invention has the following beneficial effects:
The invention provides a rat anti-mouse CD137 antibody, which has high specificity and high affinity. And the binding epitope of the antibody and the mouse CD137 molecule is clear. The identification proves that the antibody provided by the invention has blocking capability on a natural ligand and receptor at high concentration, has definite synergistic T cell activating capability, has good T cell activating effect and shows good in-vivo tumor inhibiting effect in mice. The invention not only has important significance and application potential for developing and researching CD137 monoclonal antibodies and double-targeting antibodies, but also provides a brand-new tool for researching the action of specific CD137 antibodies in a mouse tumor model and the action and new mechanism of the CD137 antibodies in combined treatment.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the purity identification result of DB-68 antibody, wherein the DB-68 antibody shown in FIG. 1 is subjected to SDS-PAGE electrophoresis after affinity chromatography purification, the left lane is a protein marker, the right lane is DB-68, and the visible antibody lane shows two bands, namely, one band is about 55KD, the other band is about 25KD, and the purity of the antibody is more than 90%;
FIG. 2 is a graph showing the result of the identification of the specificity of the DB-68 antibody, wherein the DB-68 antibody only specifically binds to the mouse CD137 molecule, does not have cross reaction with the human CD137 molecule, and has high specificity without other binding with the mouse CD137 molecule having the same label;
FIG. 3 is a graph of binding and dissociation of the Fortebio Octet assay DB-68 antibody with mouse CD137, with different lines representing different concentrations of mCD137-his, 250nM,125nM,62.5nM,31.3nM,15.6nM,7.81nM,3.9nM, respectively, from top to bottom;
FIG. 4 is a schematic diagram of a fragment of mCD137 protein, a fusion protein of 1-85 amino acids of the extracellular region of a mouse CD137 molecule and human Fc, a fusion protein of 1-117 amino acids of the extracellular region of a mouse CD137 molecule and human Fc, a fusion protein of mE4 (mE 4-hFc), a fusion protein of 118-185 amino acids of the extracellular region of a mouse CD137 molecule and human Fc, and a fusion protein of mE400 (mE 400-mFc);
FIG. 5 is a graph showing the results of identifying the expression of mCD137 protein fragment, and determining the expressed protein as the target protein by comparing the protein fragment with a protein marker and identifying the target protein in the fusion protein by a specific antibody;
FIG. 6 is a graph showing the analysis results of the region where DB-68 antibody binds mCD137, DB-68 does not bind mE3 (mE 3-hFc), i.e., does not bind to the 1-85 amino acid region of the extracellular region of mouse CD137, DB-68 antibody binds mE4 (mE 4-hFc), i.e., binds to the 1-117 amino acid region of the extracellular region of mouse CD137, DB-68 antibody does not bind mE400 (mE 400-mFc), i.e., does not bind to the 118-185 amino acid region of the extracellular region of mouse CD137, so the region where DB-68 antibody binds mouse CD137 is limited to only 86-117 amino acid regions, i.e., the III CDR region;
FIG. 7 is a graph showing the results of blocking mouse CD137 molecules with DB-68 antibodies by binding to their ligands, and showing the results of binding to mouse CD137 ligand molecules after 1. Mu.g/ml of mCD137-hF, 5. Mu.g/ml and 25. Mu.g/ml of DB-68 antibodies were mixed overnight with 1. Mu.g/ml of mCD137-hFc, respectively;
FIG. 8 is a graph showing experimental results of the synergistic activation of CD4+ T lymphocytes of mice by the DB-68 antibody, wherein the left graph shows the addition of only αCD3e (2 ng/ml) to the culture medium, the middle graph shows the addition of αCD3e (2 ng/ml) +αCD137-1 (control antibody, 2 ng/ml), and the right graph shows the addition of αCD3e (2 ng/ml) +DB-68 (2 ng/ml);
FIG. 9 is a graph showing experimental results of the synergistic activation of CD8+ T lymphocytes in mice by DB-68 antibody, wherein the left graph shows the addition of only αCD3e (2 ng/ml) to the culture medium, the middle graph shows the addition of αCD3e (2 ng/ml) +αCD137-1 (control antibody, 2 ng/ml), and the right graph shows the addition of αCD3e (2 ng/ml) +DB-68 (2 ng/ml);
FIG. 10 is a graph showing the results of anti-tumor activity of DB-68 antibody in a mouse intestinal cancer model. CT26 (mouse intestinal cancer cell line, purchased from ATCC) establishes transplanted tumor under the skin of BALB/c mouse according to 5X 105/mouse, when the tumor is 6-7mm, the treatment group starts to inject DB-68 antibody into the tumor, 5 mug/mouse is administrated every other day, three times are administrated continuously, and the control group is internally injected with PBS liquid with the same volume. The growth rate of tumors in the DB-68 antibody-treated group was significantly different from that in the control group. NS, P >0.05; P <0.05, P <0.0001. The DB-68 antibody was shown to inhibit tumor growth, although not completely eliminating mouse subcutaneous transplants.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are well explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: ALaboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I. Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.34.J.37, J.J.37, J.F.37) and PCR methods (J.37, J.F.37, J.J.F.37, J.J.J.J.F.37, J.J.J.J.J.J.J.J.F.J.J.J.J.J.F.J.J.J.L).
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a preparation method of a rat anti-mouse CD137 monoclonal antibody, which specifically comprises the following steps in sequence:
1. immunization of animals.
The immunogen is fusion protein mCD137-hFc, namely mouse CD137 molecule and human Fc tag (purchased from Beijing Yiqiao Shenzhou science and technology Co., ltd.). The 6-8 week old inbred Lewis rats were selected. On day 0, primary immunization was performed, mCD137-hFc fusion protein and complete fodder adjuvant (purchased from sigma) were prepared into a water-in-oil mixture by high frequency shaking, 100 μg protein was immunized per rat, the volume was 0.3mL, and 2 rats were immunized by intraperitoneal and subcutaneous injections in total. On day 14, a water-in-oil mixture of a second-day, mCD137-hFc fusion protein and Freund's incomplete adjuvant (purchased from sigma) was immunized with 100. Mu.g protein per rat in a volume of 0.3mL by intraperitoneal and subcutaneous injection. On days 28 and 42, three and four-way, mCD137-hFc fusion protein and incomplete foster adjuvant (sigma) water-in-oil mixture were performed, 100 μg protein per rat, 0.3mL volume, and immunized by intraperitoneal and subcutaneous injection, respectively.
On day 45, tail vein was collected, rat serum was collected by centrifugation, and serum titers were measured by ELISA. The titer of the immunized rats reaches or exceeds 1:10000, 1 is selected on day 56, final immunization is carried out, 100 mug of antigen is dissolved in 0.3mL of1 XPBS, and the cells are fused after 4 days after intraperitoneal injection impact immunization.
2. Cell fusion.
(1) Preparation of immune spleen cells after 3 days of last booster immunization, the rat is sacrificed by cervical guide, spleen is aseptically taken and the culture solution is washed once. Grinding spleen, sieving with 400 mesh stainless steel screen to obtain single cell suspension, washing cells with serum-free DMEM culture solution for 2 times, counting, and collecting 108 spleen lymphocyte suspensions for use.
(2) The cells of mouse myeloma cells P3-X63-Ag8.653 (purchased from ATCC) in logarithmic growth phase were centrifuged, washed 2 times with serum-free DMEM medium, counted and 1X 107 cells were taken for use.
(3) Fusion:
① Myeloma cells and spleen cells were mixed together at a ratio of 1:10 or 1:5, washed 1 time with serum-free DMEM in a 50mL centrifuge tube, centrifuged at 1200rpm/min for 8min, the supernatant discarded, and the residual liquid was pipetted to avoid affecting polyethylene glycol (PEG, available from sigma) concentration. The bottom of the centrifugal tube is lightly flicked to loosen the cell sediment slightly.
② 1ML of 45% PEG (molecular weight 4000) solution pre-warmed at 37℃was added over 90s with gentle shaking. The reaction was carried out in a 37℃water bath for 90s.
③ Serum-free DMEM broth pre-warmed at 37 ℃ was added to terminate PEG action, and 1mL, 2mL, 3mL, 4mL, 5mL and 6mL were added every 2min, respectively.
④ Centrifuge at 800rpm/min for 6min.
⑤ The supernatant was removed and resuspended in 20% fetal bovine serum DMEM medium containing HAT (available from sigma).
⑥ The cells were added to an existing feeder cell layer 96-well plate with 100. Mu.L of each well, and an immunophilium was inoculated into 20 96-well plates.
⑦ The plates were incubated in a 37℃5% CO2 incubator.
3. Hybridoma primary screening and secreted antibody detection.
(1) HAT screening, in which spleen cells and myeloma cells are treated by PEG to form a mixture of various cells, only the fusion body of the spleen cells and the myeloma cells can survive in a HAT-containing selective culture solution, other fusion bodies or non-fusion cells can not survive, and half-quantity liquid exchange is carried out after 10 days of maintenance culture.
(2) Antibody screening, namely, detecting whether the antibody secreted by hybridoma supernatant can bind to target protein by adopting an enzyme-linked immunosorbent assay (ELISA) method, wherein 1 mug/mL of mCD137-his (namely, fusion protein of mouse CD137 molecule and his label, purchased from Beijing Yiqiao Shenzhou technology Co., ltd.) is coated, 50ul (half amount) of supernatant of each hole of each plate after fusion is taken out after sealing, hybridoma cells are prevented from being sucked out, the supernatant is respectively added into the sealed ELISA plates, color development is carried out through HRP-goat anti-rat secondary antibody and substrate, OD value is detected, and at least 40 positive clones are primarily screened in the test.
4. Hybridoma cloning.
Cloning the positive hybrid clone, and performing at least three times to ensure the stability of the hybridoma. The cloned hybridoma cells also need to be periodically re-cloned to prevent mutations or chromosome loss of the hybridoma cells, thereby losing the ability to produce antibodies. In the experiment, the limited dilution method is adopted for cloning, and the antibodies screened in the experiment are subjected to subcloning for more than 3 times.
Feeder cell layers were prepared 1 day before cloning, 40 positive clones, the 10 clones with the strongest positivity required subcloning one 96-well plate, and the remaining 30 subcloning half 96-well plates, a total of 25 96-well plates. Hybridoma cells to be cloned were gently blown up from the culture well, counted, if one plate was subcloned, about 100 hybridoma cells (subhalf-fast plate, 50 hybridoma cells were taken), added to 10ml (subhalf-fast plate, 5 ml) of complete medium containing HT (purchased from sigma company), mixed well, dropped into 96-well plates, 100. Mu.l/well, and incubated in 37℃5% CO2 incubator with 1 cell per well as much as possible. And (3) forming cell clones in 8-9 days, cloning 25 96-well plates by hybridoma growth for about 10 days, and detecting whether antibodies in the supernatant are combined with target proteins by ELISA. Cells of the positive wells were transferred to 24 well plates for expansion culture and frozen.
5. Freezing and resuscitating hybridoma cells.
(1) Freezing hybridoma cells, wherein each ampoule for freezing hybridoma cells contains more than 1X 107, cell freezing solution, 50% calf serum, 40% DMEM culture solution and 10% DMSO (dimethyl sulfoxide, purchased from sigma). The cells are gradually cooled from room temperature to-80 ℃ by a program cooling instrument during freezing, and then are put into liquid nitrogen, and the cells can be stored for several years or longer in the liquid nitrogen.
(2) Recovery of hybridoma cells: the invention relates to a hybridoma which undergoes repeated freeze thawing process, wherein antibody secretion activity and yield are stable, and the frozen cells are thawed in a water bath at 37 ℃ by carefully taking out glass ampoule from liquid nitrogen, washing the cells twice with 20% DMEM culture solution, then transferring the cells into a culture bottle of feeder cells prepared on the first day, culturing the cells in a 37 ℃ 5% CO2 incubator, and detecting the activity of supernatant antibodies by ELISA when the cells form colonies.
Example 2
This example demonstrates the determination of antibody purity, specificity, affinity and analysis of antigen binding epitopes on the rat anti-mouse CD137 monoclonal antibody (DB 68) prepared in the above example and verifies its blocking effect on CD137 molecule binding to CD137 ligand.
1. DB-68 antibody purity
(1) DB-68 antibody production
The DB-68 hybridomas are selected to be resuscitated into a T75 culture flask, after the cells grow to be full, about 5 multiplied by 107 cells are transferred into a special roller bottle for the hybridomas, 100ml of 5 percent DMEM is added for 2 days of culture, the supernatant is collected and replaced by 100ml of serum-free DMEM, the supernatant is collected again after 5 days of culture, the two supernatants are mixed and centrifuged to remove cell fragments, and a 0.2 mu m filter membrane is used for suction filtration, so that impurities are further removed to prevent the clogging of an affinity chromatography column in the affinity chromatography process.
(2) DB-68 antibody purification
DB-68 antibody in hybridoma culture supernatant was purified according to a set procedure using AKTA Protein purification system and Protein G affinity chromatography column (GE Co., USA), the purified antibody buffer was replaced with 1 XPBS by dialysis, the antibody was concentrated to a predetermined concentration by ultrafiltration centrifugation through an ultrafiltration centrifuge tube (Millipore USA), and the resultant was stored in a sub-package at-80 ℃.
(3) Antibody purity identification
After purification, 2 mug of the antibody was mixed with a protein loading buffer, boiled in boiling water for 5 minutes, dried to room temperature, and then electrophoresed by using a 4-12% gradient SDS-PAGE gel, at constant pressure 140V for 70 minutes, and the gel was stained with Coomassie brilliant blue overnight, and then decolorized with a decolorizing solution, as shown in FIG. 1, the antibody lane was seen to show two bands, one of about 55KD and one of about 25KD. The DB-68 antibody lanes, 55KD and 25KD proteins occupy more than 90% of the lane protein bands, i.e., the antibody purity is more than 90% and the impurity protein ratio is less than 10%. Subsequent assays may be performed after the identification of antibody purity.
2. Identification of the specificity of DB-68 antibodies Using ELISA
Respectively coating mCD137-his, HCD137-his, PD-L1-his, EGFR-his, PD-1-his, CD137L-his, epcam-his fusion protein 1 μg/mL, blocking with 5% milk (BD company), adding DB-68 antibody 1 μg/mL, incubating at 37deg.C for 1 hr, adding HPR labeled goat anti-rat secondary antibody at 37deg.C, incubating for 1 hr, adding substrate for color development, and determining OD450 value.
The experimental results are shown in FIG. 2, wherein mCD137-his is a fusion protein with his tag of mouse CD137 molecule, hCD137-his is a fusion protein with his tag of human CD137 molecule, and other proteins are fusion proteins with his tag of different proteins (all purchased from Beijing Yiqiao Shenzhou science and technology Co., ltd.) and the result shows that the antibody in the embodiment 1 of the invention has strong specificity for only recognizing and binding to mouse CD137 molecule. The DB-68 antibody has high specificity, and has no cross reaction with human CD137 and no binding with other proteins with his label.
3. DB-68 antibody affinity detection
The specific method comprises the following steps:
(1) The AMC sensor (PALL, USA) was pre-wetted in equilibration solution (0.1% BSA+0.02% TWEEN20 in PBS) for 10 minutes, DB-68 antibody was diluted to 20. Mu.g/mL with equilibration solution and added to the second column of the dark 96-well plate, 200. Mu.L/well.
(2) MCD137-his was diluted from a 250nM initial fold ratio to 3.9nM and added to the fourth column of a dark 96-well plate at 200. Mu.L/well, well H4 as a blank, and 200. Mu.L equilibration solution was added.
(3) The equilibration solution was added to the first and third columns at 200 μl/well.
(4) The sensor was equilibrated in the first column for 120 seconds to obtain a base equilibration curve, and then antibody immobilized in the second column for 300 seconds using a Fortebio-ott 96 instrument test. The binding curve was obtained by conducting a further equilibration for 120 seconds in the third column and binding the antigen for 180 seconds in the fourth column, and then dissociating for 300 seconds in the third column, obtaining a dissociation curve.
(5) The curves were fit analyzed using Fortebio Octet96 analysis software to obtain affinity values.
TABLE 1Fortebio Octet analysis of PD-L1 antibody binding to PD-L1 and dissociation results
The results of the experiments are shown in FIG. 3 and Table 1, and the binding affinity of the antibodies of example 1 to mCD137-his protein is 1.02X10-10 mol/L, i.e.10-10 mol/L, as measured by the fortebio Octet molecular interaction analyzer, the smaller the affinity, the stronger the binding capacity.
The different lines in FIG. 3 represent different concentrations of mCD137-his, 250nM,125nM,62.5nM,31.3nM,15.6nM,7.81nM,3.9nM, respectively.
4. DB-68 antibody and mCD137 molecule binding epitope analysis
(1) Segmented expression of mCD137 molecule
To analyze the specific binding sites of DB-68 antibodies to mCD137 molecules, the mCD137 molecules were expressed in segments.
Firstly, designing specific primers, amplifying different sections of mCD137 by using PCR, then inserting the amplified fragments into an expression vector PcDNA3.1, and determining that the sequence of the inserted target gene fragment is completely correct by sequencing. After 293 cells are fully paved at the bottom of a 6-hole plate, the expression plasmid is transfected into the 293 cells in a transfection mode, 2ml of serum-free culture medium is added for culturing for 72 hours after the transfection is finished, the expressed target protein enters a culture supernatant in a secretion mode, and the protein in the supernatant can reach 0.5-1ug/ml.
(2) Identification of the segment proteins of mCD137 molecule
Culture supernatants were collected in 6-well plates, centrifuged at about 2ml to remove cell debris, and 15ul of a protease A or ProteinG agarose gel suspension (GE. Co. USA) was added and mixed overnight at 4 degrees to allow the human or mouse Fc tagged mCD137 fusion protein fragments in the supernatant to bind sufficiently to the protease A or ProteinG agarose gel, which then deposited on the bottom of the centrifuge tube by centrifugation of the protease A or ProteinG agarose gel, and the fusion proteins could be enriched simultaneously. Mixing the enriched protein with protein loading buffer solution, boiling for 5min, separating target protein with protein A or ProteinG agarose gel, centrifuging, and settling protein A or ProteinG agarose gel at the bottom of tube.
And (3) carrying out SDS-PAGE electrophoresis on proteins in the supernatant, transferring the proteins to a nitrocellulose membrane, blocking 5% milk, and identifying a human Fc tag of mE3-hFc and a mouse Fc tag of mE4-hFc by using a goat anti-human or goat anti-mouse secondary antibody marked by HRP, and simultaneously determining the correctness of the expressed proteins by using the comparison of the expressed proteins and protein markers.
Referring to FIG. 4, mE3, mE4, and mE400 represent the first 85 amino acids, the first 117 amino acids, and the last 68 amino acids of the mCD137 molecule, respectively. Specifically, mE3 (mE 3-hFc) represents a fusion protein of 1-85 amino acids of the extracellular region of a mouse CD137 molecule and human Fc, mE4 (mE 4-hFc) represents a fusion protein of 1-117 amino acids of the extracellular region of a mouse CD137 molecule and human Fc, and mE400 (mE 400-mFc) represents a fusion protein of 118-185 amino acids of the extracellular region of a mouse CD137 molecule and mouse Fc.
The SDS-PAGE-ECL test shows that the protein fragments are expressed in the correct size, as shown in FIG. 5.
(3) DB-68 antibody and mCD137 molecule binding epitope analysis
After 50. Mu.l+50. Mu.l of coating solution from the identified correct mE3, mE4, mE400 and control supernatant (empty vector) were mixed, they were added to ELISA plates and coated overnight at 4 ℃. The following day, after blocking, DB-68 antibody was added, incubated at 37℃for 1 hour, HRP-labeled goat anti-rat secondary antibody was added, incubated at 37℃for 1 hour, and the substrate was developed to determine the OD450 value.
The experimental results are shown in FIG. 6, in which the antibody of example 1 of the present invention has a region between 85-117 amino acids, i.e., the III-th CDR region, which recognizes and binds to mCD137 molecule.
5. Blocking of mouse CD137 binding to CD137 ligand by DB-68 antibodies.
Blocking was detected by ELISA.
The coated mCD137L-mFc (mouse CD137 ligand molecule plus mouse Fc tag, laboratory construction, expression and identification), 1. Mu.g/ml, while 5. Mu.g/ml and 25. Mu.g/ml DB-68 antibodies were mixed with 1. Mu.g/ml mCD137-hFc, respectively, and left standing for 4℃overnight. The next day, the mixture was added to ELISA plates coated with mouse CD137 ligand and incubated at 37℃for 2h, goat anti-human secondary antibodies were labeled with HRP to detect mCD137-hFc, and OD450 values were read.
The results are shown in FIG. 7, which shows that when DB-68 antibody is 5 μg/ml, only a small fraction of mCD137 molecules are blocked from binding to its ligand, while when DB-68 antibody is 25 μg/ml, 48% of mCD137 molecules are blocked from binding to its ligand.
6. Full length sequencing of hybridoma antibody nucleic acids
According toReagents (U.S. Thermo FISHER SCIENTIFIC) technical manual, total RNA was isolated from hybridoma cells, the total RNA was reverse transcribed into cDNA using isotype specific antisense primers (or universal primers) according to PRIMESECPTTM first strand cDNA synthesis kit (Takara, CAT: 610A) technical manual, antibody fragments of VH, VL, CH and CL were amplified according to the cDNA end (RACE) rapid amplification standard procedure (SOP) of GESTCRIPT, the amplified antibody fragments were cloned into standard cloning vectors, sequenced, and the variable region sequence analysis tools (i) NCBI Nucleotide BLAST, (ii) IMGT/V Quest program, (iii) NCBI Ig blast. Colony PCR was used to screen for inserts of the correct size, each fragment with inserts of the correct size was sequenced, and the different cloning sequences were aligned to obtain consistent sequencing results.
The heavy chain DNA sequencing result is shown by referring to SEQ ID NO.13, and comprises a signal peptide sequence and a termination sequence.
The result of the light chain DNA sequencing is shown by reference to SEQ ID NO.14, and comprises a signal peptide sequence and a termination sequence.
Experimental example 1
The experimental example verifies that the DB-68 antibody has the function of synergistically activating the T lymphocytes of mice.
(1) Preparation of mouse lymphocyte suspension
Mice were sacrificed by cervical scission and their spleens were removed in an ultra clean bench. 3ml of mouse lymphocyte separation medium was placed in a 35mm dish, the spleen was placed in a nylon mesh on top of the dish, and the spleen was grinded into single cells by a syringe inner piston and entered into the mouse lymphocyte separation medium below. The spleen cell-suspended isolate was immediately transferred to a 15ml centrifuge tube, gently covered with 500ul of serum-free 1640, and centrifuged at 800g for 30min. Lymphocytes from the spleen of mice were passed into the upper serum-free DMEM by gradient centrifugation, the lymphocyte layer was aspirated into new tubes, washed once with 5%1 x PBS, and the cells were resuspended in 1ml of 5% PBS and counted for a total of 3-4 x 107 cells.
(2) Analysis of mouse T lymphocyte proliferation by CFSE method
The spleens of 3-4×107 mice were resuspended in 1ml of 5% PBS. 1.1ul of 5mM CFSE stock solution was suspended in 110ul of 1 XPBS, added to the spleen cell suspension, thoroughly mixed, incubated at room temperature for 5 minutes in the dark, and cells were washed twice with 10 volumes of 5%1 XPBS. Count prior to the last centrifugation, total 1×107 cells. After centrifugation, the supernatant was discarded, 10ml 1640 of complete medium was resuspended in cells at a concentration of approximately 1X 106/ml, 1 ml/well was added to a 24-well plate for a total of 10 wells. Three wells were filled with only αCD3e (BD Co 145-2C 11) at a concentration of 2ng/ml, three wells were filled with αCD3e (2 ng/ml) +αCD137-1 (control antibody, 2 ng/ml), and three wells were filled with αCD3e (2 ng/ml) +DB-68 (2 ng/ml) and incubated in 37℃and 5% CO2.
(3) Flow detection analysis
After CFSE staining, 0h,24h,48h and 72h, 1 well cell (0 h is one well cell without any reagent) was taken and subjected to flow analysis once under the three culture conditions, respectively, 1ml of 1 XPBS was used to wash the cells once, and the supernatant was discarded by centrifugation. Adding 0.5ul dead living dye (BV 605) into 500ul 1 XPBS, mixing, adding into cell sediment, mixing, blocking light, standing at room temperature for 15min, adding 2ml 1 XPBS, washing and centrifuging twice, discarding supernatant, blocking liquid Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) 2 ul/tube, mixing, blocking light for 15min, adding PE-Rat Anti-Mouse CD4,5ul, APC-Rat Anti-Mouse-CD8a,5ul, mixing, blocking light for 30min,1ml 1 XPBS for two times, adding into 200 ul/tube 1 XPBS, and analyzing the synergistic activation ability of different Rat Anti-Mouse CD137 antibodies on Mouse CD4+ and CD8+ T lymphocytes by using a flow cytometer.
In FIG. 8, CD4+ T lymphocytes were analyzed by flow through for different proliferation after 72 hours of culture with three condition stimuli. The left panel shows the addition of αCD3e (2 ng/ml) only to the broth, the middle panel shows the addition of αCD3e (2 ng/ml) +αCD137-1 (control antibody, 2 ng/ml), and the right panel shows the addition of αCD3e (2 ng/ml) +DB-68 (2 ng/ml), where CD4+ T cells proliferated most strongly under αCD3e+DB-68 conditions, indicating that DB-68 has a stronger synergistic activation capacity than the control CD137 antibody.
In FIG. 9, CD8+ T lymphocytes were analyzed by flow through for their proliferation after 72 hours of stimulation culture under three conditions, the left panel shows the addition of only αCD3e (2 ng/ml) to the culture broth, the middle panel shows the addition of αCD3e (2 ng/ml) +αCD137-1 (control antibody, 2 ng/ml), and the right panel shows the addition of αCD3e (2 ng/ml) +DB-68 (2 ng/ml), where CD8+ T cells proliferated the strongest under αCD3e+DB-68 conditions and the synergistic activation capacity of DB-68 was stronger than that of control CD137 antibody, and CD8+ T cells proliferated the stronger than CD4+ T cells under the same conditions.
From the results shown in FIGS. 8 to 9, it is understood that the DB-68 antibody has strong synergistic activation ability, especially against CD8+ T lymphocytes of mice.
Experimental example 2
The experimental example verifies the anti-tumor activity of the DB-68 antibody in a mouse transplanted tumor model.
(1) Mouse intestinal cancer model establishment
Female BALB/c mice were selected for about 6-8 weeks, CT26 (mouse intestinal cancer cell line, purchased from ATCC) was inoculated subcutaneously in 5X 105/mouse, about 100. Mu.l, and 15 BALB/c mice were inoculated as semi-spherical as possible. On day 7 or so, tumor cells were inoculated to form 6-7mm tumors under the skin of the mice, the long and short diameters of the tumors were measured with calipers, 10 BALB/c mice with substantially the same tumor size were selected, and 5 mice per group were housed and labeled in control and treatment groups.
(2) DB-68 antibody anti-tumor treatment
On days 6-7 after inoculation, the subcutaneous transplantation tumor had a diameter of about 6-7mm, the treatment group began to inject DB-68 antibody intratumorally, 5 μg/dose, 50ul volume, three consecutive doses every other day, and the control group intratumorally injected PBS liquid of the same volume. And at the beginning of the treatment, the tumor size was measured with a caliper every other day, and the tumor area was calculated until the animal experiment ended, i.e., the tumor area exceeded 225mm2.
Results referring to fig. 10, the DB-68 antibody treated group showed a significant difference in tumor growth rate from the control group. NS, P >0.05; P <0.05, P <0.0001.DB-68 antibody can not completely eliminate mice subcutaneous transplantation tumor, but can inhibit tumor growth.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.