Anti-CD27L fully humanized antibody or antibody fragment and application thereof

Abstract

The invention relates to an anti-CD27L fully humanized antibody or antibody fragment, which comprises a heavy chain variable region and a light chain variable region, amino acid sequences of three complementary determining regions of the heavy chain variable region are respectively shown as SEQ ID NO.1-3, and amino acid sequences of three complementary determining regions of the light chain variable region are respectively shown as SEQ ID NO.5, 7 and 9; or the amino acid sequences of the three complementary determining regions of the heavy chain variable region are respectively shown as SEQ ID NO.1-2 and 4, and the amino acid sequences of the three complementary determining regions of the light chain variable region are respectively shown as SEQ ID NO.6, 8 and 10. The anti-CD27L fully-humanized antibody or the antibody fragment disclosed by the invention can be crossly combined with CD27L proteins of human, cynomolgus monkeys and mice and has blocking activity on CD27L / CD27, so that new possibility is provided for further exploring and developing a new CD27L targeting antibody and related clinical research.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a fully human anti-CD27L antibody or antibody fragment and an application thereof. Background Art

[0002] CD27L (also known as TNFSF7, CD70 or CD27LG, etc.) belongs to the Tumor Necrosis Factor Receptor Super Family and is a widely reported T and B cell immune co-stimulatory molecule. The gene encoding human CD27L is located in the chromosome 19p13.2-13.3 region. Its mature protein size is about 50kDa and it is a type II transmembrane glycosylated protein that exists in the form of a trimer. The full-length CD27L protein contains 193 amino acids, including an extracellular domain (ECD), a transmembrane region (TM), an intracellular domain (ICD) and two N-terminal glycosylation sequences; the first and third, second and fourth cysteines of its ECD each form a disulfide bond to maintain a specific spatial conformation. CD27L is widely used in animal disease models, but there are large differences between humans and mice, with only about 56% similarity between the two.

[0003] CD27L is mainly expressed on specific immune cells, including dendritic cells, activated lymphocytes, tonsils, T cells in the skin and intestines, antigen-presenting cells in the intestinal lamina propria, etc. The expression of CD27L on lymphocytes is extremely short-lived, mainly present on activated T, B and NK cells, such as T cells that produce IFN-γ are basically CD27L+, B cells that secrete immunoglobulins and are activated by antigens, etc. This highly restricted and time-regulated expression pattern is carefully mediated by co-stimulatory signals, cell activation signals, antigen stimulation and cytokines, thereby accurately and strictly regulating the timing of CD27L to exert its biological function. Under pathological conditions, CD27L is highly expressed in a variety of tumor tissues, including a variety of lymphoid malignancies, pancreatic cancer, glioblastoma, etc.; while in normal tissues, CD27L is only restrictedly expressed on activated immune cells, and the expression level is low, which makes CD27L an ideal target for immunotherapy.

[0004] It is known that CD27L binds to its only receptor CD27, which is a member of the tumor necrosis factor receptor superfamily (TNFR) and is a homodimeric transmembrane glycoprotein formed by two monomers with a molecular weight of about 55kDa connected by a disulfide bond. Studies on CD27L-CD27 signal transduction in tumors have shown that its signal transduction can induce the proliferation of malignant cells. CD27L-CD27 co-stimulation can prevent Treg cells developed in the thymus from dying, increase the number of Tregs in the spleen and lymph nodes of tumor-bearing mice, and promote T cell differentiation. The absence of CD27L-CD27 signal can inhibit the occurrence of collateral circulation in the hind limb ischemia mouse model. At the same time, studies have also shown that CD27L-CD27 co-stimulatory signals are involved in CD4+T cell-mediated autoimmune diseases. Therefore, the CD27L-CD27 interaction is crucial for regulating cellular immune responses. In addition, CD27L, as a B cell co-stimulatory molecule, interacts with CD27 in BT cell contact to promote B cell differentiation into plasma cells, thereby increasing the production of immunoglobulins and increasing the toxic effects of activated natural killer cells. There is much evidence that CD27L plays an important role in different types of tumors (such as lymphomas, solid tumors) and autoimmune diseases (such as lupus erythematosus). In some tumor cells, overexpression of CD27L can help tumors escape attacks by the immune system.

[0005] In summary, based on the discovery of these tumor-promoting and immunosuppressive mechanisms of CD27L-CD27, and the characteristics that CD27 is continuously expressed on immune cells in the steady state while CD27L is not expressed, we can develop blocking antibodies targeting CD27L to treat related diseases. There are mainly the following pathways: (1) Interfering with the signal transduction between CD27L and CD27, thereby weakening the activation and proliferation of T cells; (2) Blocking the CD27L / CD27 signaling pathway to reduce excessive activation of T cells; (3) Inducing antibody-dependent cell-mediated cytotoxicity (ADCC); (4) Antibody-mediated interference with intracellular signal transduction, which can affect the survival, proliferation and migration of tumor cells, thereby inhibiting tumor growth and spread.

[0006] At present, a variety of therapies targeting CD27L have made progress in preclinical and clinical studies, such as AbbVie's CD27L monoclonal antibody ABBV181, which is currently in the clinical trial stage. By binding to CD27L, ABBV181 blocks the interaction between CD27L and CD27, inhibiting the proliferation and survival of tumor cells. Drugs such as Cusatuzumab (ARGX-110) carry Fc receptors (FcγRIII) and activate immune effector cells and block target proteins based on antibody-dependent cellular cytotoxicity (ADCC). It has been proven to effectively eliminate leukemic stem cells (LSC) in AML patients treated with hypomethylating drugs, while blocking the differentiation of AML primitive cells and stem / progenitor cells induced by the CD27L-CD27 pathway. In addition, some CAR-T cell therapies targeting CD27L are being evaluated in phase I / II clinical trials in patients with hematological tumors and solid malignancies.

[0007] Although CD27L shows great potential in treatment and diagnosis, it also faces some challenges. For example, it is necessary to ensure the high specificity of antibodies to CD27L and avoid nonspecific binding to normal cells. In addition, most current CD27L antibodies cannot cross-bind to CD27L proteins of different species, so different antibodies need to be developed for in vivo activity studies and safety evaluation and analysis, which is costly and cannot be used for horizontal comparative immunological studies between different species. Therefore, further exploration and development of new antibodies targeting CD27L will provide new possibilities for related clinical research and applications, which will have positive significance for improving the effectiveness and safety of tumor treatment, and will also provide patients with more treatment options. Summary of the invention

[0008] 1. Technical issues to be resolved

[0009] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a fully human anti-CD27L antibody that cross-binds to the CD27L proteins of humans, cynomolgus monkeys and mice and has blocking activity against CD27L / CD27, and its application. The antibody can specifically cross-bind to the CD27L proteins of humans, cynomolgus monkeys and mice, which is conducive to conducting in vivo activity studies in animal models, improving experimental flexibility and efficiency, and is of great significance to accelerating the process of new drug research and development.

[0010] (II) Technical solution

[0011] In a first aspect, the present invention provides a fully human anti-CD27L antibody or antibody fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the three complementary determining regions CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown in SEQ ID NOs. 1-3, respectively, and the amino acid sequences of the three complementary determining regions CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NOs. 5, 7, and 9, respectively; or

[0012] The amino acid sequences of the three complementary determining regions CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown in SEQ ID NOs.1-2 and 4, respectively, and the amino acid sequences of the three complementary determining regions CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NOs.6, 8 and 10, respectively.

[0013] According to a preferred embodiment of the present invention, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.11, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.13; or, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.12, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.14.

[0014] According to a preferred embodiment of the present invention, the fully human antibody or antibody fragment has an amino acid sequence shown in SEQ ID NO.29 or SEQ ID NO.30.

[0015] In a second aspect, the present invention provides a polynucleotide encoding the above-mentioned fully human anti-CD27L antibody or antibody fragment.

[0016] According to a preferred embodiment of the present invention, the polynucleotide includes a gene fragment encoding a heavy chain variable region and a gene fragment encoding a light chain variable region, the gene fragment encoding the heavy chain variable region has a base sequence shown in SEQ ID NO.15-17, and the gene fragment encoding the light chain variable region has a gene sequence shown in SEQ ID NO.19, 21, and 23; or, the gene fragment encoding the heavy chain variable region has a base sequence shown in SEQ ID NO.15-16 and 18, and the gene fragment encoding the light chain variable region has a base sequence shown in SEQ ID NO.20, 22, and 24.

[0017] According to a preferred embodiment of the present invention, the polynucleotide includes a gene fragment encoding a heavy chain variable region and a gene fragment encoding a light chain variable region, the gene fragment encoding the heavy chain variable region has the base sequence shown in SEQ ID NO.25, and the gene fragment encoding the light chain variable region has the base sequence shown in SEQ ID NO.27; or, the gene fragment encoding the heavy chain variable region has the base sequence shown in SEQ ID NO.26, and the gene fragment encoding the light chain variable region has the base sequence shown in SEQ ID NO.28.

[0018] According to a preferred embodiment of the present invention, the polynucleotide has a base sequence as shown in SEQ ID NO.31 or SEQ ID NO.32.

[0019] In a third aspect, the present invention also provides an expression vector or cell comprising the above polynucleotide.

[0020] In a fourth aspect, the present invention relates to the use of the fully human anti-CD27L antibody or antibody fragment in the preparation of a detection kit, an antibody chip, an antibody probe or an anti-tumor therapeutic drug.

[0021] In the present application, antibody fragments of fully human antibodies against CD27L include Fab, F(ab')2, Fab', scFv, etc. Among them, Fab includes a complete light chain and heavy chain variable region and the first constant region. The Fab fragment retains a single antigen binding site. F(ab')2 is composed of two Fab fragments connected by a disulfide bond in the hinge region, has a bivalent binding ability, and can bind to two identical antigen epitopes at the same time. ScFv is a single-chain form formed by connecting VH and VL through a flexible peptide chain, which retains the antigen binding ability. The fully human antibody against CD27L is a complete immunoglobulin molecule, and an antibody fragment generally refers to a part of an antibody, which only contains an area that can bind to an antigen, and can be a fragment separated from a complete antibody by enzymatic hydrolysis or other methods. In some cases, it may be advantageous to use an antibody fragment instead of a whole antibody, for example to reduce nonspecific binding or improve tissue penetration.

[0022] (III) Beneficial effects

[0023] The fully human anti-CD27L antibody screened by the present invention is produced in a fully humanized manner and has an extremely low risk of immunogenicity. The antibody also has a good ability to bind to cross-species target molecules (humans, cynomolgus monkeys and mice), which is conducive to subsequent in vivo activity studies and safety evaluation analyses in mouse or cynomolgus monkey tumor models, improves the flexibility and efficiency of research experiments, and is of great significance for accelerating the development of new drugs. Experiments have confirmed that the fully human anti-CD27L antibody can effectively block the interaction between CD27L and CD27, thereby inhibiting the proliferation and survival of tumor cells. In addition, experiments have also confirmed that the fully human anti-CD27L antibody of the present invention has good binding activity (high affinity) to protein targets and cell targets, and its EC binding to protein targets and cell targets is high. 50 Values ​​and IC of blocking CD27L / CD27 50 The values ​​are all as low as pM level, so the antibody or antibody fragment provided by the present invention has good application prospects in the development of antibody drugs targeting CD27L. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the agarose gel electrophoresis detection result of the PCR product of the fully human CD27L single-chain antibody library fragment in Example 1.

[0025] Figure 2 The binding of positive clones 2C9 and 20D10 screened by yeast display of the fully human single-chain antibody library and FACS detection to human CD27L in Example 2 is shown.

[0026] Figure 3 The results of purity detection of two anti-CD27L human IgG1 antibodies by SDS-PAGE in Example 3 are shown.

[0027] Figure 4 These are the results of ELISA in Example 4 for detecting the binding activity of two anti-CD27L antibodies to CD27L proteins of different species.

[0028] Figure 5 This is the result of ELISA in Example 5 to detect the activity of two anti-CD27L antibodies in blocking CD27L / CD27 interaction.

[0029] Figure 6 This is the result of detecting the binding activity of two anti-CD27L antibodies to cells expressing CD27L using flow cytometry in Example 6. DETAILED DESCRIPTION

[0030] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0031] The present invention discloses an anti-CD27L antibody and application thereof, and its implementation mode will be described in detail below in conjunction with specific examples. In order to make the present invention more easily understood, some terms will be defined first.

[0032] The term "antibody" (or immunoglobulin) refers to a special class of proteins produced by the immune system, whose main function is to recognize and neutralize foreign substances, such as bacteria, viruses and other pathogens. Antibodies are produced by B cells, have high specificity and affinity, and can recognize specific antigens (specific molecules on foreign substances). Antibodies usually contain complete antibodies IgG or antibody fragments. "Antibody fragment" and "antigen binding portion" refer to the antigen-binding fragment of an antibody and antibody fragments, which usually include at least part of the antigen-binding region or variable region of the parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Examples of antibody fragments include, but are not limited to: scFv, Fab, Fab', F(ab')2, etc.

[0033] The term "heavy chain and light chain" means that an antibody is composed of two heavy chains and two light chains, which are linked together by disulfide bonds to form a Y-shaped structure.

[0034] The term "variable region" refers to the part of the antibody molecule that is responsible for recognizing and binding to a specific antigen. It is the key region for the antibody to exert its specific immune function. Within the variable region, there are three hypervariable regions (Complementarity-Determining Regions, CDRs), also known as hypervariable regions or complementary determining regions. The amino acid sequences of these regions vary greatly and are responsible for direct binding to the antigen. These regions play a decisive role in the antigen-binding specificity of the antibody, and the variable region of the antibody has a unique affinity and specificity for a specific antigen.

[0035] The term "constant region" refers to the part of the antibody molecule that does not participate in antigen recognition but determines the antibody type and function. The constant region is relatively conserved between different classes of antibodies and plays a series of important immune functions. Different antibody classes (IgA, IgD, IgE, IgG, IgM) have different heavy chain constant regions, which determine the function of the antibody and its role in the immune response.

[0036] The term "single-chain antibody" is called single-chain variable fragment (scFv) in English. It is an antibody fragment composed of the variable regions of the antibody (including the variable regions of the heavy chain and light chain, referred to as VH and VL respectively) connected by a short peptide (linker). Single-chain antibodies retain the antigen binding function of intact antibodies, but due to the lack of a constant region, their molecular weight is smaller and their structure is simpler. The short peptide connecting the two variable regions of the antibody is usually a flexible short peptide (such as a glycine-serine sequence containing multiple repetitions) to enable it to fold correctly and form an antigen binding site.

[0037] The term "fully human antibody" refers to an antibody that is completely encoded by human genes and is identical in structure and function to the antibodies produced naturally by the human body. Such an antibody does not have any non-human components in its structure, so the possibility of causing an immune response in the human body is extremely low.

[0038] The term “EC 50 ” Also known as the half-maximal effective concentration, it refers to the antibody concentration that elicits a response halfway between baseline and maximum after a specified exposure time.

[0039] The term "IC 50 ” Also known as half-maximal inhibitory concentration, it refers to the concentration of an antibody that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.

[0040] Example 1

[0041] This embodiment is a process for obtaining a fully human CD27L single-chain antibody library fragment, including: slowly adding normal human blood stored in a special anticoagulant for cell separation to a human peripheral blood lymphocyte separation solution, so that the separation solution and normal human blood are clearly separated. Place a 15mL centrifuge tube containing the blood and separation solution in a centrifuge, and centrifuge at 650g for 15 minutes at 20°C. After centrifugation, the centrifuge tube is divided into four layers from top to bottom: the first layer is the plasma layer, the second layer is the annular milky white lymphocyte layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. Carefully aspirate the annular milky white lymphocyte layer in the centrifuge tube and transfer it to a 15mL centrifuge tube. Add 10mL of cleaning solution (or 1×PBS) to the centrifuge tube, mix the cells, centrifuge at 250g for 10 minutes, and discard the supernatant. Then add 1.5 mL of cleaning solution (or 1× PBS), transfer to a 1.5 mL EP tube (sterile and enzyme-free), take a small amount for cell counting, centrifuge the remaining sample at 250 g for 10 min, discard the supernatant, add 0.5 mL of Trizol reagent to dissolve the cells, and extract total RNA using conventional methods.

[0042] The total RNA was reverse transcribed to obtain cDNA, and the heavy chain variable region and light chain variable region of the antibody were amplified by PCR using the antibody library primers designed by our company. Then, the single-chain antibody library fragments were obtained by conventional overlapping PCR technology. The agarose gel electrophoresis test results of the products were as follows: Figure 1 shown.

[0043] Example 2

[0044] This example is a screening process for antibodies that specifically bind to CD27L, specifically, by displaying a fully human single-chain antibody library in yeast, thereby screening and identifying antibodies or antigen-binding fragments (or antibody fragments) that specifically bind to CD27L. The detailed process of this example is as follows:

[0045] First, BamH I and EcoR I restriction sites were introduced into the 5' and 3' ends of the library fragment obtained in Example 1 by PCR primers. The library fragment was inserted into the pYD1-HA-Myc vector by restriction digestion and ligation reaction, and transformed into the EBY100 strain (yeast strain), and the positive transformed strains were screened by tryptophan-deficient medium to obtain the yeast display YSD-huscFv Lib library.

[0046] Before magnetic bead enrichment, the library glycerol bacteria were inoculated into YNB-CAA-glucose Medium (YCGlu) liquid culture medium and cultured at 30°C, 160rpm for 24h (cultured to OD2-3). Take 50OD of the activated bacterial solution, collect the precipitate by centrifugation, resuspend it in 100mL of YCGal (YNB-CAA-galactose Medium) culture medium (1× double antibody), and culture it at 20°C, 160rpm for 24-48h. After incubating the biotinylated CD27L (b-CD27L) protein with SA magnetic beads, add the induced YSD-huscFv Lib for incubation, incubate at 4°C for 1h, discard the supernatant, wash 3 times with PBST-1% BSA, transfer the magnetic beads to 20mL YCGlu and culture for 24-48h. After culture, take 10OD of bacterial solution in a 1.5mL EP tube, add 15% glycerol and freeze.

[0047] Take the yeast display library after magnetic bead enrichment twice, and activate and induce it according to the above method. First, incubate biotinylated human CD27L and human CD27 with excess fluorescent marker proteins SA-650 and SA-PE for 1 hour to prepare CD27L-650 and CD27-PE. Take 0.2OD / tube (1mL volume) of induced yeast cell suspension, centrifuge for 5 minutes, discard the supernatant, add 800μL FACS Buffer (1×PBS including 1% FBS) to wash once, centrifuge at 900×g for 5 minutes, and discard the supernatant. Resuspend the precipitate to 200μL, add appropriate amount of CD27L-650 and anti-Myc tag mouse monoclonal antibody, mix well, incubate for 1 hour, wash, incubate CD27-PE and Goat anti-mouse IgG pAb-488 for 1 hour. Finally, wash the cells, resuspend the yeast cells and pipette them, and load them into the flow cytometer for sorting. By selecting the target cell population, a cell population with normal expression (higher 488 fluorescence signal, higher fluorescence intensity detected at 488nm wavelength), binding signal with target CD27L (higher 650 fluorescence signal, higher fluorescence intensity detected at 650nm wavelength) and no CD27 binding signal (no PE fluorescence signal) was obtained, and a sufficient number of cells were sorted out and cultured.

[0048] After repeated rounds of FACS sorting (using different species of CD27L target proteins in turn), the target yeast cell population obtained by sorting was spread on a YCGlu plate for culture. After a single clone grew out, several single clones were selected for activation induction and incubated in a similar manner as described above (only the target human CD27L binding signal was detected, i.e., a higher 650 fluorescence signal). After the incubation was completed, the cells were tested on a machine and detected by FACS (e.g. Figure 2 As shown in the figure, positive clones with strong binding to CD27L were selected, cultured, subjected to colony PCR and sequencing, and two antibodies 2C9 and 20D10 were obtained.

[0049] Example 3

[0050] In this example, the two antibodies screened in Example 2 were constructed into human IgG1 complete antibodies by DNA recombination technology to study their functions. The detailed operation of this example is as follows:

[0051] First, the VH and VL of the two antibodies were amplified by primers, and then fused with human IgG1CH and CL-Kappa to construct expression vectors pcDNA3.1-2C9H, pcDNA3.1-2C9L, pcDNA3.1-20D10H and pcDNA3.1-20D10L. Two days before transfection, Expi293F cells (Gibco, A14527) were inoculated into 100 mL of culture medium at a density of 4-5×10 5 cell / mL, placed in a constant temperature shaker, cultured at 37°C, 110rpm, 8% CO2. On the day of transfection, the cell density was controlled at 1.5-2×10 6 cell / mL, add 50μg of the expression plasmids of Light Chain (light chain plasmids pcDNA3.1-2C9L, pcDNA3.1-20D10L) and Heavy Chain (heavy chain plasmids pcDNA3.1-2C9H, pcDNA3.1-20D10H) to the transfection reagent, then add the mixture to 100mL of cell culture and culture it at 37°C, 8% CO2 for 5-7 days. Take out the cultured cell suspension, collect the cell culture supernatant by centrifugation, and load the cell supernatant onto a Protein A affinity purification column for purification. Next, the purity of the two anti-CD27L fully human antibodies was tested under reducing conditions (Reducing conditions) and non-reducing conditions (Non-Reducing conditions) by SDS-PAGE. The test results showed that the purity was above 90%. The test results are as follows Figure 3 shown.

[0052] Finally, the purified human IgG1 intact antibody was sequenced by SDS-PAGE (amino acid sequence and encoding gene are shown in Table 1-2).

[0053] Table 1: Amino acid sequence and coding gene sequence and number of human IgG1 complete antibody

[0054]

[0055]

[0056]

[0057] Table 2: Amino acid sequences and coding gene sequences and numbers of two scFv antibody fragments

[0058]

[0059]

[0060] Note: The underlined amino acid sequences or gene fragments indicate the complementary determining regions.

[0061] Example 4

[0062] This example uses ELISA to detect the binding activity of two anti-CD27L antibodies to CD27L proteins of different species. The method of this example is as follows:

[0063] The two anti-CD27L human IgG1 complete antibodies constructed in Example 3 were serially diluted to detect their binding activity to CD27L proteins of different species. First, Streptavidin (SA, 2 μg / mL) was coated on a 96-well MediSorp ELISA plate, and after blocking with PBS containing 2% BSA (w / v) at 37°C for 2 h, biotinylated human CD27L protein (Biotinylated Human CD27L, active trimer, referred to as "hu-CD27L"), cynomolgus monkey CD27L protein (Biotinylated Cynomolgus Monkey CD27L, active trimer, referred to as "cyno-CD27L"), mouse CD27L (Biotinylated mouse CD27L, active trimer, referred to as "mu-CD27L", 1 μg / mL each (that is, 1 μg / mL of biotinylated CD27L proteins of different species were added to the Streptavidin-coated MediSorp ELISA plate and incubated for a period of time to ensure sufficient capture), captured at 37°C for 30 minutes and washed with PBST (containing 0.05% Tween20). After washing, a series of gradient concentrations of anti-CD27L antibodies were added (20nM starting, 3-fold dilution, 7+1 gradient). After incubation at 37°C for 1 hour, washed 3 times with PBST, and after washing, diluted Goat Anti-Human F(ab')2pAb-HRP (Jackson; Cat.: 109-035-006) was added at 1:5000. Finally, 50 μl of TMB substrate was added to each well for color development. After terminating the reaction, the absorbance was measured at 450nm using an enzyme-linked immunosorbent assay (MD). EC was calculated using GraphPad Prism 50 And draw a picture, such as Figure 4 As shown in Figure 2, both antibodies have good binding activity with human CD27L (hu-CD27L), cynomolgus monkey CD27L (cyno-CD27L) and mouse CD27L (mu-CD27L). 50The EC value of antibody 2C9 and cynomolgus monkey CD27L protein is 0.39 nM. 50 At 0.44nM, antibody 2C9 binds to mouse CD27LEC 50 The EC value of antibody 20D10 and human CD27L protein is 0.69nM. 50 The EC value of antibody 20D10 and cynomolgus monkey CD27L protein is 0.49 nM. 50 At 0.72nM, antibody 20D10 binds to mouse CD27LEC 50 EC 50 The smaller the value, the stronger the binding activity of the antibody to the target protein.

[0064] Example 5

[0065] This example uses ELISA to detect the blocking activity of anti-CD27L antibodies on CD27L / CD27. The method of this example is as follows:

[0066] In order to verify whether the two anti-CD27L antibodies have a blocking effect on the binding of CD27L and CD27, a competitive ELISA test was performed in this example. Human CD27L protein (2 μg / mL) diluted with PBS was added to a 96-well ELISA plate and incubated overnight at 4°C (or incubated at 37°C for 2 hours). At this time, the CD27L protein was fixed on the 96-well plate. Then the above-mentioned plate was incubated with PBS containing 2% BSA at 37°C for 2 hours, washed with PBST after incubation, and antibodies of different concentration gradients were added. At the same time, humanCD27 protein (fused with Flag tag) was added, and the plate with antibodies and CD27 protein was incubated at 37°C for 1 hour. After the incubation, wash 3 times with PBST, and add mouse anti-Flag mAb diluted 1:8000 and incubate at 37°C for 1 hour. After incubation and washing, a 1:10,000 dilution of horseradish peroxidase-labeled goat anti-mouse polyclonal antibody (Jackson ImmunoResearch) was added and incubated for 1 h. Finally, after washing with PBST and PBS for 3 times, the cells were developed with TMB and the reaction was stopped with acid. The data were read at OD450 nm using a microplate reader. Figure 5 As shown in the figure, when the concentration of the two antibodies is higher, the absorbance value at OD450 is lower, indicating that the two antibodies can block the interaction between CD27L and CD27. Among them, the IC value of antibody 2C9 blocking the interaction between CD27L and CD27 is 50 The IC of antibody 20D10 blocking the interaction between CD27L and CD27 is 0.15 nM. 50 is 0.29nM. Among them, IC 50 The smaller the value, the stronger the blocking activity of the antibody on the interaction between CD27L and CD27.

[0067] Example 6

[0068] This example uses FACS to analyze the cell binding activity of two anti-CD27L antibodies. The method of this example is as follows:

[0069] In order to verify the cell binding activity of the two anti-CD27L antibodies, this example used a lentiviral system to construct a HEK293T cell line (HEK293T-CD27L) that highly expresses human CD27L. The HEK293T-CD27L cell line was cultured in a culture dish, and HEK293T cells were cultured as a negative control. The cells were collected, centrifuged, washed twice with FACS buffer, and resuspended. After resuspension, the cells were divided into 1×10 6 cell / portion, add the cells to a V-bottom 96-well plate, centrifuge at 500g for 5 minutes, and discard the supernatant. Use FACS buffer to gradient dilute 2C9 and 20D10 antibodies, starting from 150nM and diluting 3 times, with a total of 8 gradients. Add the diluted two antibodies 2C9 and 20D10 to a V-bottom 96-well plate (100μL / hole) and incubate at 4℃ for 1h. After incubation, wash 3 times with FACS buffer, add 1:200 diluted FITC-F(ab')2 goat anti-human IgG (H+L) pAb (JacksonImmuno Research, 109-096-088, 0.75mg / mL) after washing, and incubate at 4℃ for 1h. After incubation, wash 3 times with FACS buffer, resuspend to 200μL / hole, and load to a flow analyzer (Agilent, NovoCyte-2060R) for detection. The test results are as follows Figure 6 As shown, the EC binding of antibody 2C9 to HEK293T-CD27L 50 The EC value of antibody 20D10 binding to HEK293T-CD27L is 0.82nM, which does not bind to HEK293T. 50 The results showed that both antibodies 2C9 and 20D10 had binding activity with HEK293T-CD27L cells, but had no binding activity with negative control HEK293T cells. This indicates that the antibodies screened by the present invention have high specificity and can avoid nonspecific binding to normal cells.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fully human anti-CD27L antibody or antibody fragment, characterized in that: comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the three complementary determining regions CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown in SEQ ID NOs.1-3, respectively, and the amino acid sequences of the three complementary determining regions CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NOs.5, 7 and 9, respectively; or The amino acid sequences of the three complementary determining regions CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown in SEQ ID NO.1-2 and 4, respectively, and the amino acid sequences of the three complementary determining regions CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NO.6, 8, 10, respectively.

2. The fully human anti-CD27L antibody or antibody fragment according to claim 1, characterized in that: The heavy chain variable region has the amino acid sequence shown in SEQ ID NO.11, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.13; or The heavy chain variable region has the amino acid sequence shown in SEQ ID NO.12, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.

14.

3. The fully human anti-CD27L antibody or antibody fragment according to claim 1, characterized in that: The fully human antibody or antibody fragment has the amino acid sequence shown in SEQ ID NO.29 or SEQ ID NO.

30.

4. A polynucleotide encoding the fully human anti-CD27L antibody or antibody fragment according to any one of claims 1 to 3.

5. The polynucleotide according to claim 4, characterized in that The polynucleotide comprises a gene fragment encoding a heavy chain variable region and a gene fragment encoding a light chain variable region, wherein the gene fragment encoding the heavy chain variable region has a base sequence shown in SEQ ID NOs. 15-17, and the gene fragment encoding the light chain variable region has a gene sequence shown in SEQ ID NOs. 19, 21, and 23; or, The gene fragment encoding the heavy chain variable region has the base sequence shown in SEQ ID NOs.15-16 and 18, and the gene fragment encoding the light chain variable region has the base sequence shown in SEQ ID NOs.20, 22, and 24.

6. The polynucleotide according to claim 4, characterized in that The polynucleotide comprises a gene segment encoding a heavy chain variable region and a gene segment encoding a light chain variable region, wherein the gene segment encoding the heavy chain variable region has a base sequence as shown in SEQ ID NO.25, and the gene segment encoding the light chain variable region has a base sequence as shown in SEQ ID NO.27; or The gene fragment encoding the heavy chain variable region has the base sequence shown in SEQ ID NO.26, and the gene fragment encoding the light chain variable region has the base sequence shown in SEQ ID NO.

28.

7. The polynucleotide according to claim 4, characterized in that The polynucleotide has a base sequence as shown in SEQ ID NO.31 or SEQ ID NO.

32.

8. An expression vector comprising the polynucleotide according to any one of claims 4 to 7.

9. A cell comprising the fully human anti-CD27L antibody or antibody fragment according to any one of claims 1 to 3 or the polynucleotide according to any one of claims 4 to 7.

10. Use of the fully human anti-CD27L antibody or antibody fragment according to any one of claims 1 to 3 in the preparation of a CD27L protein detection kit, an antibody chip, an antibody probe or an anti-tumor therapeutic drug.

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