A fully human anti-CD27L antibody or antibody fragment and its application

Abstract

The present invention relates to 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 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 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 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 of the light chain variable region are shown in SEQ ID NOs. 6, 8, and 10, respectively. The fully human anti-CD27L antibody or antibody fragment of the present invention can cross-bind to CD27L proteins of humans, cynomolgus monkeys, and mice and has blocking activity against CD27L / CD27, providing new possibilities for further exploration and development of new antibodies targeting CD27L 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 applications thereof. Background Art

[0002] CD27L (also known as TNFSF7, CD70, or CD27LG), a member of the Tumor Necrosis Factor Receptor Superfamily, is a widely reported co-stimulatory molecule for T and B cell immunity. The gene encoding human CD27L is located on chromosome 19p13.2-13.3. The mature protein is approximately 50 kDa in size and is a type II transmembrane glycosylated protein that exists as a trimer. The full-length CD27L protein consists of 193 amino acids and includes an extracellular domain (ECD), a transmembrane region (TM), an intracellular domain (ICD), and two N-terminal glycosylation sequences. The first and third, and second and fourth cysteines in the ECD form disulfide bonds, respectively, to maintain a specific spatial conformation. CD27L has been widely used in animal disease models, but there are significant differences between humans and mice, with only approximately 56% similarity between the two.

[0003] CD27L is primarily expressed on specific immune cells, including dendritic cells, activated lymphocytes, tonsils, T cells in the skin and intestine, and antigen-presenting cells in the intestinal lamina propria. CD27L expression on lymphocytes is extremely transient, primarily present on activated T, B, and NK cells. For example, IFN-γ-producing T cells are essentially CD27L+, as are antigen-activated B cells that secrete immunoglobulins. This highly restricted and temporally regulated expression pattern is carefully mediated by costimulatory signals, cell activation signals, antigen stimulation, and cytokines, precisely and tightly regulating the timing of CD27L's biological functions. Under pathological conditions, CD27L is highly expressed in a variety of tumor tissues, including various lymphoid malignancies, pancreatic cancer, and glioblastoma. In contrast, in normal tissues, CD27L expression is restricted to activated immune cells and at low levels, making it an ideal target for immunotherapy.

[0004] CD27L is known to bind to its sole receptor, CD27, a member of the tumor necrosis factor receptor superfamily (TNFR). It is a homodimeric transmembrane glycoprotein composed of two monomers with a molecular weight of approximately 55 kDa linked by a disulfide bond. Studies on CD27L-CD27 signaling in tumors have shown that this signaling can induce malignant cell proliferation. CD27L-CD27 co-stimulation can protect Treg cells developing in the thymus from death, increase Treg numbers in the spleen and lymph nodes of tumor-bearing mice, and promote T cell differentiation. Absence of CD27L-CD27 signaling can inhibit the development of collateral circulation in a mouse model of hindlimb ischemia. Furthermore, studies have shown that CD27L-CD27 co-stimulation is involved in CD4+ T cell-mediated autoimmune diseases. Therefore, the CD27L-CD27 interaction is crucial for regulating cellular immune responses. In addition, CD27L acts as a B cell co-stimulatory molecule. When in contact with B cells, it interacts with CD27 to promote B cell differentiation into plasma cells, thereby increasing the production of immunoglobulins and enhancing the toxic effects of activated natural killer cells. Numerous lines of evidence indicate that CD27L plays an important role in different types of tumors (such as lymphomas and solid tumors) and autoimmune diseases (such as lupus erythematosus). In some tumor cells, overexpression of CD27L can help tumors evade the immune system's attack.

[0005] In summary, based on the discovery of these tumor-promoting and immunosuppressive mechanisms of CD27L-CD27, and the fact 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. The main pathways are as follows: (1) Interfering with the signaling between CD27L and CD27, thereby weakening the activation and proliferation of T cells; (2) Blocking the CD27L / CD27 signaling pathway, reducing the excessive activation of T cells; (3) Inducing antibody-dependent cell-mediated cytotoxicity (ADCC); (4) Antibody-mediated interference with intracellular signaling, which can affect the survival, proliferation and migration of tumor cells, thereby inhibiting tumor growth and spread.

[0006] Currently, a variety of CD27L-targeting therapies have made progress in preclinical and clinical research, such as AbbVie's CD27L monoclonal antibody ABBV181, currently in clinical trials. By binding to CD27L, ABBV181 blocks the interaction between CD27L and CD27, inhibiting tumor cell proliferation and survival. Drugs such as cusatuzumab (ARGX-110), which bind to the Fc receptor (FcγRIII) and activate immune effector cells and block the target protein through antibody-dependent cellular cytotoxicity (ADCC), have been shown to effectively eliminate leukemic stem cells (LSCs) in AML patients receiving hypomethylating agents, while also blocking the differentiation of AML blasts and stem / progenitor cells induced by the CD27L-CD27 pathway. Furthermore, several CAR-T cell therapies targeting CD27L are being evaluated in Phase I / II clinical trials in patients with hematologic malignancies and solid malignancies.

[0007] While CD27L shows great potential in therapy and diagnosis, it also faces several challenges. For example, it's necessary to ensure high antibody specificity for CD27L and avoid nonspecific binding to normal cells. Furthermore, most current CD27L antibodies cannot cross-bind to CD27L proteins from different species. This necessitates the development of separate antibodies for in vivo activity studies and safety evaluations, resulting in high research costs and the inability to conduct comparative immunological studies across species. Therefore, further exploration and development of new CD27L-targeting antibodies will open new possibilities for clinical research and application, have positive implications for improving the effectiveness and safety of cancer treatment, and provide patients with more treatment options. Summary of the Invention

[0008] (1) Technical issues to be solved

[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 macaques, and mice and has blocking activity against CD27L / CD27, and its use. The antibody can specifically cross-bind to the CD27L proteins of humans, cynomolgus macaques, 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] (2) 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 complementarity 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 complementarity 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 aforementioned fully human anti-CD27L antibody or antibody fragment.

[0016] According to a preferred embodiment of the present invention, the polynucleotide includes a gene segment encoding a heavy chain variable region and a gene segment encoding a light chain variable region, the gene segment encoding the heavy chain variable region has a base sequence shown in SEQ ID NOs. 15-17, and the gene segment encoding the light chain variable region has a gene sequence shown in SEQ ID NOs. 19, 21, and 23; or, the gene segment encoding the heavy chain variable region has a base sequence shown in SEQ ID NOs. 15-16 and 18, and the gene segment encoding the light chain variable region has a base sequence shown in SEQ ID NOs. 20, 22, and 24.

[0017] According to a preferred embodiment of the present invention, the polynucleotide includes a gene segment encoding a heavy chain variable region and a gene segment encoding a light chain variable region, the gene segment encoding the heavy chain variable region has the base sequence shown in SEQ ID NO.25, and the gene segment encoding the light chain variable region has the base sequence shown in SEQ ID NO.27; alternatively, the gene segment encoding the heavy chain variable region has the base sequence shown in SEQ ID NO.26, and the gene segment 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 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 maintains antigen binding ability. The fully human antibody against CD27L is a complete immunoglobulin molecule, and an antibody fragment usually refers to a part of the antibody, which only contains the region that can bind to the antigen, and can be a fragment separated from the complete antibody by enzymatic hydrolysis or other methods. In some cases, it may be advantageous to use antibody fragments instead of whole antibodies, for example, to reduce nonspecific binding or improve tissue penetration.

[0022] (3) 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 (human, crab-eating macaque and mouse), which is conducive to subsequent in vivo activity studies and safety evaluation analysis in mouse or crab-eating macaque tumor models, improves the flexibility and efficiency of research experiments, and is of great significance to accelerating the process of new drug development. 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 IC values ​​and blocking CD27L / CD27 50 The values ​​are all as low as pM level, so the antibodies or antibody fragments provided by the present invention have 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 to human CD27L screened by yeast display of the fully human single-chain antibody library and FACS detection in Example 2 is shown.

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

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

[0028] Figure 5 These are the results of ELISA testing of the activity of two anti-CD27L antibodies in blocking CD27L / CD27 interaction in Example 5.

[0029] Figure 6 These are the results of flow cytometry analysis of the binding activity of two anti-CD27L antibodies to cells expressing CD27L 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 methods in conjunction with the accompanying drawings.

[0031] The present invention discloses an anti-CD27L antibody and its application. The implementation methods thereof will be described in detail below with reference to specific examples. To facilitate understanding of the present invention, some terms will first be defined.

[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 and 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 antibodies are 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 portion of an antibody molecule responsible for recognizing and binding to a specific antigen. It is a key region in the antibody's ability to exert specific immune function. Within the variable region are three hypervariable regions (CDRs), also known as hypervariable regions or complementarity-determining regions. These regions vary widely in amino acid sequence and are responsible for direct antigen binding. These regions play a decisive role in the antibody's antigen-binding specificity, giving the variable region of an antibody a unique affinity and specificity for a particular antigen.

[0035] The term "constant region" refers to the portion of an antibody molecule that doesn't participate in antigen recognition but determines the antibody's type and function. This constant region is relatively conserved across different antibody classes and plays a variety of important immune functions. Different antibody classes (IgA, IgD, IgE, IgG, IgM) have different heavy chain constant regions, which determine the antibody's function and role in the immune response.

[0036] The term "single-chain antibody," or single-chain variable fragment (scFv), is an antibody fragment composed of the variable regions of an antibody (including the variable regions of the heavy and light chains, abbreviated 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, they have a smaller molecular weight and a simpler structure. The short peptide connecting the two variable regions of the antibody is typically a flexible short peptide (such as one containing multiple repeats of a glycine-serine sequence), which enables it to fold correctly and form an antigen-binding site.

[0037] The term "fully human antibody" refers to antibodies that are entirely encoded by human genes and are structurally and functionally identical to antibodies produced naturally by the human body. These antibodies lack any non-human components in their structure and are therefore less likely to induce an immune response in humans.

[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, comprising: slowly adding normal human blood stored in a special anticoagulant for cell separation to a human peripheral blood lymphocyte separation fluid, so that the separation fluid and the normal human blood maintain a clear stratification. Place a 15mL centrifuge tube containing the blood and separation fluid 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 circular milky white lymphocyte layer, the third layer is the transparent separation fluid layer, and the fourth layer is the red blood cell layer. Carefully aspirate the circular 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 washing solution (or 1× PBS) and transfer to a 1.5 mL EP tube (sterile and enzyme-free). Take a small amount for cell counting and centrifuge the remaining sample at 250 g for 10 min. Discard the supernatant and 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 describes the screening process for antibodies that specifically bind to CD27L. Specifically, a fully human single-chain antibody library is displayed in yeast to screen and identify antibodies or antigen-binding fragments (or antibody fragments) that specifically bind to CD27L. The detailed process of this example is as follows:

[0045] First, PCR primers were used to introduce BamH I and EcoR I restriction sites at the 5' and 3' ends of the library fragment obtained in Example 1. Using enzyme digestion and ligation, the library fragment was inserted into the pYD1-HA-Myc vector and transformed into the EBY100 yeast strain. Positively transformed strains were screened using tryptophan-deficient medium to obtain the yeast-displayed YSD-huscFv Lib library.

[0046] Before magnetic bead enrichment, the library glycerol stock was inoculated into YNB-CAA-glucose Medium (YCGlu) and cultured at 30°C, 160 rpm for 24 h (to an OD of 2-3). 50 OD of activated bacterial suspension was collected by centrifugation, and the precipitate was resuspended in 100 mL of YCGal (YNB-CAA-galactose Medium) (1× double-antibody) and cultured at 20°C, 160 rpm for 24-48 h. Biotinylated CD27L (b-CD27L) protein was co-incubated with SA magnetic beads, followed by the addition of induced YSD-huscFv Lib and incubation at 4°C for 1 h. The supernatant was discarded, and the beads were washed three times with PBST-1% BSA. The beads were then transferred to 20 mL of YCGlu and cultured for 24-48 h. After incubation, 10 OD of bacterial suspension was transferred to a 1.5 mL EP tube, 15% glycerol was added, and cryopreservation was performed.

[0047] After two rounds of magnetic bead enrichment, the yeast display library was activated and induced as described above. First, biotinylated human CD27L and human CD27 were incubated with excess fluorescently labeled proteins SA-650 and SA-PE, respectively, for 1 hour to produce CD27L-650 and CD27-PE. A 0.2 OD / tube (1 mL volume) suspension of induced yeast cells was centrifuged for 5 minutes, the supernatant discarded, and the pellet washed once with 800 μL of FACS buffer (1× PBS including 1% FBS). The pellet was centrifuged at 900 × g for 5 minutes, and the supernatant discarded. The pellet was resuspended to 200 μL and mixed with appropriate amounts of CD27L-650 and anti-Myc tag mouse monoclonal antibody. The cells were incubated for 1 hour, washed, and then incubated with CD27-PE and goat anti-mouse IgG pAb-488 for a total of 1 hour. Finally, the cells were washed, resuspended, and transferred to a tube for sorting on a flow cytometer. 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. A sufficient number of cells were sorted 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 YCGlu plates for culture. After single clones grew, several single clones were picked for activation induction and incubated in a similar manner as described above (only the target human CD27L binding signal was detected, that is, a higher 650 fluorescence signal). After the incubation was completed, the cells were tested on the instrument and detected by FACS (such as Figure 2 (as shown) 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 complete human IgG1 antibodies using DNA recombination technology to study their functions. The detailed procedures of this example are as follows:

[0051] First, the VH and VL of the two antibodies were amplified using primers and fused with human IgG1CH and CL-Kappa to construct expression vectors pcDNA3.1-2C9H, pcDNA3.1-2C9L, pcDNA3.1-20D10H, and pcDNA3.1-20D10L, respectively. Two days before transfection, Expi293F cells (Gibco, A14527) were seeded into 100 mL of culture medium at a seeding density of 4-5×10 5 cell / mL, and culture in a constant temperature shaker at 37°C, 110 rpm, and 8% CO2. On the day of transfection, the cell density was controlled at 1.5-2×10 6 cell / mL, add 50 μg of the expression plasmids of Light Chain (light chain plasmid pcDNA3.1-2C9L, pcDNA3.1-20D10L) and Heavy Chain (heavy chain plasmid pcDNA3.1-2C9H, pcDNA3.1-20D10H) to the transfection reagent, then add the mixture to 100 mL of cell culture and culture 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 fully human anti-CD27L 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 of human IgG1 complete antibody and their numbering

[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 intact antibodies constructed in Example 3 were serially diluted to detect their binding activity to CD27L proteins from different species. First, Streptavidin (SA, 2 μg / mL) was coated on a 96-well MediSorp ELISA plate. After blocking with PBS containing 2% BSA (w / v) at 37°C for 2 h, biotinylated human CD27L protein (hu-CD27L), cynomolgus monkey CD27L protein (cyno-CD27L), mouse CD27L protein (mouse CD27L), and IgG1 antibody were captured. Trimer, referred to as "mu-CD27L", was added at 1 μg / mL each (i.e., 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). After capture at 37°C for 30 minutes, the plates were washed with PBST (containing 0.05% Tween20). After washing, a series of gradient concentrations of anti-CD27L antibodies were added (starting at 20 nM, 3-fold dilution, 7+1 gradient). After incubation at 37°C for 1 hour, the plates were washed 3 times with PBST, and then diluted Goat Anti-Human F(ab')2pAb-HRP (Jackson; Cat.: 109-035-006) was added at a dilution of 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 450 nm 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 to human CD27L (hu-CD27L), cynomolgus monkey CD27L (cyno-CD27L) and mouse CD27L (mu-CD27L). 50The EC value of antibody 2C9 for 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 for cynomolgus monkey CD27L protein is 0.49 nM. 50 At 0.72nM, antibody 20D10 binds to mouse CD27LEC 50 EC is 1.56nM. 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] To verify whether the two anti-CD27L antibodies block the binding of CD27L to CD27, a competitive ELISA test was performed in this example. Human CD27L protein (2 μg / mL) diluted in PBS was added to a 96-well ELISA plate and incubated at 4°C overnight (or 37°C for 2 hours). At this time, the CD27L protein was fixed to the 96-well plate. The plate was then incubated with PBS containing 2% BSA at 37°C for 2 hours. After incubation, the plate was washed with PBST and different concentrations of antibodies were added. At the same time, human CD27 protein (fused with a Flag tag) was added. The plate with antibodies and CD27 protein was incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST. After washing, a 1:8000 diluted mouse anti-Flag mAb was added and incubated 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 hour. Finally, after washing with PBST and PBS three times each, 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 Figure 2, 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 in 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] To verify the cell binding activity of the two anti-CD27L antibodies, this example used a lentiviral system to construct a HEK293T cell line that highly expresses human CD27L (HEK293T-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 dilution gradients. Add the two diluted antibodies 2C9 and 20D10 to a V-bottom 96-well plate (100μL / well) and incubate at 4°C for 1 hour. 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), and incubate at 4°C for 1 hour. After incubation, wash 3 times with FACS buffer, resuspend to 200μL / well, and load onto 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 for antibody 20D10 binding to HEK293T-CD27L is 0.82 nM, indicating no binding to HEK293T. 50 The concentration of the antibodies 2C9 and 20D10 was 1.17 nM, indicating no binding to HEK293T cells. The results showed that both antibodies, 2C9 and 20D10, had binding activity to HEK293T-CD27L cells, but no binding activity to negative control HEK293T cells. This demonstrates 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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 an antigen-binding fragment thereof, characterized in that: comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the three complementarity 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 complementarity 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 NOs. 1-2 and 4, respectively. 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.

2. The fully human anti-CD27L antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof according to claim 1, characterized in that: The fully human antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof according to any one of claims 1 to 3 .

5. 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 shown in SEQ ID NOs. 15-17, and the gene segment 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, wherein 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 segment encoding the heavy chain variable region has the base sequence shown in SEQ ID NO.26, and the gene segment encoding the light chain variable region has the base sequence shown in SEQ ID NO.

28.

7. The polynucleotide according to claim 4, wherein 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a CD27L protein detection kit, an antibody chip or an antibody probe.

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