A fully human single heavy chain anti-pd-l1 antibody and application thereof

Abstract

The application discloses a kind of full human source single heavy chain anti-PD-L1 antibodies and its application, it is related to PD-L1 antibody, in particular to full human source single heavy chain anti-PD-L1 antibody.The full human source single heavy chain anti-PD-L1 antibody provided in the application greatly reduces antibody molecular weight, enhances solid tumor tissue penetration ability;Significantly improve the endocytosis efficiency of antibody, so that it has the ability of high-efficiency mediated PD-L1 internalization;On the premise of maintaining strong blocking activity, realize "block+ endocytosis" dual mechanism.

Description

Technical Field

[0001] This invention relates to PD-L1 antibodies, and more particularly to fully human single-chain anti-PD-L1 antibodies. Background Technology

[0002] HLX20 is one of the most representative fully human anti-PD-L1 antibodies currently available, and its activity in blocking PD-1 / PD-L1 interaction has been verified. However, this technical approach has the following drawbacks and limitations: 1. The antibody structure is a traditional IgG1 type: HLX20 adopts a four-chain IgG structure (containing heavy and light chains), with a molecular weight of about 145kDa. Its complex structure limits its penetration ability in solid tumor tissues, making it difficult to penetrate deep into the tumor and fully contact the target cells.

[0003] 2. Low internalization efficiency: HLX20 and its ADC derivative HLX43 have low internalization efficiency (internalization rate <15% within 4 hours). When used as an antibody-drug conjugate (ADC) carrier, it is difficult to achieve efficient internalization delivery, which limits its application potential in the fields of ADC, immunostimulatory antibody-drug conjugates (ISAC).

[0004] 3. Limited flexibility in engineering modification: The Y-shaped configuration of traditional IgG structures has steric hindrance and design limitations when constructing derivatives such as multispecific antibodies and multivalent antibodies, and the degree of freedom of modification is lower than that of single-domain antibodies. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a fully human single-chain anti-PD-L1 antibody and its applications. The fully human single-chain anti-PD-L1 antibody provided by this invention significantly reduces the antibody molecular weight, enhancing its penetration ability into solid tumor tissues; it significantly improves the antibody's internalization efficiency, enabling it to efficiently mediate PD-L1 internalization; and it achieves a dual mechanism of "blocking + internalization" while maintaining strong blocking activity.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides a fully human single-chain anti-PD-L1 antibody or its antigen-binding fragment, comprising: (a) Has the amino acid sequence shown in SEQ ID NO: 1, Or (b) an amino acid sequence derived from (a) in which one or more amino acids have been substituted, deleted, or added and specifically bind to PD-L1.

[0007] In one embodiment, the antibody or antigen-binding fragment specifically binds to human PD-L1.

[0008] In another aspect, the present invention provides a method for generating the above-mentioned antibody or its antigen-binding fragment, comprising: Animals were immunized with the PD-L1 antigen, and the fully human single-chain anti-PD-L1 antibody was obtained by screening.

[0009] In one embodiment, the amino acid sequence derived from (a) is shown in SEQ ID NO: 2 to 20.

[0010] In another aspect, the present invention provides a nucleic acid encoding the aforementioned fully human single-chain anti-PD-L1 antibody or its antigen-binding fragment.

[0011] In one embodiment, the nucleic acid is cDNA.

[0012] In another aspect, the present invention also provides a carrier comprising the aforementioned nucleic acid.

[0013] In another aspect, the present invention provides a cell that expresses the aforementioned nucleic acid and / or vector.

[0014] In another aspect, the present invention provides a method for generating the above-mentioned antibody or its antigen-binding fragment, comprising: The cells were cultured under conditions sufficient to induce them to produce antibodies or antigen-binding fragments; and Collect antibodies or antigen-binding fragments produced by the cells.

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) A therapeutically effective amount of the above-mentioned fully human single-chain anti-PD-L1 antibody or its antigen-binding fragment, nucleic acid, vector and / or cells; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0016] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0017] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0018] In one embodiment, the pharmaceutical product is a vial or box.

[0019] In another aspect, the present invention also provides the use of the above-mentioned fully human single-chain anti-PD-L1 antibody or its antigen-binding fragment, nucleic acid, vector, cell, pharmaceutical composition, pharmaceutical preparation and / or pharmaceutical product in the preparation of drugs for the prevention and / or treatment of cancer.

[0020] In one embodiment, the cancer includes PD-L1-mediated cancer.

[0021] In one embodiment, the PD-L1-mediated cancers include lung cancer (particularly non-small cell lung cancer), urothelial carcinoma / bladder cancer, renal cell carcinoma, breast cancer (including triple-negative breast cancer), melanoma, head and neck squamous cell carcinoma, gastric / gastroesophageal cancer, colon cancer, hepatocellular carcinoma (liver cancer), ovarian cancer, cervical cancer, pancreatic cancer, esophageal cancer, Merkel cell carcinoma, Hodgkin lymphoma, thyroid cancer, glioblastoma, endometrial cancer, and / or soft tissue sarcoma.

[0022] In one embodiment, the PD-L1-mediated cancers include colon cancer.

[0023] In one embodiment, the colon cancer includes colonic adenocarcinoma.

[0024] In another aspect, the present invention also provides the application of the above-mentioned fully human single-chain anti-PD-L1 antibody or its antigen-binding fragment as a preparation of PD-L1 detection products.

[0025] In one embodiment, the testing product includes testing reagents, test strips, test strips, or test kits.

[0026] This invention relates only to detection for non-diagnostic purposes.

[0027] (III) Beneficial Effects This invention provides a fully human single-chain anti-PD-L1 antibody and its applications. Compared with existing technologies, it has the following advantages: 1. Fully human single-chain structure: The antibody is entirely derived from the human VH gene family, with no camel or mouse-derived sequences, resulting in extremely low immunogenicity risk. Its molecular weight is only about 15 kDa, approximately 1 / 10 of that of traditional IgG antibodies.

[0028] 2. Its blocking activity is comparable to HLX20, and its endocytosis efficiency is significantly better than that of HLX20.

[0029] 3. It has both blocking and endocytosis mechanisms: Compared with traditional IgG antibodies, the antibody of this invention can not only block the immune checkpoint pathway, but also efficiently induce PD-L1 internalization and surface downregulation, which is expected to enhance the T cell reactivation effect.

[0030] 4. Flexible engineering modification: The single-chain structure is the smallest antigen-binding unit, which is convenient for constructing various derivatives such as multivalent antibodies, bispecific antibodies, ADCs, and ISACs. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a fluorescence intensity graph of the binding activity of the anti-PD-L1 antibody to PD-L1; Figure 2 This is a fluorescence intensity graph of the anti-PD-L1 antibody's blocking activity against PD-1 / PD-L1 binding; Figure 3 This is a fluorescence intensity graph of the endocytic activity of cancer cells against PD-L1 antibody. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Terms and Definitions As used herein, the term "antibody" generally refers to an antibody that recognizes one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854 4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases.

[0035] As used in this article, the terms “anti-PD-L1 antibody” and “PD-L1 antibody” refer to antibodies that can bind to PD-L1.

[0036] As used herein, the term “antigen-binding fragment” is equivalent to “antibody fragment” or “antigen-binding antibody fragment” and can include a portion of a complete antibody, generally a binding region or variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab’, Fab’-SH, F(ab’)2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol (“PEGylated”) (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab’)2-PEG, or Fab’-PEG) (“PEG” stands for polyethylene glycol).

[0037] As used herein, the term "humanized antibody" refers to an antibody encoded by an endogenous nucleic acid present in humans (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, the human antibody is collected from humans or generated in human cell cultures (e.g., human hybridoma cells). In some embodiments, the human antibody is generated in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, the human antibody is generated in bacterial or yeast cells. In some embodiments, the human antibody is generated in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0038] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that is capable of binding specifically to an antigen.

[0039] As used in this article, when referring to antibodies, the phrases "specifically binds to" and "specifically binds to..." mean that because the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule, the antibody preferentially interacts with its target molecule relative to other molecules; in other words, the reagent recognizes and binds to molecules containing a specific structure, rather than all molecules in general. Antibodies that specifically bind to a target molecule can be called target-specific antibodies.

[0040] As used herein, the terms "vector" and "recombinant expression vector" are used interchangeably, referring to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0041] Methods well known to those skilled in the art can be used to construct expression vectors containing the PD-L1 antibody coding sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0042] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0043] Vectors containing the appropriate DNA sequence and suitable promoters or control sequences can be used to transform suitable host cells to enable them to express proteins or peptides. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include: *Escherichia coli*, *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; and animal cells.

[0044] The polynucleotides disclosed in this invention, when expressed in higher eukaryotic cells, will enhance transcription when an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0045] As used herein, the term "pharmaceutical composition" refers to a composition comprising a PD-L1 antibody or an antigen-binding fragment thereof formulated with one or more pharmaceutically acceptable carriers.

[0046] The formulation of the pharmaceutical composition can be tailored to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0047] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0048] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0049] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0050] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0051] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0052] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0053] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0055] Example 1: Preparation of anti-PD-L1 antibody: This invention employs a combination of immunization with fully human single-chain antibody transgenic mice and phage display library technology for screening. First, fully human single-chain antibody transgenic mice (carrying the human VH locus and capable of producing fully human heavy-chain antibodies, without camel-derived sequences) are immunized with recombinant human PD-L1 protein. After immunization, spleens are harvested, and total RNA from B cells is extracted, reverse transcribed into cDNA, and the single-chain variable region (VH) gene fragment is amplified to construct a phage display library. Through multiple rounds of biological panning (using human PD-L1 as the target antigen), and simultaneous screening for blocking activity and endocytosis efficiency, candidate fully human anti-PD-L1 single-chain antibodies with both strong blocking activity and ultra-high endocytosis efficiency are ultimately obtained.

[0056] 1. Antibody source: PD-L1 antigen immunization was performed using the HUGO-Nano fully human single heavy chain antibody transgenic mouse platform. This mouse carries the human VH locus and has the endogenous heavy chain CH1 domain deleted, which can directly produce a fully human single heavy chain antibody that does not contain the light chain and consists only of the variable region of the heavy chain, thus eliminating the need for camel-derived or mouse-derived framework sequences at the source.

[0057] 2. Immunization and spleen acquisition: 50 μg of human PD-L1 FC fusion recombinant protein (Kaikai Biotechnology PDL-HM210) was emulsified with a fully Freund's adjuvant and then injected subcutaneously into 6-8 week old HUGO-Nano mice.

[0058] After three booster immunizations (each with a protein immunization dose of 25ug, emulsified with incomplete Freund's adjuvant), the spleens of mice were harvested.

[0059] 3. Construction and screening of phage display library: Total RNA was extracted from spleen cells, reverse transcribed and amplified to expand the single-strand variable region gene fragment, cloned into a phage vector, and an immune phage display library was constructed.

[0060] Three rounds of biological panning were performed using human PD-L1 as the target antigen to enrich specific phage clones.

[0061] 4. Functional screening: Single clones were selected for phage ELISA to identify binding activity. After positive clones expressed antibodies, clones that blocked PD-1 / PD-L1 binding were further screened by competitive ELISA, and clones with the highest endocytosis efficiency were screened by flow cytometry. Finally, candidate antibodies with both strong blocking activity and ultra-high endocytosis capacity were obtained.

[0062] The amino acid sequences of the candidate antibodies are as follows: P5765 (SEQ ID NO: 1): QVQLQESGPGLVKPSGTLSLTCAVSGDSISSSNWWTWVRQPPGKGLEWIGEIYHTGTTNYNPSLKSRVTISVDKSKNQFSLKVTSVTAADTAVYYCARGGISMVRRLIPDALDIWGQGKMVTVSS; P6572 (SEQ ID NO: 2): EVQLVESGGGLVQPGGSLRLSCAVSGFTFSNYAMSWVRQAPGKGPEWVSVVSGSGTSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDIVAALSSDWADYWGQGTLVTVSS; P5813 (SEQ ID NO: 3): EVQLVESGGGLVQPGGSLRLSCAVSGFTFSSYAMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDIVAALSSDWADYWGQGTLVTVSS; P5800 (SEQ ID NO: 4): EVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYDMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWADYWGQGTLVTVSS; P5808(SEQ ID NO:5): EVQLVESGGDMVQPGGSLRLSCTVSGFTFKIYTMSWVRQAPGKGPEWVSAISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDIVAALSSDWADYWGQGTLVTVSS; P5793(SEQ ID NO:6): EVQLVESGGGLVQPGGSLRLSCAVSGFTFSSYAMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWADYWGQGTLVTVSS; P5828(SEQ ID NO:7): EVQLVESGGGLVQPGGSLRLSCVASGFRFSSYAMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWADYWGQGTLVTVSS; P5775(SEQ ID NO:8): EVQLVESGGGLVQRGGSLRLSCAVSGFTFRDYAMSWVRQAPGKGPEWVSVITGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDIVAALSSDWADYWGQGTLVTVSS; P5780(SEQ ID NO:9): EVQLVESGGGLVQPGGSLRLSCAVSGFTFRNYAMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCAKEEDIVAALSSNWGDYWGQGTLVTVSS; P5830(SEQ ID NO:10): EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWADYWGQGTLVTVSS; P6491(SEQ ID NO:11): EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGPEWVSVISGGGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDIVAALSSAWADYWGQGTLVTVSS; P6489(SEQ ID NO:12): EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWADYWGQGTLVTVSS; P6493(SEQ ID NO:13): EVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYDMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCAKEEDLVAALSSDWSNYWGQGTLVTVSS; P6496(SEQ ID NO:14): EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWADYWGQGTLVTVSS; P6611(SEQ ID NO:15): EVQLVESGGGLVQPGGSLRLSCAVSGFTFRDYAMSWVRQAPGKGPEWVSVISGSGGSPYYSDSVKGRFTISRDNSKNMLYLQMNNLRAEDTAVYYCAKEEDIVAALSSDWADYWGQGTLVTVSS; P5798 (SEQ ID NO: 16): EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWADYWGQGTLVTVSS; P5801 (SEQ ID NO: 17): EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWAGYWGQGTLVTVSS; P5817 (SEQ ID NO: 18): EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDIVAALSSDWADYWGQGTLVTVSS; P5819 (SEQ ID NO: 19): QVQLKESGGGLVQPGGSLRLSCAVSGFTFRNYAMSWVRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWADYWGQGTLVTVSS; P5836 (SEQ ID NO: 20): EVQLVESGGGLVQPGGSLRLSCAASGFTFRDYDMSWIRQAPGKGPEWVSVISGSGGSTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEDLVAALSSDWADYWGQGTLVTVSS.

[0063] Example 2: Binding activity of anti-PD-L1 antibody in RKO cell line: RKO cells (human colon adenocarcinoma cells, naturally expressing human PD-L1) were collected and the cell density was adjusted to 2 × 10⁻⁶ cells / year. 5 / tube, incubate with serially diluted (starting from 500 nM, 5-fold serial dilution) of the antibody of this invention or control antibody HLX20 at 4°C in the dark for 30 min. After washing, add PE-labeled anti-human IgG secondary antibody and continue incubation for 30 min. After washing, detect by flow cytometry and record the mean fluorescence intensity (MFI).

[0064] EC was calculated by fitting a four-parameter curve with antibody concentration as the x-axis and MFI as the y-axis. 50 value.

[0065] Table 1. Fluorescence intensity of anti-PD-L1 antibody on PD-L1 binding activity The results are as follows Figure 1 As shown in Table 1, the binding of the antibody P5765 of this invention to RKO cells exhibits a typical concentration-dependent saturation curve, with a half-maximum binding concentration (EC50) of 1 / 3. 50 The maximum binding signal E is 0.13 nM. max The values ​​were 212 and 521. EC50 of the control antibody HLX20 was... 50 The maximum binding signal E is 0.20 nM. max Approximately 327,967.

[0066] In comparison, the EC50 of P5765 is lower than that of HLX20, indicating that the antibody of this invention has a higher sensitivity to binding to the native conformation of PD-L1 on the cell surface. The maximum binding signal of HLX20 is about 1.5 times that of P5765. Although the Fc sequences of the two are the same and the secondary antibody recognition conditions are consistent, the difference in Emax may be due to the difference in spatial conformation of the antibody molecule after binding to PD-L1 on the cell surface, resulting in different accessibility of the secondary antibody to the Fc region.

[0067] Example 3: Blocking activity of anti-PD-L1 antibody against PD-1 / PD-L1 binding: The ability of antibodies to competitively block the binding of PD-1 to PD-L1 was detected using enzyme-linked immunosorbent assay (ELISA).

[0068] Recombinant human PD-L1-His protein (KaiKa Bio PDL-HM110) was diluted with carbonate buffer (pH 9.6) to a final concentration of 1.0 μg / mL and added to a 96-well high-binding ELISA plate (100 μL / well). The plate was then coated overnight at 4°C. After washing the plate the following day, it was blocked with PBS buffer containing 1% BSA at 37°C for 2 h.

[0069] The antibody and control antibody HLX20 of this invention were serially diluted starting from 100 nM. The specific dilution sequences corresponded to the following dilution factors: 1-fold, 10-fold, 100-fold, 300-fold, 900-fold, 2700-fold, 8100-fold, and 81000-fold (i.e., the first two gradients were 10-fold tandem dilutions, the next five gradients were 3-fold tandem dilutions, and the last gradient was a 10-fold dilution).

[0070] Each serially diluted antibody (50 μL / well) was mixed with a fixed concentration of Biotin-labeled human PD-1 protein (50 μL / well, final detection concentration 0.25 μg / mL), and then added to a blocked ELISA plate. The plate was incubated at 37°C for 1 h. After washing to remove unbound components, a 1:2000 dilution of horseradish peroxidase-labeled streptavidin was added to each well, and the plate was incubated at 37°C for 1 h. After thorough washing, TMB chromogenic substrate was added, and the plate was incubated at room temperature in the dark for 10 min. The reaction was then terminated by adding 2 M H₂SO₄.

[0071] The absorbance at 450 nm was measured using a microplate reader. A four-parameter logistic regression curve was fitted using GraphPad Prism software, with the logarithm of antibody concentration on the x-axis and OD450 value on the y-axis, to calculate the half-maximal inhibitory concentration (IC50). 50 value).

[0072] Table 2. Fluorescence intensity of anti-PD-L1 antibody against PD-1 / PD-L1 binding blocking activity The results are as follows Figure 2 As shown in Table 2, both the antibody P5765 and the control antibody HLX20 of this invention can dose-dependently block the binding of recombinant human PD-1 and PD-L1 proteins, and the blocking curves exhibit typical S-shaped competitive inhibition kinetics. Thanks to the dense 3-fold dilution point design within the core concentration range (around 0.1 nM to 1 nM), the inflection region of the competitive inhibition curve was fitted with extremely precise accuracy. Based on four-parameter curve fitting calculations, the IC50 of the control antibody HLX20... 50 The value is 0.18 nM, and the IC50 of the antibody P5765 of this invention is... 50 The value is 0.30nM. In comparison, the P5765 IC... 50 The value was slightly higher than that of HLX20, indicating that HLX20 had a slightly better competitive ability to bind in vitro than P5765 in the in vitro blocking kinetics of purified recombinant protein.

[0073] Example 4: Detection of endocytic activity of anti-PD-L1 antibody in RKO cell line: Antibody-mediated targeted endocytosis activity was detected using a commercially available antibody endocytosis assay kit (Cat. No. IGG-PZF2001). This kit utilizes a pH-sensitive red fluorescent dye to specifically label the Fc region of human IgG. When the antibody-kit complex is endocytosed into acidic endosomes or lysosomes (pH ≈ 4.5–5.5), its red fluorescence signal is significantly enhanced, while the unendocytosed extracellular complex shows almost no fluorescence in a neutral environment (pH ≈ 7.4), thus specifically and sensitively reflecting endocytosis efficiency.

[0074] The specific procedure is as follows: Reconstitute the lyophilized endocytosis kit reagents into a stock solution using sterile deionized water and allow to stand at room temperature for 30 minutes to completely dissolve. In a 96-well plate, add 25 μL / well of 4× antibody working solution (8 μg / mL) diluted with cell culture medium, followed by 25 μL / well of 4× endocytosis reagent working solution (4 μg / mL) prepared by diluting the stock solution 50 times. Incubate at room temperature for 10 minutes to form a stable antibody-fluorescent reagent detection complex.

[0075] Collect digested RKO cells, adjust the cell density to 2 cells / mL with culture medium, and take 50 μL (i.e., 1 × 10⁻⁶ cells / mL). 5 Add one cell to a well containing the above complex and incubate for endocytosis at 37°C and 5% CO2 for 2 hours (optimization can be achieved within 1-4 hours).

[0076] After incubation, cells were harvested and washed three times with FACS buffer to thoroughly remove unbound and extracellular free components. Finally, cells were resuspended in 200 μL PBS, transferred to flow cytometry tubes, and detected using a flow cytometer (excitation wavelength 643 nm, emission wavelength 660 nm), recording the average fluorescence intensity of the red fluorescence channel.

[0077] Table 3. Fluorescence intensity of cancer cells exhibiting anti-PD-L1 antibody endocytosis activity. The results are as follows Figure 3 As shown in Table 3, both the antibody P5765 and the control antibody HLX20 of this invention can be endocytosed and transported into acidic endosomes / lysosomes after co-incubation with RKO cells, stimulating the release of red fluorescence signals by the pH-sensitive dye in the kit.

[0078] Flow cytometry analysis showed that the mean fluorescence intensity (MFI) of the antibody P5765 group was 54,244, while the MFI of the control antibody HLX20 group was 22,494. The fluorescence signal of cytotoxicity mediated by P5765 was significantly higher than that of HLX20, approximately 2.4 times that of the latter.

[0079] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fully human single-chain anti-PD-L1 antibody, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid, characterized in that, Encoding the fully human single-chain anti-PD-L1 antibody as described in claim 1.

3. A carrier, characterized in that, It includes the nucleic acid described in claim 2.

4. A cell, characterized in that, It comprises the nucleic acid of claim 2 and / or the vector of claim 3.

5. A pharmaceutical composition, characterized in that, include: (1) A therapeutically effective amount of the fully human single-chain anti-PD-L1 antibody as described in claim 1; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

6. A pharmaceutical preparation, characterized in that, Includes the pharmaceutical composition according to claim 5.

7. A pharmaceutical product, characterized in that, Includes the pharmaceutical preparation described in claim 6.

8. The use of the fully human single-chain anti-PD-L1 antibody of claim 1, the pharmaceutical composition of claim 5, the pharmaceutical formulation of claim 6, or the pharmaceutical product of claim 7 as a preparation of a drug for treating PD-L1-mediated lung cancer, urothelial carcinoma, renal cell carcinoma, breast cancer, melanoma, head and neck squamous cell carcinoma, gastric cancer, colon cancer, hepatocellular carcinoma, ovarian cancer, cervical cancer, pancreatic cancer, esophageal cancer, Merkel cell carcinoma, thyroid cancer, glioblastoma, endometrial cancer, and / or soft tissue sarcoma.

9. The application of the fully human single-chain anti-PD-L1 antibody according to claim 1 in the preparation of PD-L1 detection products.

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