A pd-l1 binding molecule and uses thereof

Abstract

The application discloses a PD-L1 binding molecule and application thereof, and relates to the technical field of monoclonal antibodies. The application also discloses a preparation method of the PD-L1 binding molecule, application of the PD-L1 binding molecule, a kit containing the PD-L1 binding molecule, an immunoconjugate and a pharmaceutical composition.

Description

[0001] Priority Statement

[0002] This application claims priority to Chinese Patent Application No. 202111274164.X, filed on October 29, 2021, entitled "A PD-L1 Binding Molecule and Its Application Thereof," the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of monoclonal antibody technology, specifically to a PD-L1 binding molecule and its applications. Background Technology

[0004] Programmed cell death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274), was first discovered in 1999 by Professor Chen Lieping, a Chinese scholar, as B7-H1, the third member of the B7 family. PD-L1 protein is widely expressed on activated T cells, B cells, and macrophages. The interaction between PD-L1 and PD-1 protein on T cells inhibits T cell activation and induces T cell apoptosis, playing a negative regulatory role in the immune response. In the tumor microenvironment, tumor cells and tumor-associated antigen-presenting cells (APCs) highly express PD-L1, and tumor-infiltrating lymphocytes highly express PD-1 under long-term stimulation by tumor antigens. After binding to PD-1, PD-L1 can induce T cell apoptosis, dysfunction, and exhaustion, thereby inhibiting the activation, proliferation, and anti-tumor function of tumor antigen-specific CD8+ T cells, achieving tumor immune escape.

[0005] The successful application of monoclonal antibodies in cancer detection and targeted therapy has revolutionized tumor treatment. However, traditional monoclonal antibodies (150kD) have excessively large molecular weights, making it difficult to penetrate tissues and resulting in low effective concentrations in tumor areas, leading to insufficient therapeutic effects. Traditional antibodies also exhibit high immunogenicity, while modified antibodies struggle to achieve the same affinity. Furthermore, the long development cycle, high production costs, and insufficient stability of fully humanized traditional antibodies limit their clinical application and widespread adoption.

[0006] Nanobodies are currently the smallest antibody molecules, with a molecular weight only 1 / 10 that of ordinary antibodies. In addition to possessing the antigenic reactivity of monoclonal antibodies, nanobodies also have some unique functional characteristics, such as small molecular weight, high stability, good solubility, easy expression, weak immunogenicity, strong penetration, strong targeting, simple humanization, and low preparation cost. They almost perfectly overcome the shortcomings of traditional antibodies, such as long development cycle, low stability, and harsh storage conditions.

[0007] Although some PD-L1-binding nanobodies have been disclosed in the art, there is still a need for nanobodies with superior binding affinity and specificity. Summary of the Invention

[0008] One of the objectives of this invention is to provide an antibody that can specifically bind to PD-L1 and its application.

[0009] Specifically, this application solves the technical problems in this field through the following technical solutions.

[0010] 1. A PD-L1 binding molecule comprising at least one immunoglobulin single variable domain, said at least one immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 selected from any one of (i) to (vi):

[0011] (i) CDR1 as shown in SEQ ID NO:29, CDR2 as shown in SEQ ID NO:30, and CDR3 as shown in SEQ ID NO:31;

[0012] (ii) CDR1 as shown in SEQ ID NO:23, CDR2 as shown in SEQ ID NO:24, and CDR3 as shown in SEQ ID NO:25;

[0013] (iii) CDR1 as shown in SEQ ID NO:26, CDR2 as shown in SEQ ID NO:27, and CDR3 as shown in SEQ ID NO:28;

[0014] (iv) CDR1 as shown in SEQ ID NO:20, CDR2 as shown in SEQ ID NO:21, and CDR3 as shown in SEQ ID NO:22;

[0015] (v) CDR1 as shown in SEQ ID NO:32, CDR2 as shown in SEQ ID NO:33, and CDR3 as shown in SEQ ID NO:34; or

[0016] (vi) CDR1 as shown in SEQ ID NO:35, CDR2 as shown in SEQ ID NO:36, and CDR3 as shown in SEQ ID NO:37.

[0017] 2. As described in Project 1, the PD-L1 binding molecule, wherein the immunoglobulin has a single variable domain, VHH.

[0018] 3. The PD-L1 binding molecule as described in Project 2, wherein the amino acid sequence of the VHH is shown in any one of SEQ ID NO:7, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:9.

[0019] 4. The PD-L1 binding molecule as described in any of items 1 to 3, wherein the PD-L1 binding molecule further comprises an immunoglobulin Fc region;

[0020] Optionally, the C-terminus of the single variable domain of the immunoglobulin is connected to the N-terminus of the Fc region of the immunoglobulin.

[0021] 5. The PD-L1 binding molecule as described in Project 4, wherein the amino acid sequence of the Fc region of the immunoglobulin is shown as amino acids 120-346 of SEQ ID NO:44.

[0022] 6. The PD-L1 binding molecule as described in any of items 1 to 5, wherein the PD-L1 binding molecule comprises an amino acid sequence as shown in any of SEQ ID NO:38-43.

[0023] 7. An isolated polynucleotide encoding the PD-L1 binding molecule described in any one of items 1 to 6.

[0024] 8. An expression vector comprising the polynucleotides described in item 7.

[0025] 9. A host cell comprising the expression vector described in item 8 or the genome thereof incorporating the polynucleotides of item 7.

[0026] 10. A method for preparing the PD-L1 binding molecule of any one of items 1 to 6, comprising culturing the host cell of item 9 under conditions that allow the generation of the PD-L1 binding molecule and recovering and separating the PD-L1 binding molecule.

[0027] 11. A phage displaying nanobodies, wherein the surface of the phage displays any one of the PD-L1 binding molecules described in items 1 to 6.

[0028] 12. A kit for detecting PD-L1, comprising any of the PD-L1 binding molecules described in items 1 to 6 or the phage displaying nanobodies described in item 11.

[0029] 13. The kit as described in Project 12, the kit further comprising a solid-phase carrier in which the PD-L1 binding molecule or the phage displaying nanobodies is immobilized.

[0030] 14. The kit as described in Item 13, wherein the kit further comprises a detectable marker that can be linked to the PD-L1 binding molecule; and / or a PD-L1 standard or a PD-L1 conjugate standard; and / or a substrate corresponding to the detectable marker; and / or an enzyme-linked immunosorbent assay (ELISA) reagent;

[0031] Preferably, the detectable marker is attached to the PD-L1 binding molecule or is present separately in the kit.

[0032] 15. A method for detecting the presence of PD-L1 in a test sample for non-therapeutic purposes, wherein the PD-L1 binding molecule described in any one of items 1 to 6 or the phage displaying nanobody described in item 11 is used as the detection antibody for PD-L1, and the presence of PD-L1 in the test sample is detected by enzyme-linked immunosorbent assay (ELISA).

[0033] 16. The method described in Item 15 specifically includes the following steps:

[0034] The sample to be tested is coated on a solid support, and the presence of PD-L1 is detected by using the PD-L1 binding molecule, which carries or does not carry a detectable marker, or the phage displaying the nanobody, as the detection antibody.

[0035] 17. An immunoconjugate comprising a therapeutic agent and a PD-L1 binding molecule as described in any one of items 1 to 6 conjugated to said therapeutic agent;

[0036] Preferably, the therapeutic agent includes a toxin, a radioactive isotope, a drug, or a cytotoxic agent.

[0037] 18. A bispecific or multispecific antibody comprising any of the PD-L1 binding molecules described in items 1 to 6, and an antibody or antibody fragment having another or more antigen-binding properties functionally linked to the PD-L1 binding molecule.

[0038] 19. A pharmaceutical composition comprising any one of the PD-L1 binding molecules described in items 1 to 6; preferably, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, a carrier, or a diluent.

[0039] 20. Use of any of the PD-L1 binding molecules described in items 1 to 6, the immunoconjugates described in item 17, the bispecific or multispecific antibodies described in item 18, and the pharmaceutical compositions described in item 19 in the preparation of a medicament for treating PD-L1-mediated diseases;

[0040] The PD-L1-mediated diseases are preferably cancers, and more preferably cancers that highly express PD-L1. These cancers include, but are not limited to, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer, with breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, and melanoma being the most preferred.

[0041] 21. A method for diagnosing, treating, preventing, or alleviating PD-L1-related diseases, symptoms, or conditions in a subject, comprising administering to the subject a therapeutically effective amount of any of the PD-L1 binding molecules described in items 1-6 and / or a pharmaceutical composition described in item 19. Attached Figure Description

[0042] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0043] Figure 1 Electrophoresis diagram of total RNA extracted in Example 2.

[0044] Figure 2 Image of the second round of nested PCR electrophoresis in Example 2.

[0045] Figure 3 The flow cytometry results in Example 5 are shown in diagrams. Diagram a shows the flow cytometry results for R1014, R1015, R1016, R1017, R1018, R1019, and R1020; diagram b shows the flow cytometry results for R1021, R1022, R1023, R1024, R1025, R1026, and R1027; diagram c shows the flow cytometry results for R1028; and diagram d shows the flow cytometry results for R1148, R1149, and R1150.

[0046] Figure 4. Binding curve of the fusion protein in Example 6. Wherein, Figure 4a The bonding curve of R1015 is shown; Figure 4b The binding curve of R1016 is shown; Figure 4c The binding curve of R1019 is shown; Figure 4d The binding curve of R1148 is shown; Figure 4e The binding curve of R1149 is shown; Figure 4f The combination curve of R1150 is shown; Figure 4gThe binding curve of R0999 is shown; Figure 4h The binding curve of R0516 is shown; Figure 4i The binding curve of R0323 is shown.

[0047] Figure 5 The diagram shows the binding curves and IC50 values ​​of the fusion protein blocking the expression of human PD-L1 protein and free human PD-1 using detection method (A) in Example 7. Specifically, a shows the binding curves and IC50 values ​​of R1148, R1149, and R1150 blocking the expression of human PD-L1 protein and free human PD-1; b shows the binding curves and IC50 values ​​of R1014, R1015, R1016, R1017, R1018, R1019, and R1027 blocking the expression of human PD-L1 protein and free human PD-1.

[0048] Figure 6 The diagram shows the binding curves and IC50 values ​​of the fusion protein blocking the expression of human PD-L1 protein and free human PD-1 using detection method (B) in Example 7. Specifically, a shows the binding curves and IC50 values ​​of R1148, R1149, and R1150 blocking the expression of human PD-L1 protein and free human PD-1; b shows the binding curves and IC50 values ​​of R1014, R1015, R1016, R1017, R1018, R1019, and R1027 blocking the expression of human PD-L1 protein and free human PD-1.

[0049] Figure 7 The image shows the binding curves and IC50 values ​​of the fusion protein blocking membrane expression of human PD-L1 protein and free human CD80 using detection method (A) in Example 9.

[0050] Figure 8 The image shows the binding curves and IC50 values ​​of the fusion protein blocking membrane expression of human PD-L1 protein and free human CD80 using detection method (B) in Example 9. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0052] As used herein, "nanobody" refers to a single-domain antibody obtained by cloning the variable region (VHH) of a heavy chain antibody lacking the light chain (e.g., derived from camels). It is the smallest functional antigen-binding fragment with a relative molecular mass (Mr) of only about 15,000. Nanobodies are characterized by their small molecular weight, high stability, good solubility, ease of expression, and low immunogenicity. The VHH typically contains three hypervariable regions, called "complementarity-determining regions (CDRs)," namely CDR1, CDR2, and CDR3.

[0053] As used herein, the “immunoglobulin Fc region” or “Fc region” refers to the crystallizable fragment of an immunoglobulin antibody after papain digestion. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0054] As used herein, “detection antibody” refers to an antibody that specifically targets PD-L1 and has a detectable marker.

[0055] As used herein, "detectable markers" refer to markers located on detection antibodies used to determine the presence and amount of PD-L1 in a sample to be tested. Examples include enzymes, fluorescent labels, radionuclides, quantum dots, and colloidal gold. Preferably, the markers are selected from horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

[0056] As used herein, "substrate corresponding to the detectable label" refers to a label catalyzed by the detectable antibody to produce a colorimetric reaction, displaying a recognition signal indicating the binding of the detectable antibody to PD-L1. Examples of such substrates include: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), and ABTS for horseradish peroxidase; p-nitrophenyl phosphate (p-NPP) for alkaline phosphatase; and so on.

[0057] As used herein, the term "amino acid" refers to twenty common, naturally occurring amino acids. These include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0058] 1. Anti-PD-L1 nanobody

[0059] This invention provides a nanobody, which is screened from a camel-derived natural single-domain heavy chain antibody library.

[0060] The nanobody described in this invention can bind to PD-L1 with high affinity and specificity. In one embodiment, the nanobody of this invention has a heavy chain variable region (VHH) as shown in SEQ ID NO:1-19. In a preferred embodiment, the nanobody of this invention has a heavy chain variable region (VHH) as shown in SEQ ID NO:7, 3, 6, 2, 8, and 9. When the sequence of the heavy chain variable region of the nanobody is SEQ ID NO:7, the amino acid sequences of its CDR1, CDR2, and CDR3 are SEQ ID NO:29, 30, and 31, respectively (based on the IMGT numbering method). When the sequence of the heavy chain variable region of the nanobody is SEQ ID NO:3, the amino acid sequences of its CDR1, CDR2, and CDR3 are SEQ ID NO:23, 24, and 25, respectively (based on the IMGT numbering method). When the sequence of the heavy chain variable region of the nanobody is SEQ ID NO:6, the amino acid sequences of its CDR1, CDR2, and CDR3 are SEQ ID NO:26, 27, and 28, respectively (based on the IMGT numbering method). When the sequence of the heavy chain variable region of the nanobody is SEQ ID NO:2, the amino acid sequences of its CDR1, CDR2, and CDR3 are SEQ ID NO:20, 21, and 22, respectively (based on the IMGT numbering method). When the sequence of the heavy chain variable region of the nanobody is SEQ ID NO:8, the amino acid sequences of its CDR1, CDR2, and CDR3 are SEQ ID NO:32, 33, and 34, respectively (based on the IMGT numbering method). When the sequence of the heavy chain variable region of the nanobody is SEQ ID NO:9, the amino acid sequences of its CDR1, CDR2, and CDR3 are SEQ ID NO:35, 36, and 37, respectively (based on the IMGT numbering method). It should be noted that the antibody sequence numbering method is not limited to the IMGT method; other methods can also be used to number antibody sequences, such as the Kabat, Chothia, Martin, and AHo methods. The CDR sequences of the antibody may differ when different numbering methods are used. Unless otherwise specified, the antibodies in this application are numbered using the IMGT method.

[0061] This invention uses the IMGT numbering system to identify CDR areas, but CDR areas identified by other methods are also within the scope of protection of this invention.

[0062] This invention also includes variants, derivatives, and analogs of the nanobodies described herein. As used herein, the terms “variant,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the nanobodies of this invention. The polypeptide variants, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify this polypeptide or the original polypeptide sequence, or a fusion polypeptide). These variants, derivatives, and analogs are within the scope of knowledge of those skilled in the art as defined herein.

[0063] In addition, other amino acid sequences that do not substantially affect the activity, expression level, and stability of the nanobody described in this invention may be added to the amino or carboxyl terminus of the nanobody.

[0064] Preferably, these added amino acid sequences are conducive to expression (e.g., signal peptides), conducive to purification (e.g., 6XHis sequences), or other sequences that can promote the activity, expression level, or stability of the nanobody.

[0065] The present invention also includes DNA molecules encoding the nanobody or its variants or derivatives thereof. These DNA molecules can be entirely synthesized artificially or obtained by PCR amplification.

[0066] To further improve the expression level in host cells, the coding sequence of the nanobody of the present invention can be modified, for example, by using codons preferred by the host cells and eliminating sequences that are detrimental to gene transcription and translation.

[0067] After obtaining the DNA sequence encoding the nanobody of the present invention or its variants or derivatives, it is cloned into a suitable expression vector and then transformed into a suitable host cell. Finally, the transformed host cell is cultured, and the novel nanobody of the present invention is obtained through isolation and purification.

[0068] In some specific embodiments, the nanobody is fused with the Fc region of an immunoglobulin.

[0069] In some specific embodiments, the C-terminus of the nanobody is fused to the N-terminus of the Fc region of an immunoglobulin.

[0070] In some specific embodiments, the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, or IgE.

[0071] In some specific embodiments, the amino acid sequence of the Fc region of the immunoglobulin is shown as amino acids 120-346 of SEQ ID NO:44.

[0072] 2. Polynucleotides

[0073] The present invention provides an isolated polynucleotide encoding the aforementioned nanobody.

[0074] As used herein, the term "polynucleotide" generally refers to RNA or DNA, and polynucleotides can be single-stranded or double-stranded, but are preferably double-stranded DNA. Unless otherwise stated, a particular polynucleotide sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly stated sequences.

[0075] 3. Expression vehicle

[0076] The present invention provides an expression vector comprising the aforementioned polynucleotides.

[0077] As used herein, the term "expression vector" includes plasmids, cloning vectors, viral vectors, etc. Various vectors known in the art can be used. For example, an expression vector can be formed by using a commercially available vector and then operatively linking the nucleotide sequence encoding the nanobody of this invention to an expression regulatory sequence.

[0078] 4. Host cells

[0079] The present invention provides a host cell comprising the aforementioned expression vector or the aforementioned polynucleotides integrated into its genome.

[0080] As used herein, the term "host cell" includes both prokaryotic and eukaryotic cells. Common examples of prokaryotic host cells include *Escherichia coli* and *Bacillus subtilis*. Host cells used for expressing nanobodies include *Escherichia coli*, yeast cells, insect cells, COS cells, and CHO cells. Preferably, the host cell is a eukaryotic cell, and more preferably, a CHO cell.

[0081] After obtaining the transformed host cells, the cells can be cultured under conditions suitable for expressing the nanoantibodies of the present invention, thereby expressing the nanoantibodies; and then the expressed nanoantibodies can be isolated.

[0082] 5. Bacteriophage

[0083] The present invention provides a bacteriophage displaying nanobodies, wherein the surface of the bacteriophage displays the aforementioned nanobodies.

[0084] Phage display technology is used in the construction of nanobody libraries to insert the DNA sequence of foreign proteins or peptides into the appropriate position of the phage coat protein structural gene, so that the foreign gene is expressed along with the expression of the coat protein. At the same time, the foreign protein is displayed on the phage surface as the phage is reassembled.

[0085] 6. Reagent kit

[0086] Based on the nanobody obtained in this invention, this invention provides a kit for detecting PD-L1, which can be used for the detection of PD-L1.

[0087] The kit contains: the nanobody described in this invention or a phage (phage particle) displaying the nanobody. In some specific embodiments, the sample to be tested can be coated on a solid-phase support, and the nanobody of this invention can be used as the detection antibody for detection. The nanobody can be linked to a detectable marker, or can bind to another antibody (anti-antibody) linked to a detectable marker, thereby determining the presence of PD-L1 in the sample to be tested. It should be understood that after obtaining the nanobody of this invention, various methods known in the art can be used to perform PD-L1 detection, all of which are included in this invention.

[0088] After determining the detection antibody used in the kit of the present invention, various markers conventionally available in the art that can bind to the detection antibody for detection can be used as detectable markers. The present invention does not impose any particular limitation on the markers used; any marker that can bind to the detection antibody and, after appropriate processing, can accurately indicate the presence and amount of PD-L1 in the sample to be tested is acceptable. For example, the markers can be selected from (but are not limited to): horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylase. For example, the detection antibody is labeled with horseradish peroxidase (HRP). Antibody labeling methods are well known in the art, for example, HRP-labeled antibodies can be performed using a simple sodium periodate method or a two-step glutaraldehyde method.

[0089] When using some of the enzyme markers shown above, it is also necessary to use substrates that bind to the corresponding enzymes, so that the presence or amount of the markers can be indicated by color development or other methods. Examples of such substrates include: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), and ABTS for horseradish peroxidase; and p-nitrophenyl phosphate (p-NPP) for alkaline phosphatase.

[0090] To eliminate false positives and false negatives, a quality control (control) should be included in the testing process. This quality control can be, for example, a PD-L1 standard. Furthermore, to obtain quantitative results, multiple PD-L1 standards with known concentrations can be included in the testing process. Conventional methods can be used to set up the standards. Using these standards, the standard curve is set as follows: the OD values ​​of the standards are plotted on the ordinate (Y-axis), and the standard concentrations are plotted on the x-axis to create a quantitative standard curve for the PD-L1 kit. Therefore, based on the OD values ​​obtained from the test sample, the concentration of PD-L1 in the test sample can be calculated using the standard curve.

[0091] Furthermore, to facilitate testing, the kit of the present invention preferably also includes other auxiliary reagents. These auxiliary reagents are commonly used in enzyme-linked immunosorbent assays (ELISA), and their properties and preparation methods are well known to those skilled in the art. The reagents include: a chromogenic agent, a washing buffer, a stop solution, and a sensitizing diluent.

[0092] The coated antibody is coated on a solid support. This invention does not impose any particular limitations on the solid support used, as long as it can be coupled (linked) to the coated antibody. For example, the solid support can be selected from: microtiter plates (also known as multi-well plates, such as 96-well plates) or microspheres.

[0093] In one embodiment of the present invention, the solid support used is a microtiter plate (ELISA plate), which is a polystyrene plate with a specification of 12×8 detachable strips.

[0094] Because the nanobody used in the kit of this invention has extremely excellent binding properties (high specificity) for PD-L1, by following the above method, as long as a known concentration of antigen control is set up and a concentration standard curve is prepared, the PD-L1 content in the sample to be tested can be obtained by comparing the concentration standard curve.

[0095] 7. Immunoconjugates

[0096] The present invention provides an immune conjugate comprising a therapeutic agent and the aforementioned nanobody conjugated with said therapeutic agent;

[0097] Preferably, the therapeutic agent includes a toxin, a radioactive isotope, a drug, or a cytotoxic agent.

[0098] As used herein, the term "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or markers.

[0099] 8. Bispecific or multispecific antibodies

[0100] The present invention provides a bispecific or multispecific antibody, comprising the aforementioned nanobody, and an antibody or antibody fragment having another or more antigen-binding properties functionally linked to the nanobody.

[0101] As used herein, the term "bispecific or multispecific antibody" refers to a molecule that can bind to two or more different antigenic epitopes of the same antigen or can bind to two or more different antigens.

[0102] 9. Pharmaceutical Composition

[0103] The present invention provides a pharmaceutical composition comprising a PD-L1 binding molecule (such as the aforementioned nanobody, immunoconjugate, bispecific or multispecific antibody);

[0104] Preferably, the pharmaceutical composition includes a pharmaceutically acceptable excipient, a carrier, or a diluent.

[0105] As used herein, the term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredient is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition will be administered. In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients, carriers, or diluents, specifically, any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, etc., to extend the shelf life or potency of the antibody.

[0106] Beneficial effects include:

[0107] This invention creatively develops PD-L1 binding molecules with high affinity and specificity for PD-L1, such as PD-L1 nanobodies that can block the binding of human PD-L1 protein to free human PD-1 and free human CD80. These nanobodies and the phages displaying the nanobodies can be used to prepare kits for detecting PD-L1. Furthermore, these PD-L1 nanobodies, their immunoconjugates, bispecific or multispecific antibodies, and pharmaceutical compositions have broad application prospects in the preparation of drugs for treating PD-L1-mediated diseases.

[0108] Example

[0109] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments without specific conditions are generally performed according to conventional conditions, such as those described in J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations. For embodiments without specific conditions, conventional conditions or manufacturer's recommendations are followed. Reagents or instruments whose manufacturers are not specified are all commercially available, conventional products.

[0110] Example 1: Material Preparation

[0111] The target genes encoding human PD-L1 (NP_054862.1), monkey PD-L1 (XP_015292694.1), mouse PD-L1 (NP_068693.1), human PD-1 (NP_054862.1), and human CD80 (NP_005182.1) were cloned into plasmids containing His, human Fc, or mouse Fc, respectively. These plasmids were then transfected into HEK293 cells for transient expression, thereby obtaining hPD-L1-His (R0201), cynoPD-L1-His, mPD-L1-His, hPD-L1-hFc, hPD-1-hFc (R0479), hPD-1-mFc (R0323), and hCD80-hFc secreted proteins.

[0112] The target genes encoding human PD-L1 (NP_054862.1), monkey PD-L1 (XP_015292694.1), and mouse PD-L1 (NP_068693.1) were cloned into plasmids containing His, human Fc, or mouse Fc, and then transfected into CHO cells to express the membrane protein, thereby obtaining cell lines overexpressing PD-L1 membrane protein, namely CHO-hPD-L1, CHO-cynoPD-L1, and CHO-mPD-L1.

[0113] Based on the sequence of nanobody No. 56 in application number 201510465481.8 and the Avelumab variable region sequence found on the Drug Bank website, a target antibody for the mouse IgG1 subtype was constructed, which was abbreviated as R0999 and R0516, respectively.

[0114] Example 2: Construction of a nanobody library

[0115] The phage library displaying nanobodies was constructed by Chengdu Apak Biotechnology Co., Ltd. One alpaca was immunized with an equal volume of 1.5 mg human PD-L1 antigen-mFc (purchased from Sino Biologics) and Freund's adjuvant via intradermal and subcutaneous injection at multiple sites on the back of the alpaca. Immunization was performed weekly for a total of four weeks to stimulate B cells to express antigen-specific nanobodies. After the four immunizations, 50 ml of peripheral blood was extracted from the alpaca, and lymphocytes were separated using lymphocyte separation medium. Total RNA was extracted using Trizol RNA extraction reagent (purchased from Invitrogen), and RNA integrity was detected by 1% agarose gel electrophoresis. (See details below.) Figure 1 Total cDNA from alpacas was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

[0116] The nanobody gene was amplified using two rounds of nested PCR. The first round of nested PCR system is shown in Table 1 below:

[0117] Table 1

[0118]

[0119] in:

[0120] The primer sequence for NPR-19001 is: 5'-CTTGGTGGTCCTGGCTGC-3' (SEQ ID NO:45);

[0121] The primer sequence for NPR-19002 is: 5'-GTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO:46);

[0122] Reaction procedure: 98℃, 52 min; 98℃, 10 s; 55℃, 30 s; 72℃, 45 s, for a total of 25 cycles; 72℃, 7 min; after the reaction, gel electrophoresis was performed, and the target VHH fragment of about 700 bp was recovered by gel cutting.

[0123] The second round of nested PCR used conventional methods to design primers and reaction programs to amplify the target gene fragment, followed by 2% agarose gel electrophoresis. The results are as follows: Figure 2 As shown, approximately 500bp of VHH fragments were recovered by tapping the rubber.

[0124] The vector and the target fragment (i.e., the VHH fragment amplified and purified after the second round of nested PCR) were digested with SfiI, incubated overnight at 50°C, and then recovered. Ligation was performed using T4 ligase at a Vector:VHH = 1:3 molar ratio, incubated at 4°C for 16 h. The vector carrying the target gene fragment was transformed into competent *E. coli* TG1 cells by electroporation, and the bacterial library volume was calculated to be 2.55 × 10⁻⁶.9 CFU. Helper phage M13KO7 was added at a 20-fold multiplicity of infection (MOI) and cultured further. The phage library was purified twice using the PEG-NaCl standard purification method to construct the phage library. The phage library titer was identified as 1.41 × 10⁻⁶. 13 cfu / mL.

[0125] Example 3: Phage panning of nanobodies

[0126] hPD-L1-His antigen was coated onto ELISA plates. A solid-phase screening method was used to screen and enrich phages in six 96-well plates. Specifically bound phages were eluted with 0.2M Gly-HCl (pH 2.2), followed immediately by neutralization with Tris-HCl neutralization buffer. This process was repeated three times. Positive clones binding to human PD-L1 were screened using ELISA, and their nucleotide sequences were sequenced. The nucleotide sequences were translated into amino acid sequences using software and compared. Based on differences in CDR3 and amino acid differences in the phylogenetic tree, the sequences were divided into 19 classes. The best-performing sequences for each class are shown in Table 2 below.

[0127] Table 2

[0128]

[0129]

[0130] The amino acid sequences of the 19 nanobodies are shown in Table 3 below.

[0131] Table 3

[0132]

[0133]

[0134] The amino acid sequences of the CDRs of nanobodies (based on the IMGT numbering method) are shown in Table 4 below.

[0135] Table 4

[0136]

[0137] The screening method is as follows: First, the hPD-L1-His and mPD-L1-hFc antigens were diluted to 2 μg / mL with 0.05M carbonate buffer (pH 9.6), and coated overnight at 4°C at a rate of 100 μL / well. The coating solution was discarded, and the plates were washed three times with PBST. 300 μL of 5% skim milk was added to each well, and the plates were blocked at 37°C for 1 h. After washing three times with PBST, 100 μL / well of phage selected through three rounds of panning was added, and the plates were incubated at 37°C for 45 min. After washing five times with PBST, 100 μL / well of horseradish peroxidase-labeled goat anti-Alpaca secondary antibody (diluted 1:1W with PBS) was added, and the plates were incubated at 37°C for 45 min. The plates were washed five times with BST. 100 μL of TMB chromogenic solution was added to each well for color development, and the reaction was carried out at 37°C for 5 min. 50 μL of stop solution was added to each well to stop the reaction, and the optical density was measured at 450 nm.

[0138] Example 4: Preparation of fusion protein

[0139] The target gene sequences encoding nanobodies were cloned into plasmids containing mouse IgG1 Fc, and then transfected into HEK293 cells for transient expression, thereby obtaining the corresponding secreted proteins (i.e., fusion proteins, with the C-terminus of the VHH linked to the N-terminus of the Fc region). The fusion proteins are abbreviated as R1014, R1015, R1016, R1017, R1018, R1019, R1148, R1149, R1150, R1020, R1021, R1022, R1023, R1024, R1025, R1026, R1027, R1028, and R1029. Each fusion protein contains two identical peptide chains. The VHH sequences of each fusion protein are shown in Table 3 above. The full-length amino acid sequences of one chain of some fusion proteins are shown in any of the amino acid sequences represented by SEQ ID NO: 38-43 in Table 5 below. The amino acid sequence of one of the CH2-CH3 segments of the Fc region of immunoglobulin is shown as amino acids 120-346 of SEQ ID NO:44 (italicized portion of Table 5).

[0140] Table 5

[0141]

[0142]

[0143] Example 5: Detection of the binding activity of the fusion protein to membrane-expressed human PD-L1 protein

[0144] The binding of the fusion protein obtained in Example 4, as well as R0999 and R0516 obtained in Example 1, to cellular CHO-hPD-L1 was detected using flow cytometry (FCM). The detection method is as follows:

[0145] Add the fusion protein to be detected and the target antibody at an appropriate dilution to 2E5 / well CHO-hPD-L1 cells. After incubation at 2-8℃ for 30 min, wash once with 200 μl / well 1×PBS. Then add 100 μl / well of E-anti-hIgG fluorescent secondary antibody diluted 1:500 (diluted with 1×PBS containing 3% BSA) at 1:500. After incubation at 2-8℃ for 30 min, wash once with 200 μl / well 1×PBS. Resuspend in 100 μl / well 1×PBS and perform flow cytometry detection.

[0146] Flow cytometry results Figure 3 As shown in the results, the EC50 values ​​of R1015, R1016, R1019, R1148, R1149, and R1150 were 0.148 nM, 0.247 nM, 0.773 nM, 0.445 nM, 0.497 nM, and 0.512 nM, respectively, indicating that these six fusion proteins have good binding activity with membrane-expressed human PD-L1 protein.

[0147] Example 6: Affinity Detection of Fusion Proteins

[0148] Biomembrane interference (BLI) was used to detect the affinity of the binding positive fusion proteins (R1015, R1016, R1019, R1148, R1149, R1150) obtained in Example 5, as well as the target antibodies (R0999 and R0516) obtained in Example 1, human PD-1 protein (R0323) protein and human PD-L1 protein (R0201).

[0149] The detection method is as follows: 5 μg / ml of R0201 was cured onto the ForteBIO (Octet OK) molecular interaction analyzer. e On the Anti-Penta-HIS (HIS1K) (18-5120, Fortebio) probe, the probe was equilibrated for 90 s, then bound to an appropriately diluted analyte for 180 s, followed by dissociation for 300 s. After regeneration and neutralization of the probe in 10 mM glycine, the equilibration, binding, dissociation, and regeneration / neutralization process was repeated for the next cycle. All cycles were performed at 1000 rpm at an experimental temperature of 30 °C.

[0150] The detection results are shown in Table 6 below, and the binding curve is shown in Figure 4. The results show that R1015, R1016, R1019, R1148, R1149, and R1150 have high affinity for human PD-L1 protein.

[0151] Table 6

[0152] protein abbreviation kon(1 / Ms) koff(1 / s) KD(M) R1015 7.69E+05 8.85E-05 1.15E-10 R1016 8.44E+05 1.18E-04 1.40E-10 R1019 6.48E+05 <1.0E-07 <1.0E-12 R1148 8.26E+05 9.34E-05 1.13E-10 R1149 4.10E+05 7.54E-05 1.84E-10 R1150 3.94E+05 4.87E-05 1.24E-10 R0999 8.57E+05 2.20E-05 2.57E-11 R0516 4.61E+05 4.67E-05 1.01E-10 R0323 1.42E+05 2.12E-04 7.78E-08

[0153] Example 7: Fusion protein blocks the binding of membrane-expressed human PD-L1 protein and free human PD-1

[0154] Flow cytometry (FCM) was used to detect the binding of fusion proteins (R1015, R1016, R1019, R1148, R1149, R1150) obtained in Example 4, as well as R0999, R0516, and the binding of cell-blocking CHO-hPD-L1 and free human PD-1 proteins obtained in Example 1. Detection methods (A) and (B) are as follows:

[0155] The detection method (A) is as follows:

[0156] Add the fusion protein to be tested and the target antibody at an appropriate dilution to 2E5 / well CHO-hPD-L1 cells, incubate on ice in the dark for 30 min, then add hPD-1-hFc protein to 96-well V-type plates (50 μL / well), incubate on ice in the dark for 30 min; centrifuge (300 g / 5 min), discard the supernatant, add 200 μL FCM buffer to wash once, centrifuge (300 g / 5 min), discard the supernatant, add 1:500 diluted PE-anti-hIgG fluorescent secondary antibody at 100 μL / well, incubate on ice in the dark for 30 min; after centrifugation and washing, resuspend in 100 μL 1×PBS, and perform detection.

[0157] Test results as follows Figure 5 As shown, the results indicate that the IC50 values ​​of R1148, R1149, and R1150 are 6.431 nM, 6.330 nM, and 8.813 nM, respectively, suggesting that these three fusion proteins can block the binding of membrane-expressed human PD-L1 protein and free human PD-1.

[0158] Detection method (B) is as follows:

[0159] Add hPD-1-hFc protein to 2E5 / well CHO-hPD-L1 cells and incubate on ice in the dark for 30 min. Then add the fusion protein to be tested and the target antibody at an appropriate dilution to 96-well V-type plates (50 μL / well) and incubate on ice in the dark for 30 min. Centrifuge (300 g / 5 min), discard the supernatant, add 200 μL of FCM buffer to wash once, centrifuge (300 g / 5 min), discard the supernatant, add 1:500 diluted PE-anti-hIgG fluorescent secondary antibody at 100 μL / well, and incubate on ice in the dark for 30 min. After centrifugation and washing, resuspend in 100 μL of 1×PBS and perform detection.

[0160] Test results as follows Figure 6As shown, the results indicate that the IC50 values ​​of R1148, R1149, and R1150 are 6.072 nM, 10.64 nM, and 13.98 nM, respectively, suggesting that these three fusion proteins can block the binding of membrane-expressed human PD-L1 protein and free human PD-1.

[0161] Example 8: Analysis of Fusion Protein Binding Epitopes

[0162] Competitive ELISA was used to group the binding regions of the positive-binding fusion proteins (R1148, R1149, and R1150) obtained in Example 7 and the target antibodies (R0999 and R0516) obtained in Example 1 to human PD-L1 protein (R0201). The detection method is as follows:

[0163] 5 μg / ml of R0201 was cured onto a ForteBIO (Octet OK) molecular interaction analyzer. e On the Anti-Penta-HIS (HIS1K) (18-5120, Fortebio) probe, the probe was equilibrated for 90 seconds, then bound to the first antibody at 150 nM for 180 seconds, and then bound to the first antibody at 150 nM for 180 seconds. After the probe was regenerated and neutralized in 10 mM glycine, the equilibration, binding and regeneration neutralization were repeated in the next cycle.

[0164] The test results are shown in Table 7 below. The results show that: the epitopes of R1148, R0999 and R0516 are consistent; the epitopes of R1149 and R1150 are consistent, but inconsistent with the epitopes of R0999 and R0516.

[0165] Table 7

[0166]

[0167]

[0168] Example 9: Fusion protein blocks the binding of membrane-expressed human PD-L1 protein and free human CD80

[0169] Flow cytometry (FCM) was used to detect the binding of fusion proteins (R1148, R1149, and R1150) and R0999, R0516 obtained in Example 1, as well as the binding of cell-blocking CHO-hPD-L1 and free human CD80 proteins. Detection methods (A) and (B) are as follows:

[0170] The detection method (A) is as follows:

[0171] Add the fusion protein to be tested and the target antibody at an appropriate dilution to 2E5 / well CHO-hPD-L1 cells, incubate on ice in the dark for 30 min, then add hCD80 protein to 96-well V-type plates (50 μL / well), incubate on ice in the dark for 30 min; centrifuge (300 g / 5 min), discard the supernatant, add 200 μL FCM buffer to wash once, centrifuge (300 g / 5 min), discard the supernatant, add 100 μL of PE-anti-hIgG fluorescent secondary antibody diluted 1:500 per well, incubate on ice in the dark for 30 min; after centrifugation and washing, resuspend in 100 μL 1×PBS, and perform detection.

[0172] Test results as follows Figure 7 As shown, the results indicate that R1148, R1149, and R1150 can block the binding of membrane-expressed human PD-L1 protein and free human CD80, and the blocking effect is comparable to that of the target antibody.

[0173] Detection method (B) is as follows:

[0174] Add hCD80 protein to 2E5 / well CHO-hPD-L1 cells and incubate on ice in the dark for 30 min. Then add the fusion protein to be tested and the target antibody at an appropriate dilution to 96-well V-type plates (50 μL / well) and incubate on ice in the dark for 30 min. Centrifuge (300 g / 5 min), discard the supernatant, add 200 μL of FCM buffer to wash once, centrifuge (300 g / 5 min), discard the supernatant, add 1:500 diluted PE-anti-hIgG fluorescent secondary antibody at 100 μL / well, and incubate on ice in the dark for 30 min. After centrifugation and washing, resuspend in 100 μL of 1×PBS and perform detection.

[0175] Test results as follows Figure 8 As shown, the results indicate that R1148, R1149, and R1150 can block the binding of membrane-expressed human PD-L1 protein and free human CD80, and the blocking effect is comparable to that of the target antibody.

[0176] In summary, this invention constructed a phage-displayed nanobody library, and used ELISA to screen for positive clones that bind to human PD-L1, obtaining PD-L1-binding molecules that specifically bind to PD-L1, such as FBP002-1128, FBP002-1136, FBP002-1136, FBP002-1369, FBP002-1389, FBP002-1471, FBP002-2002, FBP002-2056, FBP002-2058, FBP002-1191, FBP002-1217, FBP002-1118, and FBP002-113. 4. FBP002-1153, FBP002-1184, FBP002-1224, FBP002-1249, FBP002-1075, FBP002-1095, R1015, R1016, R1019, R1148, R1149, R1150, R1014, R1017, R1018, R1020, R1021, R1022, R1023, R1024, R1025, R1026, R1027, R1028, and R1029 are PD-L1 binding molecules that can block the binding of human PD-L1 protein to free human PD-1 and free human CD80.

[0177] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An anti-PD-L1 nanobody, characterized in that, The CDR1, CDR2 and CDR3 of the nanobody are shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, respectively.

2. The nanobody as described in claim 1, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:

7.

3. A fusion protein, characterized in that, The fusion protein is composed of the PD-L1 nanobody as described in claim 1 or 2 and the Fc region of immunoglobulin.

4. The fusion protein as described in claim 3, characterized in that, The C-terminus of the nanobody is connected to the N-terminus of the Fc region of the immunoglobulin.

5. The fusion protein as described in claim 3, characterized in that, The amino acid sequence of the Fc region of the immunoglobulin is shown as amino acids 120-346 of SEQ ID NO:

44.

6. The fusion protein as described in claim 5, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

41.

7. An isolated polynucleotide, characterized in that, The polynucleotide encodes the nanobody of claim 1 or 2, or the fusion protein of any one of claims 3 to 6.

8. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 7.

9. A host cell, characterized in that, The host cell comprises the expression vector of claim 8 or its genome having integrated the polynucleotide of claim 7.

10. A method for preparing the nanobody of claim 1 or 2 or the fusion protein of any one of claims 3 to 6, characterized in that, It includes culturing the host cells of claim 9 under conditions that allow for the production of the nanobodies or fusion proteins, and then recovering and separating the nanobodies or fusion proteins.

11. A bacteriophage, characterized in that, The surface of the phage displays the nanobody as described in claim 1 or 2.

12. A kit for detecting PD-L1, characterized in that, It includes the nanobody as described in claim 1 or 2, the fusion protein as described in any one of claims 3 to 6, or the phage as described in claim 11.

13. The kit according to claim 12, characterized in that, The kit also includes a solid-phase carrier in which the nanobody, fusion protein or phage is immobilized.

14. The kit according to claim 13, characterized in that, The kit also includes a detectable marker that can be linked to the nanobody or fusion protein; and / or a PD-L1 standard or a PD-L1 conjugate standard; and / or a substrate corresponding to the detectable marker; and / or an enzyme-linked immunosorbent assay (ELISA) reagent.

15. The kit according to claim 14, characterized in that, The detectable marker is attached to the nanobody or fusion protein, or the detectable marker is present separately in the kit.

16. A method for detecting the presence of PD-L1 in a sample for non-therapeutic purposes, characterized in that, Using the nanobody described in claim 1 or 2, the fusion protein described in any one of claims 3 to 6, or the phage described in claim 11 as the detection antibody for PD-L1, the presence of PD-L1 in the sample to be tested is detected by enzyme-linked immunosorbent assay (ELISA).

17. The method as described in claim 16, characterized in that, Specifically, it includes the following steps: The sample to be tested is coated on a solid support, and the presence of PD-L1 is detected by using the nanobody, fusion protein or phage with or without detectable markers as detection antibodies.

18. An immunoconjugate, characterized in that, It is a nanobody conjugated with a label as described in claim 1 or 2, or a fusion protein as described in any one of claims 3 to 6.

19. A pharmaceutical composition, characterized in that, It includes the nanobody as described in claim 1 or 2, or the fusion protein as described in any one of claims 3 to 6; wherein the pharmaceutical composition includes a pharmaceutically acceptable excipient, a carrier, or a diluent.

20. Use of the nanobody of claim 1 or 2, the fusion protein of any one of claims 3 to 6, or the pharmaceutical composition of claim 19 in the preparation of a medicament for treating PD-L1-mediated diseases, wherein the PD-L1-mediated diseases are cancers, including lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, or head and neck cancer.

21. The use as described in claim 20, characterized in that, The cancers mentioned are breast cancer, lung cancer, stomach cancer, colorectal cancer, kidney cancer, or melanoma.

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