Methods for detecting PD-L1-positive exosomes

Abstract

To provide a method for sorting out patients on which an immune checkpoint inhibitor has an effect.SOLUTION: A method for detecting PD-L1 positive tumor, making it possible to directly detect a PD-L1 positive exosome with PD-L1 expressed on its surface without separating it from a biological sample. A method in which the detection of a PD-L1 positive exosome is conducted using an antibody. A method in which the antibody is an antibody that recognizes PD-L1 or an antibody that recognizes an exosome. A method in which a patient having a PD-L1 positive tumor is sorted out by any of the methods described above.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention generally relates to the field of tumor diagnosis. Specifically, the present invention relates to a method for quantifying exosomes in human body fluids, more specifically to a method for quantifying and qualifying PD-L1-positive exosomes, and an anti-PD-L1 monoclonal antibody for use therein. [Background technology]

[0002] Programmed death-1 (PD-1), discovered in 1992, is a protein receptor expressed on the surface of T cells and is involved in cellular apoptosis. PD-1 belongs to the CD28 family and shows 23% homology in amino acid sequence with cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, unlike CTLA4, PD-1 is mainly expressed on activated T cells, B cells, and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2, respectively. PD-L1 is mainly expressed on cancer cells, and PD-L2 expression is limited to antigen-presenting cells such as activated macrophages and dendritic cells.

[0003] Many tumor cells and immune cells in the tumor microenvironment express programmed death-ligand 1 (PD-L1). New studies have detected high expression of PD-L1 protein in human tumor tissues, including breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and liver cancer, and the expression level of PD-L1 is closely correlated with the clinical symptoms and prognosis of patients.

[0004] Because PD-L1 inhibits T cell proliferation using a second signal pathway, blocking the binding of PD-L1 to PD-1 has become a very promising target in the field of tumor immunotherapy (e.g., Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2013 / 173223 publication [Patent Document 2] Special publication 2016-533335 Summary of the Invention [Problem to be solved by the invention]

[0006] Drugs that target PD-1, such as Opdivo and Keytruda, have been attracting attention as immune checkpoint inhibitors (Yarchoan M., Johnson BA III, Lutz ER, Laheru DA, and Jaffee EM 2017. Targeting neoantigens to augment antitumour immunity. Nat. Rev. Cancer. 17:569 10.1038 / nrc.2017.74), but the efficacy of these drugs alone is only seen in about 20% of cases, which is not very high. This is thought to be because PD-L1, the ligand for PD-1, is not expressed in all cancer cells. For this reason, in order to prescribe Keytruda, which targets PD-1, tissue staining for PD-L1 is essential as a companion diagnostic for patient stratification (Reck M., Rodriguez-Abreu D., Robinson AG, Hui R., Csoszi T., Fulop A., Gottfried M., Peled N., Tafreshi A., Cuffe S., et al. KEYNOTE-024 Investigators. 2016. Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N. Engl. J. Med. 375:1823-1833. 10.1056 / NEJMoa1606774: Edited by the Japan Lung Cancer Society, "Guidelines for PD-L1 Testing in Lung Cancer Patients"). Tissue staining, which is based on biopsy, a method of extracting tumor tissue using an endoscope or needle, is a highly invasive test, and the burden on patients is an issue. Therefore, there is a need for the development of liquid biopsy, which uses bodily fluids such as blood to perform diagnosis and predict treatment effects instead of biopsy, thereby reducing the burden on patients. [Means for solving the problem]

[0007] It is known that PD-L1-positive exosomes are secreted from PD-L1-positive tumors (Chen G., Huang A.C., Zhang W., Zhang G., Wu M., Xu W., Yu Z., Yang J., Wang B., Sun H., et al. 2018. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 560:382-386. 10.1038 / s41586-018-0392-8). For the purpose of directly quantifying PD-L1-positive exosomes in biological samples, the inventors prepared an antibody specific to the extracellular region of PD-L1 and succeeded in constructing a measurement system based thereon.

[0008] That is, the present invention includes the following aspects. <Detection of PD-L1-positive tumor> [1] A method for detecting a PD-L1-positive tumor, which comprises directly detecting PD-L1-positive exosomes expressing PD-L1 on their surface without separating them from a biological sample. [2] The method according to [1], wherein the detection of PD-L1-positive exosomes is performed using an antibody. [3] The method according to [2], wherein the antibody is an antibody that recognizes PD-L1 and an antibody that recognizes exosomes. [4] The method according to any one of [1] to [3], wherein the detection is performed by immunoprecipitation. [5] A method for selecting a patient having a PD-L1-positive tumor by the method according to any one of [1] to [3]. [6] A method for selecting a patient in whom an immune checkpoint inhibitor is effective by the method according to [4]. <Antibody molecule> [7] (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and (ii) a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 11, the VLCDR2 amino acid sequence of SEQ ID NO: 12, and the VLCDR3 amino acid sequence of SEQ ID NO: 13 5. An isolated antibody molecule, or derivative thereof, capable of binding to PD-L1, comprising: [8] The antibody molecule according to [7], or a derivative thereof, having a heavy chain variable region including at least one framework (FW) region comprising the VH amino acid sequence of SEQ ID NO: 1, or an amino acid sequence which is at least 80% identical thereto, or an amino acid sequence which has a substitution, deletion, insertion or addition of one or several amino acid residues compared to the VH amino acid sequence of SEQ ID NO: 1. [9] An antibody molecule described in [7] or [8], or a derivative thereof, having a light chain variable region including at least one framework region comprising the VL amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 80% identical thereto, or an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the VL amino acid sequence of SEQ ID NO: 9.

[10] A derivative of an antibody molecule according to any one of [7] to [9], which is a Fab, F(ab')2, Fv or single-chain Fv fragment (scFv). <Nucleic acid>

[11] An isolated nucleic acid encoding an antibody heavy or light chain variable region of an antibody molecule described in any one of [7] to

[10] .

[12] An isolated nucleic acid encoding heavy chain CDR1 to 3, said nucleic acid comprising the nucleotide sequence of SEQ ID NO:6, 7 or 8.

[13] An isolated nucleic acid encoding light chain CDR1 to 3, said nucleic acid comprising the nucleotide sequence of SEQ ID NO: 14, 15 or 16.

[14] An expression vector comprising the nucleic acid according to any one of

[11] to

[13] .

[15]

[14] A host cell comprising the vector described in.

[16] A method for producing an antibody molecule or a fragment thereof, comprising culturing the host cell of

[14] under conditions suitable for gene expression. <Detection>

[17] (i) obtaining a biological sample from the subject; (ii) contacting the obtained biological sample with an isolated antibody molecule or a derivative thereof according to any one of [7] to

[10] ; and (iii) detecting the formation of complexes between PD-L1-positive exosomes and antibody molecules or their derivatives; 1. A method for detecting PD-L1-positive exosomes in a biological sample, comprising:

[18]

[17] A method for selecting a patient who will respond to an immune checkpoint inhibitor, comprising selecting a patient in which PD-L1-positive exosomes are detected by the method described in the above.

[19] A kit for quantifying PD-L1-positive exosomes, comprising an isolated antibody molecule or a derivative thereof according to any one of [7] to

[10] .

[20] The kit described in

[19] , further containing a fusion protein of human PD-L1 and human CD9 protein having the amino acid sequence shown in SEQ ID NO: 19, or a fusion protein of human PD-L1 and human CD63 having the amino acid sequence shown in SEQ ID NO: 21 as a standard reagent. [Brief description of the drawings]

[0009] [Figure 1] Figure 1A shows the results of Western blot analysis using cell extracts from the G418-resistant line, and Figure 1B shows the results of Western blot analysis using exosomes from the PD-L1-expressing line. [Diagram 2]Figure 2 is a schematic diagram of the sandwich ELISA measurement system for quantifying PD-L1-positive exosomes. [Diagram 3] Figure 3 shows a graph comparing the performance of anti-PD-L1 antibodies using ELISA to measure PD-L1-positive exosomes. [Figure 4] Figure 4 is a graph confirming the quantitativeness of PD-L1-positive exosome measurements in ELISA using clone A1-9F7 as the labeled antibody. [Diagram 5] Figure 5 shows the standard curves of ELISA using PD-L1 / CD9 or PD-L1 / CD63 fusion proteins.

[0010] [Figure 6] FIG. 6 shows the results of comparing the performance of clone A1-9F7 antibody with a commercially available antibody (MIH5) in exosome ELISA. [Figure 7] Figure 7 shows the structure of the VH chain variable region of the anti-PD-L1 monoclonal antibody (clone A1-9F7). [Figure 8] Figure 8 shows the structure of the VL chain variable region of the anti-PD-L1 monoclonal antibody (clone A1-9F7). [Figure 9] Figure 9A is a graph showing the results of an ELISA recovery test in which 0, 2.5, 5, 10, 25, and 50 μL of human pooled serum and 2, 6, and 16 μg / mL of PD-L1-positive exosomes were added per well to an ELISA with an immobilized anti-CD9 antibody, and the ratio (recovery rate) of the signal obtained when serum was added at each concentration to the signal obtained when 0 μL of serum was added (no addition) is calculated. Figure 9B is a graph showing the results of an ELISA recovery test calculated in the same manner as in Figure 9A, except that human pooled plasma was used instead of human pooled serum. [Figure 10]FIG. 10 is a graph showing quantitative values obtained by comparing the quantification of PD-L1-positive exosomes by immunoprecipitation (left) and ELISA (right) using the culture supernatant of lung cancer cell line PC9. The expression of PD-L1-positive exosomes was induced by adding γ-interferon to the culture medium. γ-interferon (−) and (+) indicate the absence and presence of γ-interferon, respectively. B-B(0) is the value obtained by subtracting the background value of the culture medium alone from the absorbance measurement value of each sample.

DETAILED DESCRIPTION OF THE INVENTION

[0011] <Detection of PD-L1-Positive Tumors> One aspect of the present invention relates to a method for detecting a PD-L1-positive tumor, which is characterized by directly detecting PD-L1-positive exosomes expressing PD-L1 on their surface without separating them from a biological sample. As used herein, the term "PD-L1-positive exosome" means an exosome expressing PD-L1 on its surface. PD-L1-positive exosomes are mainly released from PD-L1-positive tumors. Their existence is described in Chen G., et al. 2018. Nature. 560:382-386. 10.1038 / s41586-018-0392-8.

[0012] Exosomes are microvesicles with a size of 30 to 120 nm and are usually actively secreted into body fluids such as blood via the exocytosis pathway. Exosomes carry membrane and cytoplasmic proteins involved in many cellular functions in addition to major histocompatibility complex proteins (MHC I, MHC II) and proteins involved in antigen presentation. Exosomes are secreted from various cell types such as dendritic cells, lymphocytes, mast cells, and epithelial cells under specific physiological conditions.

[0013] Exosomes are prepared from cell culture supernatants and body fluids by multiple centrifugation steps. However, a problem in obtaining useful in vivo data on exosomes is the low level of efficiency of currently available methods for obtaining exosomes for the preparation of exosomes from human body fluids, particularly blood samples. To provide a solution to these currently faced challenges, the present invention provides a simple and reliable method for detecting and quantifying exosomes from body fluids, particularly human blood samples.

[0014] In this embodiment, the present invention uses antibodies, preferably antibodies that recognize PD-L1 and antibodies that recognize exosomes, to detect PD-L1-positive exosomes. More preferably, the method uses the antibodies of the present invention. Specifically, the method uses at least one anti-human PD-L1 monoclonal antibody selected from A1-7C11, A1-7E1, A1-9F7, A1-13A11, A1-13G7, A1-14A5, and A1-14E9 as the antibody that detects PD-L1-positive exosomes, and an anti-CD9 monoclonal antibody (manufactured by Cosmo Bio Co., Ltd.), an anti-CD63 monoclonal antibody (manufactured by Cosmo Bio Co., Ltd.), or an anti-CD81 monoclonal antibody (manufactured by Cosmo Bio Co., Ltd.) as the antibody that recognizes exosomes.

[0015] In the present invention, "PD-L1 positive tumor" means a tumor expressing PD-L1 on its cell surface. PD-L1 binds to PD-1 and B7-1 and inhibits the immune system, but many tumor cells and immune cells in the tumor microenvironment express PD-L1. New studies have detected high expression of PD-L1 protein in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and liver cancer, and the expression level of PD-L1 is closely correlated with the clinical symptoms and prognosis of patients. In addition, PD-L1 expression has been found in many cancers, including human lung, ovarian and colon cancers and various myelomas, and has been reported to be often associated with poor prognosis (Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53). PD-L1 has been suggested to play a role in tumor immunity by increasing apoptosis of antigen-specific T cell clones (Dong et al. (2002) Nat Med 8:793-800), and may also be involved in intestinal mucosal inflammation, and inhibition of PD-L1 has been suggested to suppress debilitating disease associated with colitis (Kanai et al. (2003) J Immunol 171:4156-63).

[0016] The method for detecting PD-L1-positive tumors of the present invention is characterized in that PD-L1-positive exosomes are directly detected without being separated from a biological sample. The biological sample refers to a body fluid, particularly a blood sample such as blood, plasma, or serum, and may also be ascites, cerebral fluid, bone marrow, urine, feces, or bronchoalveolar lavage fluid.

[0017] When PD-L1-positive purified exosomes were quantified by ELISA using the antibodies of the present invention, concentration-dependent measurements were obtained, demonstrating that these ELISAs are suitable for quantifying PD-L1-positive exosomes (Figure 4). In addition, whether the solid-phase antibody was CD9 or CD63, it was found that the ELISA using the A1-9F7 antibody of the present invention was able to detect PD-L1-positive exosomes with significantly higher sensitivity than the commercially available antibody (Figure 6). Furthermore, in an exosome ELISA in which anti-CD9 antibody was used as the solid-phase antibody to quantify PD-L1-positive purified exosomes, an additive recovery test was performed to evaluate the binding specificity of the antibody and exosomes in the presence of serum or plasma, and the recovery rate was approximately 100%, demonstrating that the present invention can directly detect PD-L1-positive exosomes without isolating them from biological samples (Figures 9A and 9B). As for a system that can directly detect exosomes, reliable measurements can be obtained as long as the amount of the biological sample, particularly blood samples, is 25 μL or less, more preferably 20 μL or less, and particularly preferably 10 μL or less. In particular, CD9 / PD-L1 ELISA can provide reliable measurements with sample volumes of 10 μL or less. Of course, the sample volume must be at least large enough to provide a detection level or higher.

[0018] In a preferred embodiment of the present invention, PD-L1-positive exosomes can be detected by immunoprecipitation. In immunoprecipitation, soluble antigens react specifically with antibodies to insolubilize them, and it is possible to detect, separate, and purify antigens under relatively mild conditions. For example, a large structure is insolubilized by crosslinking a large number of substrates with antibodies. Generally, antibodies are bound to carriers such as Sepharose beads, but a method using magnetic beads is also often performed. Polyclonal antibodies are easier to immunoprecipitate than monoclonal antibodies. In immunoprecipitation, nonspecifically adsorbed components may be removed by centrifugation. When magnetic beads are used, separation by magnetic beads is performed instead of centrifugation.

[0019] In detail, PD-L1-positive exosomes are contacted with an exosome-specific antibody to form an immunoprecipitated complex. Then, when the PD-L1-positive exosomes are contacted with the immobilized specific antibody, the antibody is usually bound to a solid support to form an immunoprecipitated complex. The solid support is a material that can be modified to include a suitable site for antibody attachment or association. The material of the solid support includes plastics including acrylic resin, polystyrene, and copolymers of styrene with other substances, polypropylene, polyethylene, polybutylene, polyurethane, or Teflon J, nylon, nitrocellulose, polysaccharides; silica, or silicon; and metals, inorganic glass, and the like. The size and shape of the solid support are not limited as long as it can bind PD-L1-positive exosomes. The solid support may be a flat surface, a well (hole), i.e., a plate with 364 holes, or the solid support may be beads. In some embodiments, the solid support is a well of a plate with multiple walls. In other embodiments, the solid support is preferably a bead, more preferably a magnetic bead.

[0020] As shown in the following examples, it has been demonstrated that the antibody of the present invention can be used for immunoprecipitation, and that it is possible to directly detect PD-L1-positive exosomes without isolating them from a biological sample. In addition, in the case of ELISA, the size of the plate wells is limited, and the amount of sample that can be processed is also limited, whereas in immunoprecipitation, there is no limit to the amount of sample, and it has been demonstrated that exosomes can be concentrated in a relatively large amount of sample. Thus, even when the total amount of the sample itself is relatively large, or even when the amount of PD-L1-positive exosomes present in the sample is small, i.e., the concentration is low, immunoprecipitation can provide the advantage of enabling quantification.

[0021] The method for detecting PD-L1-positive tumors of the present invention can be applied to a method for selecting patients with PD-L1-positive tumors, and such an embodiment is also within the scope of the present invention. The method for selecting patients with PD-L1-positive tumors of the present invention can be applied to a method for selecting patients for whom immune checkpoint inhibitors are effective, and such an embodiment is also within the scope of the present invention. "Immune checkpoint inhibitors" refer to antibody drugs used in new cancer immunotherapy, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, and in particular, in the present invention, anti-PD-1 antibodies and anti-PD-L1 antibodies are relevant as "immune checkpoint inhibitors." Tumor cells express PD-L1 to escape attacks from immune cells, and when this binds to PD-1 on immune cells, the function of the immune cells is suppressed. Anti-PD-1 antibodies bind to PD-1 on immune cells and inhibit binding to PD-L1, and anti-PD-L1 antibodies bind to PD-L1 expressed by cancer cells and inhibit binding to PD-1. Specific examples of commercially available "immune checkpoint inhibitors" include anti-PD-1 antibodies such as Opdivo (nivolumab) from Ono Pharmaceutical Co., Ltd. / Bristol-Myers Squibb (USA) and Keytruda (pembrolizumab) from Merck (MSD) (USA), and anti-PD-L1 antibodies such as Bavencio (avelumab) from Merck (Germany) / Pfizer (USA), Tecentriq (atezolizumab) from Roche (Switzerland) / Chugai Pharmaceutical, and Imfinzi (durvalumab) from AstraZeneca (UK).

[0022] Immunotherapy using immune checkpoint inhibitors has been recognized as one of the standard therapies for cancer. These drugs are recognized as an excellent treatment that exerts long-term therapeutic effects on intractable cancers that are refractory to existing standard therapies and extends overall survival. However, there are many challenges to overcome with immune checkpoint inhibitors, such as the occurrence of immune-related adverse events and the identification of optimal treatment regimens. In particular, the response rate of current immune checkpoint inhibitors as a single agent is insufficient at about 10-30%, so the development of composite cancer immunotherapy aimed at further improving the therapeutic effect is being actively promoted around the world. Composite cancer immunotherapy is mainly performed in combination with anti-PD-1 / PD-L1 antibodies, and therapies that simultaneously inhibit multiple immune checkpoint molecules and the combination of immune checkpoint inhibitors and drugs that activate immune stimulatory molecules are being considered. In addition, as a combination with non-immunotherapy, many clinical trials are being conducted using not only cytotoxic anticancer drugs, molecular targeted drugs, and angiogenesis inhibitors, but also radiation therapy in combination with anti-PD-1 / PD-L1 antibodies. In any case, the reason why the efficacy rate of immune checkpoint inhibitors as a single agent is insufficient at about 10-30% is thought to be because PD-L1 is not expressed in all cancer cells. Therefore, taking into consideration the burden on patients, it is desirable to develop a liquid biopsy that reduces the burden on patients by using body fluids such as blood to perform diagnosis and predict treatment effects instead of biopsy, and the method of the present invention meets this requirement.

[0023] <Antibody molecule> In another aspect, the present invention provides a method for producing a composition comprising: (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and (ii) a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 11, the VLCDR2 amino acid sequence of SEQ ID NO: 12, and the VLCDR3 amino acid sequence of SEQ ID NO: 13 The present invention relates to an isolated antibody molecule, or derivative thereof, capable of binding to PD-L1, comprising: The three light chain CDRs are referred to as LCDR1, LCDR2 and LCDR3, and the three heavy chain CDRs are referred to as HCDR1, HCDR2 and HCDR3.

[0024] In the present invention, "isolated" refers to a sample that is isolated from its original natural environment. For example, a polynucleotide or polypeptide of natural origin present in a living animal is not isolated, but the same polynucleotide or polypeptide that is artificially separated from some or all of the sample with which it coexists in the natural system is isolated.

[0025] In the present invention, the term "derivative of an antibody molecule" refers to diabodies and single chain molecules, as well as "antigen-binding fragments". The term "antigen-binding fragments" refers to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and scFv fragments that bind to human PD-L1 and can include one or more CDR regions in the antibody defined in the present invention. An Fv fragment is the smallest antibody fragment that contains all of the antigen-binding site without the heavy chain variable region, the light chain variable region, and the constant region. In general, an Fv antibody further contains a polypeptide linker between the VH and VL domains to form the structure required for antigen binding. Also, different linkers are used to link the variable regions of two antibodies to form a polypeptide named single chain antibody or single chain Fv (scFv). In the present specification, the term "binds to PD-L1" means capable of interacting with human PD-L1. In the present specification, the term "antigen-binding site" refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0026] In another aspect, the present invention relates to an antibody molecule of the present invention, or a derivative thereof, having a heavy chain variable region comprising at least one framework (FW) region comprising the VH amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical thereto, or an amino acid sequence that has one or several amino acid residue substitutions, deletions, insertions or additions compared to the VH amino acid sequence of SEQ ID NO: 1, and an antibody molecule of the present invention, or a derivative thereof, having a light chain variable region comprising at least one framework region comprising the LH amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 80% identical thereto, or an amino acid sequence that has one or several amino acid residue substitutions, deletions, insertions or additions compared to the LH amino acid sequence of SEQ ID NO: 9. The VH and VL regions can be subdivided into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions, termed "framework regions" (FR or FW).

[0027] In some embodiments, the light or heavy chain variable framework of the anti-PD-L1 antibody molecule may comprise a light or heavy chain variable framework that comprises at least 80%, 85%, 87%, 90%, 93%, 95%, 97%, 99% of the light or heavy chain variable framework residues derived from a human consensus sequence.

[0028] <Nucleic acid> In another aspect, the present invention relates to an isolated nucleic acid encoding an antibody heavy or light chain variable region of an antibody molecule of the present invention, in particular an isolated nucleic acid encoding heavy chain CDR1 to 3 comprising the nucleotide sequence of SEQ ID NO: 6, 7 or 8, or an isolated nucleic acid encoding light chain CDR1 to 3 comprising the nucleotide sequence of SEQ ID NO: 14, 15 or 16. As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. In further aspects, the invention relates to an expression vector comprising a nucleic acid of the invention, and a host cell comprising a vector of the invention.

[0029] In some embodiments, the vector comprises a nucleotide sequence encoding an antibody molecule as described herein. In some embodiments, the vector comprises a nucleotide sequence as described herein. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). Additionally, cells in which the DNA has stably integrated into the chromosome may be selected by introducing one or more markers that allow for the selection of transfected host cells. The markers may confer, for example, prototrophy, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper, to an auxotrophic host. The selectable marker gene may be directly linked to the DNA sequence to be expressed or may be introduced by co-transformation into the same cell. Additionally, additional elements may be necessary for optimal synthesis of mRNA. These elements may include splice signals, transcription promoters, enhancers, and termination signals.

[0030] In one embodiment, the host cell is genetically modified to contain a nucleic acid encoding an antibody molecule. In one embodiment, the host cell is genetically modified by the use of an expression cassette. The expression "expression cassette" refers to a nucleotide sequence capable of affecting expression of a gene in a host with which the sequence is compatible. Such a cassette may include a promoter, an open reading frame with or without an intron, and a termination signal. Additional elements necessary to effect expression may be used, such as an inducible promoter. The cell may be a eukaryotic cell, a bacterial cell, an insect cell or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, CHO cells, etc. Suitable insect cells include, but are not limited to, Sf9 cells. In another embodiment, the invention relates to a method of producing an antibody molecule or a fragment thereof, comprising culturing a host cell of the invention under conditions suitable for gene expression.

[0031] <Detection> The present invention relates to a further aspect, (i) obtaining a biological sample from the subject; (ii) contacting the obtained biological sample with an isolated antibody molecule or derivative thereof of the present invention; and (iii) detecting the formation of complexes between PD-L1-positive exosomes and antibody molecules or their derivatives; The present invention relates to a method for detecting PD-L1-positive exosomes in a biological sample, comprising: Similar to the method for detecting PD-L1-positive tumors of the present invention, the method for detecting PD-L1-positive exosomes of the present invention is characterized in that PD-L1-positive exosomes are directly detected without being separated from a biological sample. The biological sample refers to a body fluid, particularly blood, plasma, or serum, and may also be ascites, cerebral fluid, bone marrow, urine, feces, or bronchoalveolar lavage fluid. The method for detecting PD-L1-positive exosomes of the present invention can be applied to a method for selecting patients with PD-L1-positive tumors, and such an embodiment is also within the scope of the present invention. The method for selecting patients with PD-L1-positive tumors of the present invention can be applied to a method for selecting patients who respond to immune checkpoint inhibitors, and such an embodiment is also within the scope of the present invention.

[0032] The method for detecting the formation of a complex between PD-L1-positive exosomes and antibody molecules or their derivatives involves binding an antibody that recognizes exosomes, such as an anti-CD9 antibody, to a carrier such as a plastic plate or magnetic beads, and then contacting a biological sample containing exosomes to bind the exosomes to the antibody on the carrier. Furthermore, a luminescent substrate is applied to the complex formed by binding the exosomes to an anti-PD-L1 antibody labeled with a label such as horseradish peroxidase, and the complex is quantified by detecting the luminescence intensity using a plate reader or the like.

[0033] In another aspect, the present invention relates to a kit for quantifying PD-L1-positive exosomes, comprising the isolated antibody molecule or derivative thereof of the present invention. The kits of the invention may contain one or more of the following other components: instructions for use; other reagents, e.g., labels, or agents for labeling the antibody. The kit of the present invention preferably contains a standard reagent, such as a fusion protein of human PD-L1 and human CD9 protein having the amino acid sequence as set forth in SEQ ID NO:19, and a fusion protein of human PD-L1 and human CD63 protein having the amino acid sequence as set forth in SEQ ID NO:21.

[0034] The present invention will be described in detail below with reference to examples. However, it should be noted that these do not limit the scope of the present invention and are merely illustrative. EXAMPLES

[0035] Example 1 Preparation of anti-PD-L1 monoclonal antibodies 1-1: Preparation of antigen A cDNA (SEQ ID NO: 17) was synthesized in which the Fc region of rabbit immunoglobulin was linked to the carboxyl terminus of Met1-Thr239 of the extracellular domain of human PD-L1 protein (registered in the National Center for Biotechnology Information database under accession number BC069381) and inserted downstream of the cytomegalovirus promoter in the expression vector pD603 (ATUM). This plasmid was introduced into 293-6E cells using the FreeStyle 293 Expression System (Thermo Fisher Scientific) for transient expression, and then purified to a purity of approximately 95% using a Monofinity A Resin Prepacked Column (GenScript) to prepare the PD-L1 antigen as an Fc fusion protein of PD-L1. For reference, the full-length DNA sequence of PD-L1, including the extracellular domain as well as the transmembrane and intracellular domains, is shown in SEQ ID NO:18.

[0036] 1-2: Preparation of monoclonal antibodies An emulsion was prepared as an immunogen by mixing the PD-L1 Fc fusion protein with equal amounts of complete Freund's adjuvant (Becton Dickinson) for the first immunization and incomplete Freund's adjuvant (Becton Dickinson) for the second and subsequent immunizations. The monoclonal antibody was prepared by the method described in K. Watanabe et al., Vasohibin as an endothelium-driven negative feedback regulator of angiogenesis, J. Clin. Inves. 114(2004), 898-907. Specifically, 100 μg of Fc fusion protein per mouse was administered subcutaneously and intraperitoneally in equal amounts to 5-week-old female A / J mice. After the sixth immunization, the spleen was removed from the mice that had received the final booster (4 days before cell fusion), and splenocytes were prepared. Cell fusion between splenocytes and myeloma cells (P3U1) was performed by the method described in K. Watanabe et al., Vasohibin as an endothelium-driven negative feedback regulator of angiogenesis, J. Clin. Inves. 114(2004), 898-907.

[0037] 1-3: Evaluation of monoclonal antibodies The antibody titers of the antisera and hybridoma supernatants were evaluated (primary evaluation) by the ELISA method described below. Specifically, the antisera or hybridoma supernatants were added to a 96-well microplate with goat anti-mouse IgG antibody immobilized thereon, and then PD-L1 protein biotinylated with a biotin labeling kit (Dojindo) and europium-labeled streptavidin (PerkinElmer) were mixed and stirred at room temperature for 2 hours. After the reaction, the plates were washed three times with a washing solution (saline containing 0.01% Tween 20 and 0.1% ProClin 150), and 100 μl of enhancement reagent (Delfia Enhancement Solution, PerkinElmer) was added and stirred for 5 minutes. The time-resolved fluorescence intensity was measured using Nivo (PerkinElmer). A signal intensity more than three times that obtained without the addition of antisera or hybridoma was judged to be positive, and 7 clones of antibodies were selected (Table 1).

[0038] Table 1: Selected anti-human PD-L1 monoclonal antibodies [Table 1]

[0039] Example 2 Confirmation of reactivity of anti-PD-L1 monoclonal antibodies with PD-L1-positive exosomes: Secondary evaluation (exosome ELISA) 2-1: Preparation of PD-L1-positive exosomes A cDNA encoding the entire region of human PD-L1 and tagged with His was synthesized (outsourced to Eurofins Genomics), inserted into the expression vector pD603 (ATUM), and transfected into HEK293 cells using GenePORTER Gold transfection reagent (Cosmo Bio). Several stable transfectants were selected in the presence of 1 mg / mL G418. Extracts of these cell lines were prepared, and cell lines with high transgene expression were selected from the stable transfectants by Western blotting using an anti-His tag antibody (Proteintech) (Figure 1A). The cell lines were expanded to ten 150 mm culture dishes, and exosomes were prepared from the culture medium in serum-free medium (Advanced DMEM, Thermo Fisher Scientific) by ultracentrifugation (100,000 × g for 70 minutes at 4°C). A cell line (clone number 8) expressing high levels of PD-L1 on exosomes was identified (Figure 1B). PD-L1-positive exosomes prepared from this cell line were used to establish the following ELISA.

[0040] 2-2: Construction of sandwich ELISA A sandwich ELISA measurement system for quantifying exosomes expressing PD-L1 was constructed using the seven anti-PD-L1 monoclonal antibody clones prepared in Examples 1-3, an anti-CD9 monoclonal antibody (manufactured by Cosmo Bio), and an anti-CD63 monoclonal antibody (manufactured by Cosmo Bio) (Figure 2). In detail, first, a 96-well microplate was prepared with anti-CD9 monoclonal antibody and anti-CD63 monoclonal antibody immobilized. Each antibody solution, prepared at 5 μg / mL with Tris-HCl buffer, was added to the 96-well microplate at 100 μL / well and reacted overnight. Thereafter, the reaction solution was removed, and the plate was washed once with a washing solution (phosphate buffered saline containing 0.01% Tween 20 and 0.1% ProClin 150), after which 200 μL / well of 2% Block Ace (manufactured by Sumitomo Dainippon Pharma) was added and left at 4°C overnight for blocking. Meanwhile, all of the antibodies shown in Table 1 were reacted with 20 times the molar amount of Sulfo-NHS-LC-Biotin (manufactured by Thermo Fisher) at room temperature for 2 hours, and gel filtration was performed with a PD-10 column (manufactured by GE Healthcare) to prepare labeled antibodies.

[0041] 100 μL of PD-L1 positive exosomes (10 μg / mL) were added to the plate on which the antibody was immobilized, and the plate was stirred and reacted at room temperature for 2 hours. After that, the reaction solution was removed, and the plate was washed three times with the above washing solution, and 100 μL of the labeled antibody prepared above at 1 μg / mL was added, stirred, and reacted at room temperature for 2 hours. After washing three times, 100 μL of HRP (Horseradish Peroxidase)-labeled streptavidin (manufactured by R&D) was added and reacted at room temperature for 20 minutes. After washing three times in the same manner, 100 μL of TMB solution (Colorburst Blue, manufactured by ALerCHEK) was added and reacted at room temperature for 20 minutes. 50 μL of 2N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm of each well was measured (Figure 3). As a result, it was found that when clone A1-9F7 of the seven anti-PD-L1 antibodies was used as the labeled antibody, the strongest signal was detected regardless of whether the solid-phase antibody was anti-CD9 or anti-CD63.

[0042] 2-3: Exosome concentration dependence of ELISA measurements In an ELISA measurement system using anti-CD9 monoclonal antibody (Cosmo Bio) and anti-CD63 monoclonal antibody (Cosmo Bio) as the solid-phase antibody and anti-PD-L1 monoclonal antibody (clone A1-9F7) prepared in Example 1-3 and screened in Example 2-2 as the labeled antibody, 0, 0.3125, 0.625, 1.25, 2.5, 5.0, and 10 μg / mL of PD-L1-positive purified exosomes prepared by ultracentrifugation (centrifugation at 100,000 × g for 70 minutes at 4℃) from the culture supernatant of clone number 8 cell line were quantified. All of the measurements were concentration-dependent, indicating that these ELISAs are suitable for quantifying PD-L1-positive exosomes (Figure 4).

[0043] 2-4: Standard reagents Purified exosomes have low storage stability, so if they are used as standard reagents to draw standard curves, it is difficult to obtain the same standard curve in different tests, and they cannot be used to correct differences between kit lots or between tests. Therefore, it is desirable to use purified recombinant proteins with high storage stability as standard reagents when drawing standard curves. For this reason, we prepared recombinant proteins in which the extracellular domains of CD9 or CD63 were fused with the extracellular domain of PD-L1 as standard reagents for PD-L1-positive exosome ELISA. That is, a plasmid was constructed by inserting a cDNA encoding a fusion protein in which the carboxyl-terminal side of Met1 to Arg258 of human PD-L1 was linked to a His tag sequence from Gly110 to Phe193 of human CD9 protein or Ala103 to Leu204 of human CD63 protein into the expression vector pD603, and the plasmid was introduced into HEK293 cells for transient forced expression, and the PD-L1 / CD9 or PD-L1 / CD63 fusion protein secreted from the cells was purified using His Mag Sepharose excel (GE Healthcare Life Sciences). These fusion proteins were measured at concentrations of 0, 0.625, 1.25, 2.5, 5.0, 10 and 20ng / mL in an ELISA system using anti-CD9 monoclonal antibody or anti-CD63 monoclonal antibody as the solid-phase antibody and anti-PD-L1 monoclonal antibody (clone A1-9F7) as the labeled antibody. The measured values ​​were concentration-dependent, indicating that these fusion proteins can be used as standard reagents in the measurement system (Figure 5). The amino acid and DNA sequences of the fusion proteins in which Gly110 to Phe193 of human CD9 protein is linked to the carboxyl-terminal side of Met1 to Arg258 of human PD-L1 are shown in SEQ ID NOs: 19 and 20, respectively. The amino acid and DNA sequences of the fusion proteins in which Ala103 to Leu204 of human CD63 protein is linked to the carboxyl-terminal side of Met1 to Arg258 of human PD-L1 are shown in SEQ ID NOs: 21 and 22, respectively.

[0044] 2-5: Performance comparison of the antibody of the present invention with commercially available antibodies The anti-PD-L1 monoclonal antibody (clone A1-9F7) prepared as described in Examples 1-3 was compared in performance with a commercially available anti-PD-L1 monoclonal antibody (clone MIH5, Invitrogen) used as a labeled antibody in Chen G., Huang AC, Zhang W., Zhang G., Wu M., Xu W., Yu Z., Yang J., Wang B., Sun H., et al. 2018. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 560:382-386. 10.1038 / s41586-018-0392-8. Specifically, these two types of monoclonal antibodies were labeled with biotin as the labeled antibody, and anti-CD9 antibody (Cosmo Bio) or anti-CD63 antibody (Cosmo Bio) was used as the solid-phase antibody to quantify PD-L1-positive purified exosomes. The sensitivity of each detection system was compared based on the slope of the obtained calibration curve in an exosome ELISA. As a result, in both cases where the solid-phase antibody was CD9 or CD63, the A1-9F7 antibody of the present invention gave a significantly stronger signal than the commercially available antibody, demonstrating that this ELISA can detect PD-L1-positive exosomes with more than twice the sensitivity (Figure 6).

[0045] Example 3 Structural analysis of monoclonal antibody A1-9F7 for detecting PD-L1-positive exosomes Structural analysis of the VH chain variable region and VL chain variable region of the anti-PD-L1 monoclonal antibody (clone A1-9F7), which showed the highest reactivity in the ELISA detecting PD-L1-positive exosomes, was performed using the bioinformatics analysis system of Repertoire Genesis Co., Ltd. Specifically, cDNA was synthesized using messenger RNA extracted from the hybridoma producing the monoclonal antibody clone A1-9F7 as a template, and the gene sequences of the VH chain variable region and VL chain variable region were determined using a next-generation sequencer, and each sequence was replaced with an amino acid sequence (VH chain variable region: Figure 7, SEQ ID NO: 1 and SEQ ID NO: 2; VL chain variable region: Figure 8, SEQ ID NO: 9 and SEQ ID NO: 10).

[0046] Example 4 Recovery test in ELISA In an exosome ELISA in which anti-CD9 antibody is used as a solid-phase antibody to quantify PD-L1-positive purified exosomes, an addition recovery test was performed to evaluate the binding specificity of the antibody and exosomes in the presence of serum or plasma. Specifically, 0, 2.5, 5, 10, 25, and 50 μL of human pooled serum (Cosmo Bio) and PD-L1-positive exosomes at concentrations of 2, 6, and 16 μg / mL were added to an ELISA in which anti-CD9 antibody (Cosmo Bio) was solid-phased, and measurements were performed. The ratio of the signal obtained when serum was added at each concentration to the signal obtained when 0 μL of serum (no addition) was calculated (recovery rate). The ratio was also calculated in the same way for human pooled plasma (Cosmo Bio). As a result, when the amount of serum added was 10 μL or less, the recovery rate was good, at 83.5 to 113.4% (Figure 9A). Even in the case of plasma, when the added amount was 10 μL or less, the recovery rate was good, ranging from 92.3 to 107.5% (FIG. 9B).

[0047] Example 5 Exosome enrichment by immunoprecipitation 5-1: Preparation of anti-CD9 antibody solid-phase magnetic beads 100μg of anti-CD9 antibody (clone 12A12, Cosmo Bio) was reacted with amino group biotin labeling reagent (Thermo Fisher) at room temperature for 2 hours to be biotinylated. Biotinylated antibody was added to 1mg of streptavidin solid-phase magnetic beads (Tamagawa Seiki) and binding reaction was carried out in PBS at 4℃ for 1 hour. After washing with 10mM HEPES-NaOH (pH 7.9), 50mM KCl, 1mM EDTA, 10% glycerol, it was stored in the same solution at a concentration of 0.1mg / 20μL.

[0048] 5-2: Immunoprecipitation of exosomes in culture supernatant of lung cancer cell line PC9 Human lung cancer cell line PC9 (obtained from The Jikei University School of Medicine) was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum until subconfluent. 100 nM human gamma interferon was added to induce PD-L1 expression (Predictive Markers for the Efficacy of Anti-PD-1 / PD-L1 Antibodies in Lung Cancer. J Thorac Oncol. 2016 Jul; 11(7): 976-88.) and the culture medium was then collected after two more days of culture. As a control, culture medium without gamma interferon was also collected. To 1 mL of each of these culture solutions, 50 μg / 10 μL of the anti-CD9 antibody solid-phase magnetic beads prepared in Example 5-1, which had been washed and suspended in 20 mM HEPES-NaOH (pH 7.9), 150 mM KCl, 1 mM MgCl2, 0.2 mM CaCl2, 0.2 mM EDTA, and 10% glycerol in the presence of 0.1% polyoxyethylene 10-phenyl ether, were added, and the mixture was rotated and mixed overnight at 4° C. The beads were then magnetically separated, washed three times with the same solution, and suspended in 50 μL. In addition, the same treatment was performed on 1 mL of the culture solution before adding it to the cells as a blank sample.

[0049] 5-3: Detection of PD-L1 on immunoprecipitated exosomes 200μg of anti-PD-L1 antibody (clone A1-9F7) prepared in Example 1-3 and screened in Example 2-2 was labeled with HRP using a peroxidase labeling kit (Dojindo Chemical Industries, Ltd.) to prepare a labeled antibody. 50μL of the labeled antibody, diluted to 2μg / mL with the same solution as the immunoprecipitation, was added to an equal amount of immunoprecipitated beads, and reacted at room temperature for 2 hours. The beads were magnetically separated and washed four times with the same solution. After confirming color development by adding 100μL of HRP substrate solution (Moss), 50μL of 2N sulfuric acid was added to the solution from which the beads were removed by magnetic separation, and the absorbance at a wavelength of 450nm was measured. The measured value (B) was subtracted from the measured value (B(0)) of the blank sample to obtain a value (BB(0)). The sample in which PD-L1 expression was induced with gamma interferon had a significantly higher value than the sample in which PD-L1 expression was not induced (Figure 10, left). This indicates that immunoprecipitation can distinguish between the presence and absence of PD-L1-positive exosomes in the samples of this example.

[0050] When 100 μL of the same sample was measured by ELISA (sandwich ELISA in Example 2) using anti-CD9 antibody (clone 12A12, Cosmo Bio) as the solid phase and anti-PD-L1 antibody (clone A1-9F7) as the HRP-labeled antibody, almost no difference was observed in the measured values ​​depending on the presence or absence of gamma interferon, i.e., the measured values ​​indicating the presence or absence of PD-L1-positive exosomes (Figure 10, right). This indicates that ELISA cannot be applied to samples in which the presence or absence of PD-L1-positive exosomes could be distinguished by immunoprecipitation. As described above, in this example, we compared the quantification feasibility of immunoprecipitation and ELISA using the same samples. As a result, it was found that immunoprecipitation can detect PD-L1-positive exosomes even in samples with low expression levels.

Claims

1. A method for detecting PD-L1-positive tumors, comprising directly detecting PD-L1-positive exosomes expressing PD-L1 on their surface using an antibody that recognizes PD-L1, without isolating the exosomes from a biological sample, comprising: The antibody that recognizes PD-L1 is (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and (ii) a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 11, the VLCDR2 amino acid sequence of SEQ ID NO: 12, and the VLCDR3 amino acid sequence of SEQ ID NO: 13 or an isolated antibody molecule capable of binding to PD-L1, comprising: ２ , Fv or single chain Fv fragment (scFv).

2. The method of claim 1, wherein the antibody recognizing PD-L1 is an antibody molecule having a heavy chain variable region comprising at least one framework (FW) region comprising the VH amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical thereto, or an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the VH amino acid sequence of SEQ ID NO:

1.

3. 3. The method of claim 1 or 2, wherein the antibody that recognizes PD-L1 is an antibody molecule having a light chain variable region comprising at least one framework region comprising the VL amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 80% identical thereto, or an amino acid sequence that has one or several amino acid residue substitutions, deletions, insertions or additions compared to the VL amino acid sequence of SEQ ID NO:

9.

4. The method according to any one of claims 1 to 3, further comprising using an antibody that recognizes exosomes.

5. The method according to any one of claims 1 to 4, wherein the detection is carried out by immunoprecipitation.

6. (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and (ii) a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 11, the VLCDR2 amino acid sequence of SEQ ID NO: 12, and the VLCDR3 amino acid sequence of SEQ ID NO: 13 or an isolated antibody molecule capable of binding to PD-L1, comprising: ２ , Fv or single chain Fv fragment (scFv).

7. 7. The antibody molecule of claim 6, having a heavy chain variable region comprising at least one framework (FW) region comprising the VH amino acid sequence of SEQ ID NO: 1, or an amino acid sequence which is at least 80% identical thereto, or an amino acid sequence which has one or several amino acid substitutions, deletions, insertions or additions compared to the VH amino acid sequence of SEQ ID NO: 1, or an isolated Fab, F(ab') fragment of said antibody molecule capable of binding to PD-L1. ２ , Fv or single chain Fv fragment (scFv).

8. 8. The antibody molecule of claim 6 or 7, having a light chain variable region comprising at least one framework region comprising the VL amino acid sequence of SEQ ID NO: 9, or an amino acid sequence which is at least 80% identical thereto, or an amino acid sequence which has one or several amino acid substitutions, deletions, insertions or additions compared to the VL amino acid sequence of SEQ ID NO: 9, or an isolated Fab, F(ab') fragment of said antibody molecule capable of binding to PD-L1. ２ , Fv or single chain Fv fragment (scFv).

9. (i) combining a biological sample obtained from a subject with an isolated antibody molecule of any one of claims 6 to 8, or an isolated Fab, F(ab') or F(ab') antibody molecule thereof capable of binding to PD-L1; ２ , Fv or single chain Fv fragment (scFv), and (ii) PD-L1-positive exosomes and an antibody molecule, or an isolated Fab, F(ab') molecule of the antibody molecule capable of binding to PD-L1. ２ , detecting the formation of a complex between an Fv or a single chain Fv fragment (scFv) 1. A method for detecting PD-L1-positive exosomes in a biological sample, comprising:

10. A method for selecting a patient who will respond to an immune checkpoint inhibitor, comprising selecting a patient in whom PD-L1-positive exosomes are detected by the method according to claim 9.

11. 9. The isolated antibody molecule of any one of claims 6 to 8, or an isolated Fab, F(ab') or F(ab') antibody molecule thereof capable of binding to PD-L1. ２ A kit for quantifying PD-L1-positive exosomes, including Fv or single-chain Fv fragments (scFv).

12. 12. The kit of claim 11, further comprising a fusion protein of human PD-L1 and human CD9 protein having the amino acid sequence set forth in SEQ ID NO: 19, or a fusion protein of human PD-L1 and human CD63 protein having the amino acid sequence set forth in SEQ ID NO: 21 as a standard reagent.

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