Nanobodies targeting egfr, drug conjugates and uses thereof

Abstract

The present application discloses anti-EGFR nanobodies, encoding sequences thereof and uses thereof. Specifically, the present application discloses a class of specific nanobodies against human EGFR and VHH chains thereof. Encoding sequences encoding the above-mentioned nanobodies or VHH chains thereof, corresponding expression vectors and host cells, and methods for producing the nanobodies of the present application are also disclosed. The nanobodies of the present application have high endocytosis activity and high specificity, and can be used for detection and targeted treatment against EGFR.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of November 30, 2023, the application number of CN202311625877.5, and the invention name of "Nanobody targeting EGFR, drug conjugate and use thereof". TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology, and specifically relates to a nanobody targeting EGFR and its application. BACKGROUND

[0003] Cancer is still one of the biggest threats to human health worldwide, causing more than 10 million deaths worldwide each year. Although there are many cancer treatment methods including surgery, radiotherapy and chemotherapy, these traditional treatment methods still have many problems to some extent, such as limited treatment effect and large side effects.

[0004] Compared with traditional treatment methods, the latest method based on targeted therapeutic drugs (such as antibodies) has smaller side effects, and after coupling the cytotoxic drugs or toxins with the antibodies, the treatment effect can be greatly improved through intracellular internalization. These antibody-drug conjugates are also called antibody-drug conjugates (ADC).

[0005] Nanobody is a heavy chain antibody variable region naturally lacking light chain discovered by scientists in 1989, which is derived from animals of the Camelidae family. Its protein crystal structure is 4.0 nm in length and 2.5 nm in diameter, and it is currently the smallest antibody known in terms of molecular weight, so it is called nanobody. Compared with traditional antibodies, nanobody has high stability and stronger tolerance, in addition, it also has the advantages of easy expression and easy genetic engineering. Based on its advantages in stability, penetration, etc., nanobody is widely concerned in the fields of disease treatment, diagnosis and material detection.

[0006] Phage display technology is to insert foreign genes into the genome of filamentous phage, so that the protein or polypeptide encoded by the target gene is displayed on the surface of the phage in the form of a fusion protein. Compared with traditional antibodies, nanobody naturally lacks light chain, does not need light-heavy chain assembly pairing, and has smaller molecular weight, so it is easier to express and display on the surface of phage. Combined with the huge library advantage of phage display technology, it is easier to find sequence differentiated and function differentiated nanobody molecules in the development of nanobody.

[0007] Epidermal growth factor receptor (EGFR) is a transmembrane receptor encoded by c-erbB proto-oncogene, with a molecular weight of about 170KD, which is the receptor of the epidermal growth factor family (EGF family) members of extracellular protein ligand; EGFR forms dimerization after binding with its specific ligands including epidermal growth factor (EGF) and transforming growth factor alpha (TGFa), further stimulates intracellular protein tyrosine kinase activity and initiates downstream signal transduction cascade, resulting in DNA synthesis and cell proliferation. EGFR also participates in phenotype regulation such as cell migration, adhesion and proliferation.

[0008] EGFR overexpression is associated with many malignant tumors, including bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer and kidney cancer, etc., and in these cancers, the overexpression of EGFR is also significantly associated with the poor prognosis of patients.

[0009] Currently, the development of EGFR target drugs is mainly based on the form of functional blockers, which can be divided into two categories: one belongs to small molecule drugs, which are based on intracellular signal transduction synthesis of tyrosine kinase inhibitors (TKI), including gefitinib, erlotinib, etc.; the other is ligand blocker, which is mainly based on the development of monoclonal antibody drugs to block extracellular ligand signal transduction, such as cetuximab, panitumumab, etc.; however, for cancer treatment, there are still many defects in the current products, such as limited efficacy, high drug resistance, large toxic and side effects and many other problems, and the social benefits brought by them are also very limited.

[0010] The action mechanism of the ADC based on the EGFR target point is different from that of the traditional therapy by inhibiting the receptor signal pathway. The ADC can directly deliver the cytotoxin to the tumor cells with high expression of EGFR, and the cytotoxin is brought into the tumor cells through intracellularization, so as to enhance the killing effect of the drug and improve the effectiveness of the targeted therapy. Therefore, theoretically, the ADC has a wider indication and a more powerful efficacy than the traditional therapy. At present, most of the ADCs based on the EGFR target point are in the clinical research stage, for example, AVID-100, ABT-414 and ABBV-221, and the antibody forms disclosed in the related patents such as CN106470697, CN114585391 and CN111295201 are all traditional monoclonal antibodies. The traditional monoclonal antibodies usually have many defects such as large molecular weight, poor stability, difficult preparation process, high immunogenicity and the like. In addition, two related forms of ADCs IMGN-289 and AMG-595 with the EGFR as the target point are also forced to terminate in the clinical research due to the problems such as toxicity (such as skin toxicity, gastrointestinal toxicity and the like). Based on the toxicity problem, AstraZeneca AZD9592 (patent number: US20230183358A1) also makes a design of weakening the affinity at the EGFR end, aiming to reduce the tissue toxicity driven by the EGFR. Therefore, the research on the ADC with the EGFR as the target point still has many challenges in the effectiveness and safety.

[0011] The nanobody has a smaller molecular weight, a smaller volume, higher stability, lower immunogenicity, stronger tissue penetration and lower production cost than the traditional antibody.

[0012] Therefore, it is of great significance to develop the nanobody and the antibody related drug targeting the EGFR based on the llama nanobody.

[0013] Therefore, it is urgent to develop a nanobody and a nanobody conjugated drug capable of targeting the EGFR, having high specificity, low affinity, high endocytosis activity and strong tumor cell killing activity. SUMMARY

[0014] The application provides a nanobody and a nanobody conjugated drug capable of targeting the EGFR, having high specificity, low affinity, high endocytosis activity and strong tumor cell killing activity.

[0015] In a first aspect of the application, an anti-EGFR nanobody is provided, wherein the complementarity determining region (CDR) of the VHH chain of the nanobody is one or more selected from the following group:

[0016] (1) CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 6 and CDR3 shown in SEQ ID NO: 9.

[0017] (2) CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, CDR3 as shown in SEQ ID NO: 10;

[0018] (3) CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 8, CDR3 as shown in SEQ ID NO: 11.

[0019] In another preferred embodiment, the CDR region of the VHH chain of the nanobody comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above-mentioned sequences.

[0020] In another preferred embodiment, any of the above-mentioned amino acid sequences further comprises a derivative sequence optionally having at least one amino acid added, deleted, modified and / or substituted, and capable of retaining the EGFR binding affinity.

[0021] In another preferred embodiment, the number of the added, deleted, modified and / or substituted amino acid is 1-3, preferably 1-2, more preferably 1.

[0022] In another preferred embodiment, the VHH chain of the nanobody further comprises a framework region (FR).

[0023] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0024] In another preferred embodiment, the framework region FR is of human, murine, rabbit or camel origin.

[0025] In another preferred embodiment, the nanobody binds to EGFR of human, murine or monkey origin.

[0026] In another preferred embodiment, the nanobody is capable of endocytosis in cells expressing the EGFR antigen.

[0027] In another preferred embodiment, the VHH chain of the nanobody targeting EGFR has an amino acid sequence having ≥ 85%, ≥ 90%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, or ≥ 99% homology to the amino acid sequence as shown in SEQ ID NO: 1-3.

[0028] In another preferred embodiment, the VHH chain of the nanobody targeting EGFR has one or more of the amino acid sequences as shown in SEQ ID NO: 1-3.

[0029] In another preferred embodiment, the anti-EGFR Nanobody comprises a monomer, a bivalent (diabody), a tetravalent (tetrabody), and / or a multivalent (multibody).

[0030] In another preferred embodiment, the amino acid sequence of the VHH chain of the Nanobody is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

[0031] In a second aspect of the present application, a Nanobody fusion protein is provided, which has a structure as shown in Formula I from N-terminus to C-terminus:

[0032] Z1-Z2-L-Z3(Formula I)

[0033] In the formula,

[0034] Z1 is a VHH chain of an anti-EGFR Nanobody as described in the first aspect of the present application;

[0035] Z2 is an Fc fragment of an immunoglobulin;

[0036] L is a linker sequence;

[0037] Z3 is an immunomodulatory molecule moiety.

[0038] In a third aspect of the present application, a polynucleotide is provided, which encodes a protein selected from the group consisting of the anti-EGFR Nanobody as described in the first aspect of the present application.

[0039] In another preferred embodiment, the polynucleotide comprises DNA, RNA or cDNA.

[0040] In a fourth aspect of the present application, an expression vector is provided, which contains the polynucleotide as described in the third aspect of the present application.

[0041] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof.

[0042] In another preferred embodiment, the expression vector comprises a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus.

[0043] In a fifth aspect of the present application, a host cell is provided, which contains the expression vector as described in the fourth aspect of the present application, or has the polynucleotide as described in the third aspect of the present application integrated into its genome.

[0044] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0045] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, a yeast cell, a mammalian cell, a bacteriophage, or a combination thereof.

[0046] In a sixth aspect of the present application, there is provided a method of producing an anti-EGFR nanobody, comprising the steps of:

[0047] (a) culturing the host cell of the fifth aspect of the present application under conditions suitable for production of a nanobody, thereby obtaining a culture comprising the anti-EGFR nanobody; and

[0048] (b) isolating or recovering the anti-EGFR nanobody from the culture; and

[0049] (c) optionally, purifying and / or modifying the anti-EGFR nanobody obtained in step (b).

[0050] In a seventh aspect of the present application, there is provided an immunoconjugate comprising:

[0051] (a) the anti-EGFR nanobody of the first aspect of the present application; and

[0052] (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

[0053] In another preferred embodiment, the conjugating moiety is a drug or a toxin.

[0054] In another preferred embodiment, the drug is a cytotoxic drug.

[0055] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

[0056] In another preferred embodiment, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, exemplary cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[l,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0057] In another preferred embodiment, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureus, maytansinoids, ricin, ricin A-chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, phytolaccin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoid, or combinations thereof.

[0058] In another preferred embodiment, the conjugation moiety is a detectable label.

[0059] In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biological toxin, a cytokine (e.g., IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.

[0060] In another preferred embodiment, the immunoconjugate comprises a multivalent (e.g., bivalent) anti-EGFR nanobody according to the first aspect of the present application. The multivalent means that the amino acid sequence of the immunoconjugate comprises multiple repeats of the anti-EGFR nanobody according to the first aspect of the present application.

[0061] In an eighth aspect of the present application, there is provided a use of an anti-EGFR nanobody according to the present application for the preparation of (a) a reagent for detecting an EGFR molecule; (b) a medicament for treating a tumor.

[0062] In another preferred embodiment, the detecting comprises flow detection, cell immunofluorescence detection.

[0063] In a ninth aspect of the present application, there is provided a use of one or more anti-EGFR nanobody according to the present application:

[0064] (i) for detecting a human EGFR molecule;

[0065] (ii) for flow detection;

[0066] (iii) for cell immunofluorescence detection;

[0067] (iv) for treating a tumor;

[0068] (v) for tumor diagnosis.

[0069] In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

[0070] In a tenth aspect of the present application, there is provided a multispecific antibody comprising an anti-EGFR nanobody according to the first aspect of the present application.

[0071] In another preferred embodiment, the multispecific antibody further comprises an Fc fragment of an antibody.

[0072] In an eleventh aspect of the present application, there is provided a recombinant protein having:

[0073] (i) a sequence of the Nanobody as described in the first aspect of the present application; and

[0074] (ii) an optional Fc fragment; and

[0075] (iii) an optional tag sequence to assist expression and / or purification.

[0076] In another preferred embodiment, the tag sequence comprises a 6His tag and a HA tag

[0077] In another preferred embodiment, the recombinant protein specifically binds to EGFR protein.

[0078] In a twelfth aspect of the present application, there is provided a use of the Nanobody as described in the first aspect of the present application, or the immunoconjugate as described in the seventh aspect of the present application, in the manufacture of a medicament, a reagent, an assay plate or a kit;

[0079] wherein the reagent, assay plate or kit is used for detecting EGFR protein in a sample;

[0080] wherein the medicament is used for treating or preventing a tumor expressing EGFR protein (i.e. EGFR positive).

[0081] In another preferred embodiment, the tumor comprises bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, or kidney cancer.

[0082] In a thirteenth aspect of the present application, there is provided a pharmaceutical composition comprising:

[0083] (i) the anti-EGFR Nanobody as described in the first aspect of the present application, or the immunoconjugate as described in the seventh aspect of the present application, or the recombinant protein as described in the eleventh aspect of the present application; and

[0084] (ii) a pharmaceutically acceptable carrier.

[0085] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0086] In another preferred embodiment, the pharmaceutical composition is used in the manufacture of a medicament for treating a tumor, which comprises a cancer selected from the group consisting of bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer, or a combination thereof.

[0087] In another preferred embodiment, the pharmaceutical composition is used in the manufacture of a medicament for treating a tumor, which comprises a cancer selected from the group consisting of lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer, or a combination thereof.

[0088] In another preferred embodiment, the conjugating moiety of the immunoconjugate is a drug, a toxin, and / or a therapeutic isotope.

[0089] In another preferred embodiment, the pharmaceutical composition further comprises another drug for treating immune system diseases or tumor diseases.

[0090] In another preferred embodiment, the another drug for treating immune system diseases or tumor diseases is selected from the group consisting of budesonide, fluticasone, beclometasone, mometasone furoate, salbutamol, theophylline, formoterol, tiotropium bromide, sulfasalazine, methotrexate, cyclophosphamide, fluorouracil, bleomycin, anastrozole, or a combination thereof.

[0091] In a fourteenth aspect of the present application, there is provided a use of the anti-EGFR Nanobody according to the first aspect of the present application, or the immunoconjugate according to the seventh aspect of the present application, or the recombinant protein according to the eleventh aspect of the present application, or the pharmaceutical composition according to the thirteenth aspect of the present application, for:

[0092] (a) preparing a medicament for preventing and / or treating a disease associated with EGFR; and / or

[0093] (b) preparing a reagent, a plate or a kit for detecting EGFR.

[0094] In another preferred embodiment, the EGFR is human EGFR.

[0095] In another preferred embodiment, the reagent is a diagnostic reagent.

[0096] In another preferred embodiment, the diagnostic reagent is a contrast agent.

[0097] In another preferred embodiment, the reagent is used for detecting EGFR protein or a fragment thereof in a sample.

[0098] In another preferred embodiment, the detecting comprises flow detection, cell immunofluorescence detection.

[0099] In another preferred embodiment, the use is diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic.

[0100] In a fifteenth aspect of the present application, there is provided a method for detecting EGFR protein in a sample, the method comprising the steps of:

[0101] (1) contacting the sample with the Nanobody according to the first aspect of the present application;

[0102] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of EGFR protein in the sample.

[0103] In a sixteenth aspect of the present application, there is provided a method of treating a disease, comprising administering to a subject in need thereof a Nanobody according to the first aspect of the present application or an immunoconjugate according to the seventh aspect of the present application.

[0104] In another preferred embodiment, the subject is a mammal, such as a human.

[0105] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0106] In a seventeenth aspect of the present application, there is provided an EGFR protein detection reagent, comprising:

[0107] (i) an anti-EGFR Nanobody according to the first aspect of the present application or an immunoconjugate according to the seventh aspect of the present application or a recombinant protein according to the eleventh aspect of the present application; and

[0108] (ii) a detectably acceptable carrier.

[0109] In another preferred embodiment, the conjugating moiety of the immunoconjugate is a diagnostic isotope.

[0110] In another preferred embodiment, the detectably acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0111] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of an isotope tracer, a contrast agent, a flow cytometry reagent, a cellular immunofluorescence reagent, a nano-magnetic particle and an imaging agent.

[0112] In another preferred embodiment, the detection reagent is for in vivo detection.

[0113] In another preferred embodiment, the detection reagent is in a dosage form selected from the group consisting of a liquid and a powder (e.g. an aqueous solution, a syringe, a lyophilized powder, a tablet, a buccal, an inhalant).

[0114] In an eighteenth aspect of the present application, there is provided a kit for detecting an EGFR protein, comprising an immunoconjugate according to the seventh aspect of the present application or a detection reagent according to the seventeenth aspect of the present application, and an instruction manual.

[0115] In another preferred embodiment, the instruction manual describes that the kit is for non-invasively detecting the expression of EGFR in a subject.

[0116] In a nineteenth aspect of the present application, there is provided the use of an immunoconjugate according to the seventh aspect of the present application for the preparation of a contrast agent for in vivo detection of an EGFR protein.

[0117] In another preferred embodiment, the detection is for the diagnosis or prognosis of a disease or condition associated with EGFR.

[0118] In a twentieth aspect of the present application, there is provided a method for treating a disease associated with EGFR, comprising administering to a subject in need thereof the anti-EGFR Nanobody of the first aspect of the present application, or the immunoconjugate of the seventh aspect of the present application, or the recombinant protein of the eleventh aspect of the present application, or the pharmaceutical composition of the thirteenth aspect of the present application.

[0119] In another preferred embodiment, the subject comprises a human or a non-human mammal.

[0120] In another preferred embodiment, the non-human mammal comprises a rodent (e.g., mouse, rabbit), a non-human primate (e.g., monkey).

[0121] It should be understood that, within the scope of the present application, all the technical features described above and in the following (e.g., in the examples) of the present application can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0122] Figure 1 The general technical route of the present application is shown.

[0123] Figure 2 The electrophoresis of EGFR-His protein is shown.

[0124] Figure 3 The verification of the binding activity of EGFR antigen and BMK is shown.

[0125] Figure 4 The detection of the llama serum titer is shown.

[0126] Figure 5 The technical route of constructing the llama Nanobody library is shown.

[0127] Figure 6 The panning of the EGFR-targeted Nanobody is shown.

[0128] Figure 7 The technical route of Nanobody expression and preparation is shown.

[0129] Figure 8 The SEC detection of the Nanobody after purification is shown.

[0130] Figure 9A The ELISA activity detection of KY303-01 to KY303-10 Nanobodies is shown.

[0131] Figure 9B The ELISA activity detection of KY303-11 to KY303-20 Nanobodies is shown.

[0132] Figure 9C KY303-21 to KY303-30 Nanobody ELISA activity assay is shown.

[0133] Figure 9D KY303-31 to KY303-40 Nanobody ELISA activity assay is shown.

[0134] Figure 9E KY303-41 to KY303-50 Nanobody ELISA activity assay is shown.

[0135] Figure 9F KY303-51 to KY303-57 Nanobody ELISA activity assay is shown.

[0136] Figure 10A KY303-01 to KY303-10 Nanobody FACS activity assay is shown.

[0137] Figure 10B KY303-11 to KY303-20 Nanobody FACS activity assay is shown.

[0138] Figure 10C KY303-21 to KY303-30 Nanobody FACS activity assay is shown.

[0139] Figure 10D KY303-31 to KY303-40 Nanobody FACS activity assay is shown.

[0140] Figure 10E KY303-41 to KY303-50 Nanobody FACS activity assay is shown.

[0141] Figure 10F KY303-51 to KY303-57 Nanobody FACS activity assay is shown.

[0142] Figure 11A KY303-01 to KY303-30 Nanobody specific binding assay is shown.

[0143] Figure 11B KY303-31 to KY303-57 Nanobody specific binding assay is shown.

[0144] Figure 12A Nanobody endocytosis rate assay on HCT116 cells is shown.

[0145] Figure 12B Nanobody endocytosis rate assay on 769-P cells is shown.

[0146] Figure 12CThe nanobody endocytosis rate detection on 786-O cells is shown.

[0147] Figure 13 The nanobody conjugation MMAE technical route is shown.

[0148] Figure 14A The ADC killing effect on NCI-H1975 cells is shown.

[0149] Figure 14B The ADC killing effect on HCT116 cells is shown.

[0150] Figure 14C The ADC killing effect on BxPC-3 cells is shown.

[0151] Figure 14D The ADC killing effect on MDA-MB-468 cells is shown.

[0152] Figure 14E The ADC killing effect on NCI-H1993 cells is shown.

[0153] Figure 14F The ADC killing effect on HT29 cells is shown.

[0154] Figure 14G The ADC killing effect on MDA-MB-231 cells is shown.

[0155] Figure 15A The tumor volume change of a mouse HCT116 model is shown.

[0156] Figure 15B The body weight change of a HCT116 model mouse is shown.

[0157] Figure 15C The tumor volume change of a mouse NCI-H1975 model is shown.

[0158] Figure 15D The body weight change of a NCI-H1975 model mouse is shown. DETAILED DESCRIPTION

[0159] The present inventors, through extensive and in-depth research, unexpectedly obtained an anti-EGFR nanobody with excellent endocytosis activity and specificity through a large number of screenings.

[0160] The experimental results show that the nanobody of the present application has good binding activity and high endocytosis functional activity. In addition, the nanobody of the present application has strong tumor cell killing activity after conjugation with a cytotoxin (MMAE), can significantly inhibit tumor growth, and has good in vivo safety. On this basis, the present application is completed.

[0161] The present application is based on the development of a nanobody molecule targeting EGFR using alpaca nanobody and phage display technology. The molecule has high binding and endocytosis function activity to tumor cell lines, and has good tumor cell killing effect when developed as an antibody conjugated drug, and has the potential to be developed as an ADC anti-tumor drug. The general technical route of the present application is shown in Figure 1

[0162] As used herein, the terms "the present nanobody", "the present anti-EGFR nanobody", "the present EGFR nanobody" are used interchangeably, and all refer to a nanobody that specifically recognizes and binds to EGFR (including human EGFR). Particularly preferred is a nanobody whose amino acid sequence of the VHH chain is as shown in any one of SEQ ID NO.: 1-3.

[0163] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons having the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regular interval intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0164] As used herein, the terms "single domain antibody (VHH)", "nanobody" have the same meaning, which refers to the cloning of the variable region of the heavy chain of the antibody, and the construction of a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with complete function. Usually, after obtaining an antibody naturally lacking light chain and heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0165] ​As used herein, the term "variable" refers to certain portions of the variable region of an antibody that differ in sequence among antibodies and are responsible for the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light chain and the heavy chain variable regions. The more highly conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, form the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647- 669 (1991)). The constant regions of the antibodies are not directly involved in binding to an antigen but exhibit various effector functions, such as participation in antibody- dependent cellular cytotoxicity.

[0166] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates of drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the antibodies or fragments thereof of the present application. The present application also includes cell surface markers or antigens conjugated to the anti-EGFR protein antibodies or fragments thereof.

[0167] As used herein, the term "heavy chain variable region" is used interchangeably with "V H ".

[0168] As used herein, the term "variable region" is used interchangeably with "complementarity determining region" (CDR).

[0169] In a preferred embodiment of the present application, the heavy chain variable region of the antibody comprises three complementarity determining regions, CDR1, CDR2, and CDR3. There are currently several methods for the partitioning of CDRs, including the IMGT method, the Kabat method, the Chothia method, the VBASE2 method, etc. The CDR partitioning method referred to in the present patent uses the IMGT method.

[0170] In a preferred embodiment of the present application, the heavy chain of the antibody comprises the heavy chain variable region described above and a heavy chain constant region.

[0171] In the present application, the terms "antibody of the present application", "protein of the present application", or "polypeptide of the present application" are used interchangeably and refer to a polypeptide that specifically binds to an EGFR protein, such as a protein or polypeptide having a heavy chain variable region. They can or can not contain the initial methionine.

[0172] The present application also provides other proteins or fusion expression products having the antibodies of the present application. In particular, the present application includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugates and fusion expression products) having a heavy chain comprising a variable region, provided that the variable region is identical to or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibodies of the present application.

[0173] Generally, the antigen binding properties of an antibody can be described by three specific regions of the variable region of the heavy chain, called the complementarity determining regions (CDRs), which are interspersed with four framework regions (FRs) whose amino acid sequences are relatively conserved and do not directly participate in binding interactions. The CDRs form loops or "hot spots" on the surface of the variable region, and are in close proximity to each other through the FRs that form a beta sheet. The CDRs on the heavy chain and the corresponding CDRs on the light chain together form the antigen binding site of the antibody. Which amino acids constitute the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same class.

[0174] The variable regions of the heavy chains of the antibodies of the present application are of particular interest because at least some of them are involved in binding the antigen. Thus, the present application includes those molecules having the variable region of the heavy chain of the antibody with the CDRs, provided that the CDRs are at least 90% homologous, preferably at least 95% homologous, and most preferably at least 98% homologous to the CDRs identified herein.

[0175] The present application includes not only intact antibodies, but also fragments of the antibodies that are immunologically active or fusion proteins of the antibodies with other sequences. Thus, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0176] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. The polypeptide fragments, derivatives, or analogs of the present application can be (i) polypeptides having one or more conservative or non-conservative amino acid substitutions (preferably conservative amino acid substitutions) of the amino acid residues, where such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) polypeptides having a substitution group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide to another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing additional amino acid sequences to the polypeptide sequence (such as a leader or secretory sequence, or a sequence for purification of the polypeptide, or a proprotein sequence, or a fusion protein with a 6His tag). These fragments, derivatives, and analogs are within the scope of one skilled in the art in light of the teachings herein.

[0177] The term "antibody" of the present application refers to a polypeptide having the binding activity to EGFR protein, which comprises the CDR regions described above. The term also includes variants of the polypeptide comprising the CDR regions described above, which have the same function as the antibody of the present application. These variants include, but are not limited to, deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with similar or identical amino acids usually does not change the function of the protein. For another example, addition of one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibody of the present application.

[0178] The variants of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA hybridizing to the coding DNA of the antibody of the present application under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present application.

[0179] The present application also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to the almost full-length polypeptides, the present application also includes fragments of the nanobodies of the present application. Usually, the fragments have at least about 50 consecutive amino acids of the antibody of the present application, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, most preferably at least about 100 consecutive amino acids.

[0180] In the present application, "conservative variants of the antibody of the present application" refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by similar or identical amino acids compared to the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table 1.

[0181] Table 1

[0182]

[0183]

[0184] The present application also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0185] Polynucleotides encoding the mature polypeptides of the present application include: coding sequences that encode only the mature polypeptide; coding sequences that encode the mature polypeptide and various additional coding sequences; coding sequences that encode the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0186] The term "polynucleotide encoding a polypeptide" can be a polynucleotide that includes only coding sequence that encodes the polypeptide, or it can be a polynucleotide that includes additional coding and / or non-coding sequences.

[0187] The present application also relates to polynucleotides that hybridize to the sequences described above and that have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS at 60°C; or (2) hybridization in the presence of a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll at 42°C; or (3) hybridization only when the identity between the two sequences is at least 90%, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0188] The nucleotide full-length sequence of the antibody of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is short. Generally, a long fragment can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0189] Once the relevant sequence is obtained, it can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules that exist in an isolated form.

[0190] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present application by chemical synthesis.

[0191] The present application also relates to vectors that contain the appropriate DNA sequences described above and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express the protein.

[0192] The host cell can be a prokaryote, such as a bacterial cell, or a eukaryote, such as a yeast cell, or a higher eukaryote, such as a mammalian cell. Representative examples of useful host cells include: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0193] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation, if necessary, followed by electroporation. When the host is a eukaryote, transformation can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical procedures such as microinjection, electroporation, liposome packaging, etc.

[0194] The resulting transformant can be cultured in conventional nutrient media to express the polypeptide encoded by the gene of the application. The culture conditions, such as temperature, pH and the like, are those previously determined to be appropriate for the host cell used. When the host cell has reached an appropriate cell density, the selected promoter is induced by the appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period.

[0195] The recombinant polypeptide of the above method can be expressed intracellularly, on the cell membrane, or secreted from the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0196] The antibodies of the application can be used alone or in combination or conjugation with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties, or any combination of these.

[0197] Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing detectable products.

[0198] Therapeutic agents that can be conjugated or coupled to the antibodies of the present application include, but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2 and the like; 4. Gold nanoparticles / nanorods; 5. Virus particles; 6. Liposomes; 7. Magnetic nanoparticles; 8. Prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or benzyl-hydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles and the like.

[0199] Pharmaceutical compositions

[0200] The present application also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or topical administration.

[0201] The pharmaceutical composition of the present application can be directly used to bind to EGFR protein molecules, and thus can be used to treat tumors. In addition, other therapeutic agents can also be used simultaneously.

[0202] The pharmaceutical composition of the present application comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned nanobody (or conjugate thereof) of the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, using normal saline or an aqueous solution containing glucose and other adjuvants by conventional methods. The pharmaceutical composition such as the needle, solution should be manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0203] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account the administration route, the patient's health status and the like, which are within the skill of the skilled physician.

[0204] Labeled nanobodies

[0205] In a preferred embodiment of the application, the nanobodies are labeled with a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label or a fluorescent label.

[0206] The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the application, the anti-EGFR nanobodies are labeled with colloidal gold to obtain colloidal gold-labeled nanobodies.

[0207] The anti-EGFR nanobodies of the application have good specificity and high titer.

[0208] Detection method

[0209] The application also relates to a method for detecting EGFR protein. The steps of the method are as follows: obtaining a cell and / or tissue sample; dissolving the sample in a medium; detecting the level of EGFR protein in the dissolved sample.

[0210] The application also relates to a method for detecting EGFR protein. The steps of the method are as follows: obtaining a cell and / or tissue sample; dissolving the sample in a medium; detecting the level of EGFR protein in the dissolved sample.

[0211] In the detection method of the application, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservative.

[0212] Kit

[0213] The application also provides a kit containing the antibody (or fragment thereof) or detection plate of the application. In a preferred embodiment of the application, the kit further comprises a container, instructions for use, a buffer, etc.

[0214] The application also provides a detection kit for detecting the level of EGFR, which comprises an antibody that recognizes EGFR protein, a lysis medium for dissolving the sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0215] Application

[0216] As described above, the nanobodies of the application have wide biological and clinical application value, and their applications involve diagnosis and treatment of diseases related to EGFR, basic medical research, biological research, etc. A preferred application is for clinical diagnosis and targeted therapy against EGFR.

[0217] The main advantages of the application are:

[0218] (a). The anti-EGFR nanobodies developed based on the llama nanobodies of the application have high endocytosis activity.

[0219] (b) The nanobody conjugated drug developed by the present application can directly and efficiently kill tumor cells.

[0220] (c) Compared with the conventional antibodies currently developed on the market, the nanobody developed by the present application has smaller molecular weight, lower immunogenicity in human body, is easy to modify, has higher stability, is easy to engineer, can further reduce the cost in the commercialization process, and brings better social benefits.

[0221] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0222] Example 1. Preparation of antigen

[0223] The hEGFR-His protein expression vector was constructed, and the hEGFR-His protein was prepared by inducing expression and purification in 293 cell lines. The purified hEGFR-His protein was verified by electrophoresis, and the verification of the protein purity is shown in Figure 2 : The protein purity is > 90%.

[0224] Verification of the activity of the purified EGFR protein: The hEGFR-His protein was diluted to 1 μg / mL, the enzyme-labeled plate was coated overnight, the BMK molecule was diluted to 4 μg / mL, and the binding activity was detected according to the 4-fold ratio 8 gradient; wherein BMK1: cetuximab; BMK2: regeneron, patent number US10047160B2; BMK3: abbvie, patent number US20200405878A1, and the experimental results are shown in Figure 3 : The hEGFR-His protein has good binding activity with BMK1 and BMK3.

[0225] Example 2. Immunization of hEGFR-His protein in llama

[0226] The llama was immunized according to the llama immunization strategy in Table 1;

[0227] Table 1 Llama immunization strategy

[0228] Period Experiment Dose Day 1 First immunization 800 μg Day 14 Second immunization 400 μg Day 28 Third immunization 400 μg Day 42 Fourth immunization 800 μg Day 56 Fifth immunization 800 μg

[0229] The first immunization used Freund's complete adjuvant, the second to fifth immunization used Freund's incomplete adjuvant, and a total of five immunizations were performed. After each immunization, blood was taken to prepare antiserum for serum titer detection:

[0230] Titer test: The hEGFR-His protein was diluted to 1 μg / mL, and the antiserum was diluted to 1:1000 for titer detection according to 8 gradient dilutions of 3 times. Im.0: pre-immune alpaca serum; Im.2: alpaca serum after the second immunization; Im.3: alpaca serum after the third immunization; Im.4: alpaca serum after the fourth immunization. The detection results are shown in Figure 4 : The alpaca obtained a high serum titer after four immunizations.

[0231] Example 3. Blood was taken from the alpaca after the fifth immunization to separate PBMC, and the technical method was as follows:

[0232] 1. The alpaca was subjected to jugular vein blood collection to obtain 100 mL of peripheral blood;

[0233] 2. An equal volume of 100 mL of normal saline was added to dilute the peripheral blood;

[0234] 3. PBMC separation solution was added to the bottom of the centrifuge tube, and the diluted peripheral blood was added to the upper layer.

[0235] 4. The PBMC cells were separated by centrifugation at room temperature.

[0236] 5. After centrifugation, the PBMC layer (i.e., the white membrane layer) was carefully aspirated and transferred to a 50 mL centrifuge tube.

[0237] 6. 30 mL of normal saline was added, and the cells were collected by centrifugation. The residual liquid was removed by washing to obtain the alpaca peripheral blood PBMC.

[0238] Example 4. Alpaca RNA extraction and cDNA preparation:

[0239] Alpaca RNA was extracted from alpaca PBMC and reverse transcribed into cDNA;

[0240] The alpaca RNA extraction was performed using a commercial kit (name: NucleoSpin RNA Plus, brand: MN, item number: 740984.5), and the RNA extraction steps were performed according to the kit steps.

[0241] The cDNA preparation was performed using PrimeScript TM 1st Strand cDNA Synthesis Kit (brand: Takara, item number: 6110A), and the cDNA preparation process was performed according to the kit instructions. The cDNA preparation was completed.

[0242] Example 5: Nanobody library construction

[0243] According to the method of Example 1, the alpaca was immunized with the hEGFR-His protein to obtain the alpaca serum, and the serum was used to screen the nanobody library. Figure 5The technical route shown is used to construct a llama nanobody library. Specifically, it includes:

[0244] 1. Designing upstream and downstream primers for llama nanobodies to amplify the llama VHH fragment, and obtaining the llama VHH fragment;

[0245] 2. Preparing a phagemid vector and simultaneously digesting the VHH fragment and the vector;

[0246] 3. Recovering and ligating the digested VHH fragment and the vector to prepare a VHH ligation product;

[0247] 4. Preparing E. coli electrotransformation competent cells and electrotransforming the ligation product to complete the construction of the nanobody library.

[0248] Example 6: Library panning and screening of target EGFR nanobodies

[0249] According to the technical route shown, the panning and screening of target EGFR nanobodies are carried out. Figure 6

[0250] Specific implementation steps:

[0251] 1. Culture and amplify the nanobody library, and infect the helper phage to package the library;

[0252] 2. Purify the packaged library to obtain a phage library;

[0253] 3. Use EGFR protein solid-phase coating to enrich and select to obtain a positive library;

[0254] 4. Select positive library monoclonal strains for monoclonal ELISA supernatant verification;

[0255] 5. Perform gene sequencing on positive monoclonal strains to obtain target EGFR nanobody sequences.

[0256] Experimental results: The monoclonal ELISA detection results are shown in Table 2, and the values of 0.3 or more positive clones are selected for sequencing;

[0257] Table 2A ELISA results of monoclonal B1 plate

[0258] B1 1 2 3 4 5 6 7 8 9 10 11 12 A 0.8358 0.0579 0.036 0.1334 0.0288 0.5615 0.4965 0.0356 0.3721 0.6512 0.0968 0.2204 B 0.0472 0.6588 0.6131 0.0328 0.0678 0.1606 0.1237 0.043 0.7434 0.0265 0.041 0.029 C 0.0645 0.6575 0.7096 0.9659 0.0246 0.028 0.0306 0.6614 0.227 0.036 0.0307 0.0839 D 0.0565 0.0396 0.4899 0.042 0.0255 0.9596 0.9169 0.0318 0.187 0.1097 0.053 0.5277 E 0.628 0.4396 0.0256 0.0255 0.6302 0.0604 0.4198 0.0701 0.0619 0.0293 0.0264 0.2821 F 0.0494 0.5302 0.3798 0.0253 0.0267 0.0266 0.5539 0.0236 0.0378 0.4246 0.0943 0.0416 G 0.0826 0.0373 0.059 0.0289 0.0793 0.028 0.8253 0.7954 0.2232 1.2109 0.0301 0.045 H 2.0952 0.6646 0.057 0.8941 0.3753 0.0344 2.2762 0.8531 0.6971 0.8992 0.8509 0.0454

[0259] Table 2B ELISA results of monoclonal B2 plate

[0260] B2 1 2 3 4 5 6 7 8 9 10 11 12 A 1.0699 0.0295 0.0169 0.0129 1.2256 0.0134 1.2001 0.0254 0.0204 1.2186 0.0198 0.0184 B 0.0268 1.2621 0.019 1.2931 1.2687 1.3181 1.2335 1.232 1.3101 0.0186 0.0187 0.0139 C 1.2778 0.0201 0.0123 0.0167 0.014 0.0195 1.1854 0.0149 1.206 0.0121 0.0117 0.015 D 0.0278 1.2144 1.2362 0.012 0.013 0.0197 1.2263 0.0117 0.02 0.0131 0.0131 0.0159 E 1.2819 0.023 0.0225 0.0142 0.0213 0.0139 0.0143 1.2032 0.0122 1.2236 0.0122 0.0165 F 1.1998 1.2078 1.1738 1.206 0.0197 0.0128 1.1763 0.0159 0.0172 0.0172 0.0121 0.0152 G 0.0172 1.2286 0.0195 0.0143 1.1887 0.0131 1.1581 0.0145 0.0219 1.1399 0.0199 0.0421 H 1.1312 0.038 0.0204 0.0257 0.0271 1.2099 0.0234 0.017 1.1917 0.0204 0.0179 0.0438

[0261] Table 2C ELISA results of monoclonal B3 plate

[0262] B3 1 2 3 4 5 6 7 8 9 10 11 12 A 0.0228 1.3234 0.0163 1.4661 0.0154 1.4029 0.0115 1.4554 0.016 0.016 1.378 0.0945 B 0.0198 0.0244 0.0165 1.286 1.4205 0.0239 0.0139 1.2047 1.4229 0.0133 0.0121 1.4311 C 0.0182 1.1727 1.3418 0.0164 0.0148 1.2393 0.0145 1.1813 0.0248 0.0148 0.0138 0.0165 D 1.36 0.0246 0.0249 0.0237 0.0259 0.0229 1.2731 1.2955 1.2676 0.0163 1.2319 0.0178 E 0.7862 0.0211 0.015 0.0149 1.392 1.3779 0.0243 0.1391 1.2565 1.3445 0.02 0.0165 F 0.0299 0.0154 0.0242 0.0163 0.0248 1.2882 0.0165 0.0166 1.3415 0.0193 0.0155 0.0178 G 0.0225 1.3481 0.0175 1.2672 0.017 0.0153 1.26 0.0145 1.2378 1.3023 0.0156 0.0395 H 0.0306 1.3523 0.0342 0.026 0.0225 0.0239 0.0198 0.0275 1.2926 1.3669 0.0296 0.0532

[0263] ​Table 2D Monoclonal B4 plate ELISA results

[0264] B4 1 2 3 4 5 6 7 8 9 10 11 12 A 1.3937 0.0998 1.4029 1.4371 0.02 1.1429 0.026 0.0336 0.0343 0.6302 1.4793 1.6108 B 1.3725 1.5723 0.4543 0.0509 1.1362 0.1054 0.0273 0.0539 0.0443 1.7301 0.9597 1.0655 C 0.0771 0.0322 0.0504 0.0946 0.0633 0.0384 0.0526 1.2999 1.4369 0.0451 1.3102 0.115 D 1.5908 1.1695 1.0614 0.0776 1.4472 1.4836 0.1105 0.1119 0.0211 0.0316 0.8277 0.0468 E 0.0531 1.3958 0.0646 0.0476 0.0571 0.8217 0.0209 0.9121 0.1253 1.4179 1.2701 1.4456 F 1.6853 1.4822 1.1447 0.2767 1.4296 1.4325 1.5065 1.1178 0.1478 1.2886 0.0383 1.4705 G 1.7481 0.0728 0.5414 0.0527 0.1422 0.9068 1.6295 1.4971 1.218 1.4568 1.5908 0.114 H 1.2997 1.5126 1.8319 1.7626 0.1041 1.3744 0.0767 0.0546 0.4076 0.0687 0.1562 0.1458

[0265] Example 7

[0266] The expression and preparation of nanobodies were carried out using a mammalian cell expression system, and the technical route is shown in Figure 7 .

[0267] According to the above route, 57 strains of nanobodies were prepared by expression, and 20 mL system was expressed using CHO cell system, and nanobodies were obtained after purification; the information of nanobodies after purification is shown in Table 3.

[0268] Table 3 Nanobody expression and purification

[0269]

[0270]

[0271] The results of SEC purity detection of 57 strains of nanobodies are shown in Figure 8 .

[0272] Example 8: ELISA detection of binding activity of 57 strains of nanobodies

[0273] Principle of experiment: The hEGFR-His protein is connected to the solid carrier, and after the antibody to be tested is combined with the antigen, it is combined with the enzyme-labeled secondary antibody to form an antigen-antibody to be tested-enzyme-labeled secondary antibody complex, and the amount of complex formation is proportional to the amount of antibody to be tested;

[0274] Specific experimental process:

[0275] 1. The target protein hEGFR-His was diluted to 1 μg / mL, and 100 μL was added to each well of the enzyme-labeled plate for overnight coating;

[0276] 2. The nanobody to be tested was diluted to 4 μg / mL, and 100 μl was added to each well at a gradient of 4 times 6;

[0277] 3. Incubate at 37°C for 60 minutes;

[0278] 4. After diluting the enzyme-labeled secondary antibody 1:10,000, 100 μL of enzyme-labeled secondary antibody was added to each well, and incubated at 37°C for 30 minutes;

[0279] 5. Add 100 μL of TMB color developing liquid to each well, color develop at room temperature for 5 minutes, add 50 μL of stop solution, and read after stopping;

[0280] The experimental results are shown in Figure 9AResults analysis see Table 4: Nanobody ELISA binding EC50.

[0281] Results analysis see Table 4: Nanobody ELISA binding EC50.

[0282] Table 4 Nanobody ELISA binding EC50

[0283] Antibody KY303-01 KY303-02 KY303-03 KY303-04 KY303-05 KY303-06 KY303-07 KY303-08 KY303-09 KY303-10 EC50 0.8665 0.8917 0.8658 0.7792 0.8285 0.8619 0.8521 0.8765 0.914 0.9620 Antibody KY303-11 KY303-12 KY303-13 KY303-14 KY303-15 KY303-16 KY303-17 KY303-18 KY303-19 KY303-20 EC50 0.7795 0.8829 0.8697 0.8153 1.009 0.4923 0.4848 0.8350 0.8312 0.7642 Antibody KY303-21 KY303-22 KY303-23 KY303-24 KY303-25 KY303-26 KY303-27 KY303-28 KY303-29 KY303-30 EC50 0.8452 0.8292 1.1690 0.8513 0.8460 1.7990 0.9715 0.5505 0.6071 0.8749 Antibody KY303-31 KY303-32 KY303-33 KY303-34 KY303-35 KY303-36 KY303-37 KY303-38 KY303-39 KY303-40 EC50 0.3598 0.7144 0.4236 0.4112 0.5816 0.6529 0.5571 0.8380 0.3271 0.4079 Antibody KY303-41 KY303-42 KY303-43 KY303-44 KY303-45 KY303-46 KY303-47 KY303-48 KY303-49 KY303-50 EC50 0.8922 0.7665 0.6611 0.5964 0.8014 0.8470 0.7894 0.8519 0.7821 0.6329 Antibody KY303-51 KY303-52 KY303-53 KY303-54 KY303-55 KY303-56 KY303-57 BMK2 EC50 0.7032 0.7915 0.7786 0.8238 0.8072 0.8247 0.7394 0.8175

[0284] Example 9: FACS detection of 57 Nanobodies binding activity

[0285] The specific implementation steps are as follows:

[0286] 1. Preparation before experiment: Prepare EGFR-CHO-K1 cells in logarithmic growth phase in advance;

[0287] 2. Cell treatment: Digest and collect cells, resuspend cells with PBS, and adjust the cell number to a concentration of 3E6 / mL;

[0288] 3. Cell plating: Add 100 μL / well of cell suspension to a 96-well (V-bottom) cell culture plate;

[0289] 4. Antibody preparation: Dilute the antibody in advance with PBS+1% BSA buffer, 20 μg / mL in the first well, and 4-fold gradient dilution, a total of 8 concentration gradients;

[0290] 5. Incubation of primary antibody: Add 100 μL / well of antibody diluent to the cell plate with cell suspension, mix well with a blow gun after adding, and incubate at 4°C for 60 minutes in the dark;

[0291] 6. Washing: After incubation, centrifuge at 500g for 5 minutes, discard the supernatant. Wash the cells twice with 200 μL / well of PBS+1% BSA;

[0292] 7. Incubation of secondary antibody: Add 100 μL / well of PE-anti-human-Fc antibody diluent (1:100 dilution), mix well after blowing, and incubate at 4°C for 30 minutes in the dark;

[0293] 8. Washing: After incubation, centrifuge at 500g for 5 minutes, discard the supernatant. Wash the cells twice with 200 μL / well of PBS+1% BSA;

[0294] 9. Resuspension: Resuspend the cells in each well with 200 μL of PBS+1% BSA;

[0295] 10. FACS detection: Median-PE of fluorescence intensity expressed in the cells to be tested, curve according to fluorescence intensity;

[0296] The experimental results are shown in Figure 10A -F are shown: among them, the nanobodies KY303-12, KY303-16, KY303-20, KY303-23, KY303-24, KY303-25, KY303-26, KY303-27, KY303-28, KY303-29, KY303-30, KY303-31, KY303-54 have no binding at the FACS level, and the rest have strong binding.

[0297] The result analysis is shown in Table 5: nanobody FACS binding EC50.

[0298] Table 5 Nanobody FACS binding EC50

[0299] Antibody KY303-01 KY303-02 KY303-03 KY303-04 KY303-05 KY303-06 KY303-07 KY303-08 KY303-09 KY303-10 EC50 0.0867 0.0934 0.3647 0.0894 1.4400 0.5697 0.3633 0.6145 0.4715 0.9081 Antibody KY303-11 KY303-12 KY303-13 KY303-14 KY303-15 KY303-16 KY303-17 KY303-18 KY303-19 KY303-20 EC50 0.7651 / 1.0420 0.7982 0.02631 / 0.7553 0.1986 0.0455 / Antibody KY303-21 KY303-22 KY303-23 KY303-24 KY303-25 KY303-26 KY303-27 KY303-28 KY303-29 KY303-30 EC50 0.7292 2.1510 / / / / / / / / Antibody KY303-31 KY303-32 KY303-33 KY303-34 KY303-35 KY303-36 KY303-37 KY303-38 KY303-39 KY303-40 EC50 / 0.3191 0.3576 1.7710 0.4261 0.5227 0.2982 0.4126 1.8820 0.9408 Antibody KY303-41 KY303-42 KY303-43 KY303-44 KY303-45 KY303-46 KY303-47 KY303-48 KY303-49 KY303-50 EC50 1.1030 1.0120 0.7126 0.7766 0.5865 0.2845 0.8605 1.5050 0.7054 5.0920 Antibody KY303-51 KY303-52 KY303-53 KY303-54 KY303-55 KY303-56 KY303-57 BMK1 EC50 1.3010 0.6983 0.8519 / 0.8785 0.6381 0.3918 0.06213

[0300] Example 10: FACS specific binding detection of 57 nanobodies

[0301] The specific implementation steps are as follows:

[0302] 1. Preparation before experiment: Prepare CHO-K1 cells in logarithmic growth phase in advance;

[0303] 2. Cell treatment: digest and collect the cells, resuspend the cells with PBS, and adjust the cell number to a concentration of 3E6 / mL;

[0304] 3. Cell plating: add 100 μL / well of cell suspension to a 96-well (V-bottom) cell culture plate;

[0305] 4. Antibody preparation: dilute the antibody in advance with PBS+1% BSA buffer. Dilute the antibody to 20 μg / mL;

[0306] 5. Incubation of primary antibody: add 100 μL / well of antibody diluent to the cell plate with cell suspension, mix well with a blow gun after adding, and incubate at 4°C in the dark for 60 minutes;

[0307] 6. Washing: centrifuge at 500g for 5 minutes after incubation, discard the supernatant. Add 200 μL / well of PBS+1% BSA to wash the cells twice;

[0308] 7. Incubation of secondary antibody: add 100 μL / well of PE-anti-human-Fc antibody diluent (1:100 dilution), mix well after blowing, and incubate at 4°C in the dark for 30 minutes;

[0309] 8. Wash: 500g centrifuge for 5 minutes, discard supernatant; add 200 μL / well PBS + 1% BSA to wash cells twice;

[0310] 9. Resuspend: resuspend cells with 200 μL PBS + 1% BSA per well;

[0311] 10. FACS detection: Median-PE of the fluorescence intensity expressed by the cells to be tested.

[0312] The experimental results are shown in Table 1 Figure 11A -B, wherein KY303-25, KY303-28, KY303-29, KY303-30 have non-specific binding with CHO-K1.

[0313] Example 11: Detection of endocytosis activity of EGFR high-binding activity nanobodies in different tumor cell lines

[0314] 1. Prepare the cells to be tested (HCT116, 769-P, 786-O) in the logarithmic growth phase in advance;

[0315] 2. Dilute the cells to a concentration of 2E6 / mL and transfer them to 1.5 mL EP tubes;

[0316] 3. Add 100 μL of the cell suspension to each well of a 96-well plate, centrifuge at 500g for 5 minutes, and discard the supernatant;

[0317] 4. Prepare the antibody to be tested: dilute the antibody to be tested to 10 μg / mL with complete medium, add 100 μL / well of the antibody to be tested, incubate at 4°C for 60 minutes, centrifuge at 500g for 5 minutes, and discard the supernatant;

[0318] 5. Add 200 μL of PBS + 1% bovine serum albumin (BSA) per well to wash away the unbound antibody, centrifuge at 500g for 5 minutes, discard the supernatant, and repeat 2 times;

[0319] 6. Add 100 μL / well of complete medium to each well, and incubate the cells for 2 hours at 37°C to detect the internalization efficiency, and incubate the control cells in the refrigerator at 4°C as a control;

[0320] 7. Centrifuge at 500g for 5 minutes, and discard the supernatant;

[0321] 8. Add 100 μL / well of PE-anti-human-Fc antibody diluted with complete medium to each well;

[0322] 9. Incubate at 4°C in the dark for 30 minutes, centrifuge at 500g for 5 minutes, and discard the supernatant;

[0323] 10. Add 200 μL of PBS + 1% bovine serum albumin (BSA) to each well to wash the cells, centrifuge at 500g for 5 minutes, discard the supernatant, and repeat twice;

[0324] 11. Add 200 μL of PBS + 1% bovine serum albumin (BSA) to each well to resuspend the cells;

[0325] 12. Use FACS to detect the mean fluorescence intensity (MFI) of the expression in the test cells.

[0326] The experimental results are shown in Figure 12A As shown in -C, KY303-39, KY303-50, and KY303-52 exhibit high endocytic activity on cells.

[0327] Example 12: Detection of the binding affinity between three highly endocytic nanobodies and EGFR protein

[0328] The results are shown in Table 6: the affinity of KY303-39 is 2.61E-8; the affinity of KY303-50 is 2.50E-7; and the affinity of KY303-52 is 2.72E-7.

[0329] Table 6. Affinity Detection of Highly Endocytotic Nanobodies

[0330] Antibody Ka (1 / Ms) Kd (1 / Ms) KD (M) KY303-39 1.47E+5 3.82E-3 2.61E-8 KY303-50 2.36E+3 5.88E-4 2.50E-7 KY303-52 1.06E+4 2.89E-3 2.72E-7

[0331] The sequences of the three highly endocytic nanobodies KY303-39, KY303-50, and KY303-52 are shown in Table 7.

[0332] Table 7. Nanobody sequences of KY303-39, KY303-50, and KY303-52

[0333]

[0334] Example 13: Three highly endocytic nanobodies KY303-39, KY303-50, and KY303-52 conjugated with MMAE

[0335] Nanobody-conjugated MMAE technology route such as Figure 13 As shown.

[0336] The specific implementation steps are as follows:

[0337] 1. Antibody reduction: The reduction system was fixed and reduced in a metal bath at 37°C and 200 rpm for 4 hours;

[0338] 2. Toxin Coupling: With a fixed amount of Vc-MMAE, the mixture was reacted in a metal bath at 4°C and 200 rpm for 2 hours;

[0339] 3. Reaction termination: L-Cysteine solution was added to terminate the reaction;

[0340] 4. ADC purification: the solution after conjugation was repeatedly centrifuged with 10 kDa ultrafiltration tube to replace the solution;

[0341] 5. Activity detection: DAR value and free toxin detection.

[0342] The experimental results are shown in Table 8: the DAR value of the nanobody KY303-39-MMAE after conjugation is 2.76; the DAR value of the nanobody KY303-50-MMAE after conjugation is 2.93; and the DAR value of the nanobody KY303-52-MMAE after conjugation is 2.99.

[0343] Table 8: Conjugation of MMAE with KY303-39, KY303-50, KY303-52 nanobodies and BMK3 antibody

[0344]

[0345] Example 11: In vitro killing activity detection of three strains of nanobodies conjugated with MMAE

[0346] Specific implementation steps:

[0347] 1. Experimental preparation: prepare the cells to be tested in the logarithmic growth phase in advance;

[0348] 2. Cell plating: collect and process the cells to be tested by digestion, and prepare a cell suspension with complete culture medium. Add 100 μL / well of cell suspension to a 96-well black transparent flat-bottom plate. Discard the edge wells of the cell plate and add 100 μL / well of PBS. Place the cell plate with plated cells in an incubator overnight to allow the cells to adhere;

[0349] 3. ADC preparation: dilute the ADC with complete culture medium. The first well is 120 μg / mL, and the next four wells are diluted by 4 times, for a total of 6 concentrations;

[0350] 4. Incubation of ADC: add 20 μL / well of ADC dilution to the 96-well cell culture plate and incubate at 37°C, 5% CO2 for 6 days;

[0351] 5. Detection: after incubation, add 100 μL / well of detection solution to the 96-well cell culture plate, and let it stand for 15 minutes until the cells are completely lysed;

[0352] 6. Plate reading: detect the measured luminescence value with an enzyme marker, calculate the cell viability according to the luminescence value, make a curve, and calculate the IC50.

[0353] 7. The experimental results are shown in Figure 14A -G: three strains of nanobody-conjugated drugs have strong killing activity on seven tumor cells;

[0354] The results are shown in Table 9: the IC50 of 3 ADCs against 7 tumor cells, KY303-39-MMAE, KY303-50-MMAE and KY303-52-MMAE are better than BMK3-MMAE in killing NCI-H1975, BxPC-3, MDA-MB-468, NCI-H1993 cells.

[0355] Table 9: IC50 of 3 ADCs against 7 tumor cells

[0356]

[0357] Example 12

[0358] The in vivo efficacy of three ADCs was evaluated, and the technical details are as follows:

[0359] 1. The tumor cells HCT116 and NCI-H1975 (China Academy of Sciences Typical Culture Preservation Committee Cell Library) were inoculated into female NCG mice (purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd., 5-6 weeks old, 18-21 g);

[0360] 2. When the tumor grew to 100-150 mm 3 around, the ADC and control PBS were injected into the tail vein;

[0361] 3. The mice inoculated with HCT116 tumor cell strain were injected with nanobody conjugated drug at a dose of 4.5 mg / kg, BMK drug at a dose of 0.9 mg / kg, and PBS; the mice inoculated with NCI-H1975 tumor cell strain were injected with nanobody conjugated drug at a dose of 3 mg / kg, BMK drug at a dose of 5.6 mg / kg, and PBS;

[0362] 4. The change in tumor volume was measured and the tumor inhibition rate was calculated;

[0363] The test results are shown in Figure 15A -D: On Day 18, the HCT116 tumor growth was significantly inhibited at a dose of 4.5 mg / kg, with an inhibition rate of 60.2%, and the mouse body weight had no significant change; on Day 14, the NCI-H1975 tumor growth inhibition rate was 93.5% at a dose of 3 mg / kg, and the mouse body weight had no significant change.

[0364] The above results show that KY303-39-MMAE, KY303-50-MMAE, KY303-52-MMAE can significantly inhibit tumor growth and have good in vivo safety, and have the potential to be developed as ADC anti-tumor drugs.

[0365] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to those skilled in the art upon consideration of this disclosure or can be learned from practice of the application. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced therein.

Claims

1. An anti-EGFR nanobody, characterized in that, The complementarity determining regions (CDRs) of the VHH chain of the nanobody are as follows: CDR1 as set forth in SEQ ID NO: 5, CDR2 as set forth in SEQ ID NO: 7, CDR3 as set forth in SEQ ID NO:

10.

2. The anti-EGFR nanobody of claim 1, wherein The amino acid sequence of the VHH chain of the nanobody is SEQ ID NO:

2.

3. A polynucleotide, comprising, The polynucleotide encodes a protein selected from the group consisting of the anti-EGFR nanobody of claim 1.

4. The polynucleotide of claim 3, wherein, The polynucleotide comprises DNA, RNA, or cDNA.

5. An expression vector, characterized by, The expression vector contains the polynucleotide of claim 3.

6. A host cell, characterized in that, The host cell contains the expression vector of claim 5, or has integrated into its genome the polynucleotide of claim 3.

7. The host cell of claim 6, wherein The host cell comprises a prokaryotic cell or a eukaryotic cell.

8. The host cell of claim 6, wherein, The host cell is selected from the group consisting of E. coli, a yeast cell, a mammalian cell, a bacteriophage, or a combination thereof.

9. A method of producing an anti-EGFR nanobody, characterized in that, The method comprises the steps of: (a) culturing the host cell of claim 6 under conditions suitable for production of the nanobody, thereby obtaining a culture containing the anti-EGFR nanobody; and (b) isolating or recovering the anti-EGFR nanobody from the culture; and (c) purifying and / or modifying the anti-EGFR nanobody obtained in step (b).

10. An immunoconjugate, comprising, The immunoconjugate contains: (a) the anti-EGFR nanobody of claim 1; and (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, or a combination thereof.

11. A recombinant protein, characterized in that, The recombinant protein has: (i) the sequence of the nanobody of claim 1; and (ii) a tag sequence that facilitates expression and / or purification.

12. The recombinant protein of claim 11, wherein, The tag sequence comprises a 6His tag and a HA tag.

13. The recombinant protein of claim 11, wherein, The recombinant protein specifically binds to the EGFR protein.

14. A pharmaceutical composition, characterized by, The pharmaceutical composition contains: (i) the anti-EGFR nanobody of claim 1, or the immunoconjugate of claim 10, or the recombinant protein of claim 11; and (ii) a pharmaceutically acceptable carrier.

15. Use of the anti-EGFR Nanobody according to claim 1, or the immunoconjugate according to claim 10, or the recombinant protein according to claim 11, or the pharmaceutical composition according to claim 14, characterized in that, for: (a) preparing a medicament for preventing and / or treating an EGFR-positive tumor; and / or (b) preparing a reagent, a plate, or a kit for detecting EGFR; wherein the tumor is selected from the group consisting of bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer, or a combination thereof.

Images