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 case is a divisional application of Chinese invention patent application filed on November 30, 2023, with application number CN202311625877.5 and invention title "Nanobodies Targeting EGFR, Drug Conjugates and Their Uses". Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to nanobodies that can target EGFR and their applications. Background Technology

[0003] Cancer remains one of the greatest threats to human health worldwide, causing more than 10 million deaths annually. Although many cancer treatments exist, including surgery, radiotherapy, and chemotherapy, these traditional treatments still have many problems, such as limited effectiveness and significant side effects.

[0004] Compared to traditional treatments, the latest approaches based on targeted therapies (such as antibodies) have fewer side effects. Furthermore, conjugating antibodies to cytotoxic drugs or toxins can greatly improve treatment efficacy through cell internalization. These antibody-drug conjugates are also known as antibody-drug conjugates (ADCs).

[0005] Nanobodies, discovered by scientists in 1989, are heavy chain antibodies derived from camels and naturally lacking their light chains, containing variable regions. Their protein crystal structure is 4.0 nm in length and 2.5 nm in diameter, making them the smallest known antibodies, hence the name nanobody. Compared to traditional antibodies, nanobodies exhibit higher stability and stronger tolerability. Furthermore, they are easily expressed and readily genetically engineered. Based on their advantages in stability and penetration, nanobodies are currently attracting widespread attention in disease treatment, diagnosis, and substance detection.

[0006] Phage display technology involves inserting a foreign gene into the genome of a filamentous phage, allowing the protein or polypeptide encoded by the target gene to be displayed on the phage surface as a fusion protein. Compared with traditional antibodies, nanobodies are easier to express and display on the phage surface because they naturally lack light chains, do not require light and heavy chain assembly and pairing, and have smaller molecular weights. Combined with the huge library advantage of phage display technology, it is easier to discover nanobody molecules with sequence differences and functional differences during the development of nanobodies.

[0007] The epidermal growth factor receptor (EGFR), encoded by the c-erbB proto-oncogene, is a transmembrane receptor with a molecular weight of approximately 170 kDa. It is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Upon binding to its specific ligands, including epidermal growth factor (EGF) and transforming growth factor α (TGFα), EGFR dimers, further stimulating intracellular protein tyrosine kinase activity and triggering downstream signaling cascades, leading to DNA synthesis and cell proliferation. EGFR also participates in phenotypic regulation of 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. In these cancers, EGFR overexpression is also significantly associated with poor prognosis.

[0009] Currently, EGFR-targeted drug development is mainly based on functional blockers, which can be divided into two main categories: one is small molecule drugs, namely tyrosine kinase inhibitors (TKIs) synthesized based on intracellular signal transduction, including gefitinib and erlotinib; the other is ligand blockers, mainly monoclonal antibody drugs developed based on traditional antibodies to block extracellular ligand signal transduction, such as cetuximab and panitumumab. However, for cancer treatment, current products still have many shortcomings, such as limited efficacy, high drug resistance, and significant toxic side effects, resulting in very limited social benefits.

[0010] EGFR-targeted ADCs differ from those that act by inhibiting receptor signaling pathways. ADCs can directly deliver cytotoxins to EGFR-overexpressing tumor cells, where they are internalized, enhancing the drug's killing effect and improving the effectiveness of targeted therapy. Therefore, theoretically, ADCs have broader indications and more potent efficacy than traditional therapies. Currently, most EGFR-targeted ADCs are in clinical trials, such as AVID-100, ABT-414, and ABBV-221. Related patents, such as CN106470697, CN114585391, and CN111295201, disclose antibody forms that are traditional monoclonal antibodies. Traditional monoclonal antibodies typically have many drawbacks, including large molecular weight, poor stability, difficult manufacturing processes, and high immunogenicity. Furthermore, two related ADC forms targeting EGFR already exist. IMGN-289 and AMG-595 were also forced to terminate their clinical trials due to toxicity issues (such as skin toxicity and gastrointestinal toxicity). Based on toxicity issues, AstraZeneca's AZD9592 (patent number: US20230183358A1) also incorporated a design to reduce affinity at the EGFR end in order to reduce EGFR-driven tissue toxicity. Therefore, research on ADCs targeting EGFR still faces many challenges in terms of efficacy and safety.

[0011] Nanobodies with smaller molecular weights are superior to traditional antibodies in many aspects, such as small size, high stability, low immunogenicity, stronger tissue penetration, and low production cost.

[0012] Therefore, the development of EGFR-targeting nanobodies and antibody-related drugs based on alpaca nanobodies is of great significance.

[0013] Therefore, there is an urgent need in this field to develop a nanobody and nanobody-conjugate that can target EGFR with high specificity, low affinity, high endocytic activity, and strong tumor cell killing activity. Summary of the Invention

[0014] This invention provides a nanobody and nanobody-conjugated drug that can target EGFR with high specificity, low affinity, high endocytic activity, and strong tumor cell killing activity.

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

[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 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:10;

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

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

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

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

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

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

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

[0025] In another preferred embodiment, the nanobody is bound to human, mouse, or monkey-derived EGFR.

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

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

[0028] In another preferred embodiment, the VHH chain of the anti-EGFR nanobody has one or more 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 (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[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, and SEQ ID NO:3.

[0031] In a second aspect of the invention, a nanoantibody fusion protein is provided, the nanoantibody fusion protein having a structure from the N-terminus to the C-terminus as shown in Formula I:

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

[0033] In the formula,

[0034] Z1 is the VHH chain of the anti-EGFR nanobody as described in the first aspect of the present invention;

[0035] Z2 is the Fc segment of an immunoglobulin;

[0036] L represents the connector sequence;

[0037] Z3 is the immunomodulatory molecular component.

[0038] In a third aspect of the invention, a polynucleotide is provided that encodes a protein selected from the group consisting of the anti-EGFR nanobody described in the first aspect of the invention.

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

[0040] In a fourth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide described in the third aspect of the invention.

[0041] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

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

[0043] In a fifth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the fourth aspect of the invention, or having the polynucleotide described in the third aspect of the invention integrated into its genome.

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

[0045] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0046] In a sixth aspect of the invention, a method for generating anti-EGFR nanobodies is provided, comprising the steps of:

[0047] (a) Culturing the host cells described in the fifth aspect of the invention under conditions suitable for the production of nanobodies, thereby obtaining a culture containing the anti-EGFR nanobodies; and

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

[0049] (c) Optionally, purify and / or modify the anti-EGFR nanobody obtained in step (b).

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

[0051] (a) the anti-EGFR nanobody as described in the first aspect of the present invention; and

[0052] (b) The coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0053] In another preferred embodiment, the coupling portion is a drug or 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: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0056] In another preferred embodiment, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and microtubule inhibitors. Typical 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[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0057] In another preferred embodiment, the toxin is selected from the group consisting of: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, methamphetamine, pyrine, pyrine A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxychloroquine. Anthraxone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachomycin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0058] In another preferred embodiment, the coupling portion is a detectable marker.

[0059] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0060] In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) anti-EGFR nanobody as described in the first aspect of the invention. The multivalent meaning refers to the presence of a plurality of repeating anti-EGFR nanobodies as described in the first aspect of the invention within the amino acid sequence of the immunoconjugate.

[0061] In an eighth aspect of the invention, the use of the anti-EGFR nanobody of the present invention is provided, which is used to prepare (a) a reagent for detecting EGFR molecules; and (b) a drug for treating tumors.

[0062] In another preferred embodiment, the detection includes flow cytometry and cell immunofluorescence detection.

[0063] In a ninth aspect of the invention, one or more uses of an anti-EGFR nanobody are provided:

[0064] (i) Used for detecting human EGFR molecules;

[0065] (ii) Used for flow cytometry detection;

[0066] (iii) Used for cell immunofluorescence detection;

[0067] (iv) Used to treat tumors;

[0068] (v) Used for tumor diagnosis.

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

[0070] In a tenth aspect of the present invention, a multispecific antibody is provided, the multispecific antibody comprising: the anti-EGFR nanobody described in the first aspect of the present invention.

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

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

[0073] (i) the sequence of the nanobody as described in the first aspect of the invention; and

[0074] (ii) Optional Fc segment; and

[0075] (iii) Optional tag sequences to assist in expression and / or purification.

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

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

[0078] In a twelfth aspect of the invention, the use of nanobodies as described in the first aspect of the invention, or immunoconjugates as described in the seventh aspect of the invention, is provided for use in the preparation of pharmaceuticals, reagents, detection plates, or kits.

[0079] The reagents, detection plates, or kits are used to detect EGFR protein in samples.

[0080] The drug is used to treat or prevent tumors that express EGFR protein (i.e., EGFR positive).

[0081] In another preferred embodiment, the tumor includes: 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 invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

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

[0084] (ii) Pharmaceutically acceptable carriers.

[0085] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0086] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating tumors, said tumors including cancers 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 combinations thereof.

[0087] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating tumors, said tumors including cancers selected from the group consisting of lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer, or combinations thereof.

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

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

[0090] In another preferred embodiment, the other drugs for treating immune system diseases or tumor diseases are selected from the group consisting of: budesonide, fluticasone, beclomethasone, mometasone furoate, salbutamol, theophylline, formoterol, tiotropium bromide, sulfasalazine, methotrexate, cyclophosphamide, fluorouracil, bleomycin, anastrozole, or combinations thereof.

[0091] In a fourteenth aspect of the invention, use is provided for an anti-EGFR nanobody as described in a first aspect of the invention, or an immunoconjugate as described in a seventh aspect of the invention, or a recombinant protein as described in an eleventh aspect of the invention, or a pharmaceutical composition as described in a thirteenth aspect of the invention, for:

[0092] (a) To prepare medicines for the prevention and / or treatment of EGFR-related diseases; and / or

[0093] (b) Prepare reagents, test plates or kits for detecting EGFR.

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

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

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

[0097] In another preferred embodiment, the reagent is used to detect EGFR protein or fragments thereof in a sample.

[0098] In another preferred embodiment, the detection includes flow cytometry and 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 invention, a method for detecting EGFR protein in a sample is provided, the method comprising the steps of:

[0101] (1) Contact the sample with the nanobody described in the first aspect of the present invention;

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

[0103] In a sixteenth aspect of the invention, a method for treating a disease is provided, the method comprising administering to a desired subject the nanobody described in the first aspect of the invention or the immunoconjugate described in the seventh aspect of the invention.

[0104] In another preferred embodiment, the object includes mammals, such as humans.

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

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

[0107] (i) the anti-EGFR nanobody described in the first aspect of the present invention, or the immunoconjugate described in the seventh aspect of the present invention, or the recombinant protein described in the eleventh aspect of the present invention; and

[0108] (ii) A detectable carrier.

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

[0110] In another preferred embodiment, the detection-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 isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

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

[0113] In another preferred embodiment, the test reagent is in liquid or powder form (such as aqueous solution, injection, lyophilized powder, tablet, lozenge, or inhaler).

[0114] In an eighteenth aspect of the invention, a kit for EGFR protein is provided, the kit containing the immunoconjugate described in the seventh aspect of the invention or the detection reagent described in the seventeenth aspect of the invention, and instructions.

[0115] In another preferred embodiment, the instruction manual states that the kit is used for non-invasive detection of EGFR expression in a test subject.

[0116] In a nineteenth aspect of the invention, the use of the immunoconjugate described in the seventh aspect of the invention is provided for the preparation of a contrast agent for in vivo detection of EGFR protein.

[0117] In another preferred embodiment, the test is used for the diagnosis or prognosis of EGFR-related diseases or conditions.

[0118] In a twentieth aspect of the invention, a method for treating EGFR-related diseases is provided, the method comprising administering to a desired subject an anti-EGFR nanobody as described in the first aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention, or a recombinant protein as described in the eleventh aspect of the invention, or a pharmaceutical composition as described in the thirteenth aspect of the invention.

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

[0120] In another preferred embodiment, the non-human mammals include rodents (such as mice and rabbits) and non-human primates (such as monkeys).

[0121] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0122] Figure 1 This illustrates the overall technical approach of the present invention.

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

[0124] Figure 3 The binding activity of EGFR antigen to BMK was demonstrated.

[0125] Figure 4 The results show the alpaca serum titer assay.

[0126] Figure 5 The technical route for constructing alpaca nanobody libraries is shown.

[0127] Figure 6 The panning for EGFR-targeting nanobodies is shown.

[0128] Figure 7 The technical route for nanobody expression and preparation is shown.

[0129] Figure 8 The purified nanobody was detected by SEC assay.

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

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

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

[0133] Figure 9D The activity assay of KY303-31 to KY303-40 nanoantibodies by ELISA is shown.

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

[0135] Figure 9F The activity assay of KY303-51 to KY303-57 nanoantibodies was shown.

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

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

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

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

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

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

[0142] Figure 11A The specific binding detection of KY303-01 to KY303-30 nanobodies is shown.

[0143] Figure 11B The specific binding detection of KY303-31 to KY303-57 nanobodies is shown.

[0144] Figure 12A The endocytosis rate of nanobodies on HCT116 cells is shown.

[0145] Figure 12B The endocytosis rate of nanobodies on 769-P cells is shown.

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

[0147] Figure 13 The nanobody-conjugated MMAE technology route is shown.

[0148] Figure 14A The study demonstrated the cytotoxic effect of ADC on NCI-H1975 cells.

[0149] Figure 14B The study demonstrated the cytotoxic effect of ADC on HCT116 cells.

[0150] Figure 14C The study demonstrated the cytotoxic effect of ADCs on BxPC-3 cells.

[0151] Figure 14D The study demonstrated the cytotoxic effect of the ADC on MDA-MB-468 cells.

[0152] Figure 14E The study demonstrated the cytotoxic effect of ADC on NCI-H1993 cells.

[0153] Figure 14F The study demonstrated the cytotoxic effect of ADC on HT29 cells.

[0154] Figure 14G The study demonstrated the cytotoxic effect of the ADC on MDA-MB-231 cells.

[0155] Figure 15A The changes in tumor volume in the mouse HCT116 model are shown.

[0156] Figure 15B The changes in body weight of the HCT116 model mice are shown.

[0157] Figure 15C The changes in tumor volume in the mouse NCI-H1975 model are shown.

[0158] Figure 15D The changes in body weight of the NCI-H1975 model mice are shown. Detailed Implementation

[0159] Through extensive and in-depth research and screening, the inventors unexpectedly obtained an anti-EGFR nanobody with excellent endocytic activity and specificity.

[0160] Experimental results show that the nanobodies of this invention possess good binding activity and high endocytic activity. Furthermore, the nanobodies of this invention, when conjugated with cytotoxic agents (MMAE), exhibit strong tumor cell killing activity, significantly inhibiting tumor growth, and demonstrating good in vivo safety. Based on these findings, this invention was completed.

[0161] This invention develops an EGFR-targeting nanobody molecule based on alpaca nanobodies and phage display technology. This molecule exhibits high binding and endocytic activity to tumor cell lines and, when developed as an antibody-drug conjugate (ADC), demonstrates good tumor cell killing activity, showing potential for development into an ADC anti-tumor drug. The overall technical route of this invention is as follows: Figure 1 As shown.

[0162] As used herein, the terms "nanobody of the present invention," "anti-EGFR nanobody of the present invention," and "EGFR nanobody of the present invention" are used interchangeably and all refer to nanobodies that specifically recognize and bind to EGFR (including human EGFR). Particularly preferred are nanobodies with VHH chain amino acid sequences as shown in any of SEQ ID NO.:1-3.

[0163] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0164] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning: to clone the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies that are naturally missing the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain 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 the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0166] As those skilled in the art will recognize, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens that bind to the described anti-EGFR protein antibody or fragments thereof.

[0167] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably. H "They can be used interchangeably."

[0168] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0169] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions (CDR1, CDR2, and CDR3). Various methods exist for partitioning CDRs, including the IMGT method, Kabat method, Chothia method, and VBASE2 method. The CDR partitioning methods mentioned in this patent all use the IMGT method.

[0170] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0171] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to EGFR proteins, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0172] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0173] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0174] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0175] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0176] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0177] The term "antibody of the present invention" refers to a polypeptide having EGFR protein-binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0178] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

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

[0180] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0181] Table 1

[0182]

[0183]

[0184] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0185] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0186] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0187] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0188] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0189] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0190] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0191] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0192] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[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*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0194] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0195] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0196] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

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

[0198] Therapeutic agents that can bind to or conjugate with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0199] Pharmaceutical Composition

[0200] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are 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 may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0201] The pharmaceutical compositions of the present invention can be directly used to bind to EGFR protein molecules, and therefore can be used to treat tumors. Furthermore, other therapeutic agents can be used simultaneously.

[0202] The pharmaceutical compositions of the present invention contain 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-described nanobody (or conjugate thereof) of the present invention, 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 formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0203] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0204] Labeled nanobodies

[0205] In a preferred embodiment of the invention, the nanobody carries a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

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

[0207] The anti-EGFR nanobody of the present invention has excellent specificity and high potency.

[0208] Detection methods

[0209] The present invention also relates to a method for detecting EGFR protein. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of EGFR protein in the dissolved samples.

[0210] Book

[0211] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0212] Reagent test kit

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

[0214] This invention also provides a detection kit for detecting EGFR levels, comprising an antibody that recognizes the EGFR protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0215] application

[0216] As described above, the nanobody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of EGFR-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of EGFR.

[0217] The main advantages of this invention are:

[0218] (a) The EGFR-targeting nanobody developed in this invention based on alpaca nanobody has high endocytic activity.

[0219] (b) The nanoantibody-conjugated drug developed in this invention can directly and efficiently kill tumor cells.

[0220] (c) Compared with traditional antibodies currently on the market, the nanoantibodies developed in this invention have smaller molecular weights, lower immunogenicity in the human body, are easier to modify, have higher stability, and are easier to engineer. In the process of commercialization, they can further reduce costs and bring better social benefits.

[0221] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0222] Example 1. Antigen Preparation

[0223] An hEGFR-His protein expression vector was constructed, and its expression and purification were induced in the 293 cell line to prepare the purified hEGFR-His protein. The purified hEGFR-His protein was verified by electrophoresis, and the protein purity was confirmed as shown in the figure. Figure 2 Protein purity > 90%.

[0224] Verification of purified EGFR protein activity: hEGFR-His protein was diluted to 1 μg / mL and coated onto an ELISA plate overnight. BMK molecules were diluted to 4 μg / mL, and binding activity was detected using a 4-fold to 8-gradient assay. BMK1: cetuximab; BMK2: Regeneron (patent number US10047160B2); BMK3: AbbVie (patent number US20200405878A1). Experimental results are shown below. Figure 3 hEGFR-His protein exhibits good binding activity with BMK1 and BMK3.

[0225] Example 2. Immunization of alpacas with hEGFR-His protein

[0226] Alpaca immunization was carried out according to the alpaca immunization strategy in Table 1.

[0227] Table 1. Alpaca Immune 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, while the second to fifth immunizations used Freund's incomplete adjuvant, for a total of five immunizations. Blood was collected after each immunization to prepare antiserum for serum titer determination.

[0230] Titer assay: hEGFR-His protein was diluted to 1 μg / mL, and antiserum was diluted to 1:1000. Titer was determined using a 3-to-8 serial dilution series, where Im.0: pre-immunization alpaca serum; Im.2: post-second immunization alpaca serum; Im.3: post-third immunization alpaca serum; Im.4: post-fourth immunization alpaca serum. Results are shown below. Figure 4 The alpaca achieved a high serum titer after four immunizations.

[0231] Example 3. Blood samples were collected from alpacas after their fifth immunization to separate PBMCs. The technical method is as follows:

[0232] 1. Blood was drawn from the jugular vein of the alpaca to obtain 100 mL of peripheral blood;

[0233] 2. Add an equal volume of 100 mL of physiological saline to dilute the peripheral blood;

[0234] 3. Add PBMC separation solution to the bottom of the centrifuge tube, and add diluted peripheral blood to the top layer.

[0235] 4. Centrifuge at room temperature to separate PBMC cells.

[0236] 5. After centrifugation, carefully aspirate the PBMC layer (i.e., the white film layer) and transfer it to a 50mL centrifuge tube.

[0237] 6. Add 30 mL of physiological saline, centrifuge to collect cells, wash to remove residual liquid, and obtain alpaca peripheral blood PBMCs.

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

[0239] Alpaca RNA was extracted from alpaca PBMCs and reverse transcribed into cDNA.

[0240] Alpaca RNA extraction was performed using a commercial kit (name: NucleoSpin RNA Plus, brand: MN, catalog number: 740984.5), and all RNA extraction steps were performed according to the kit's instructions.

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

[0242] Example 5: Construction of Nanobody Library

[0243] according to Figure 5The illustrated technical route is used to construct an alpaca nanobody library. Specifically, it includes:

[0244] 1. Design upstream and downstream primers for alpaca nanobody to amplify alpaca VHH fragment and obtain alpaca VHH fragment;

[0245] 2. Prepare phage vectors and simultaneously digest the VHH fragment and the vector with enzymes;

[0246] 3. The enzyme-digested VHH fragment and vector were recovered and ligated to prepare the VHH ligation product;

[0247] 4. Prepare E. coli electroporation competent cells, and construct nanobody libraries using the electroporation ligation products.

[0248] Example 6: Library screening and EGFR-targeting nanobody screening;

[0249] according to Figure 6 The technical route shown is used for the panning and screening of EGFR-targeting nanobodies.

[0250] Specific implementation steps:

[0251] 1. Culture and amplify the nanobody library, and infect it with helper phages for library packaging;

[0252] 2. The packaged library was purified by phage to obtain a phage library;

[0253] 3. Positive libraries were obtained by enrichment and panning using EGFR protein solid-phase coating;

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

[0255] 5. Gene sequencing was performed on positive monoclonal strains to obtain EGFR-targeting nanobody sequences.

[0256] Experimental results: The results of monoclonal ELISA detection are shown in Table 2. Positive clones with a value of 0.3 or higher were selected for sequencing.

[0257] Table 2A shows the ELISA results from the single-clone 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 Results of Single-clone B2 Plate ELISA

[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 Single-clone B3 plate ELISA results

[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 Single-clone 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] Nanobodies were prepared using a mammalian cell expression system; the technical route is described in [link to technical details]. Figure 7 .

[0267] Fifty-seven nanobodies were prepared according to the above route, expressed in 20 mL CHO cell systems, and purified to obtain nanobodies; information on the purified nanobodies is shown in Table 3.

[0268] Table 3 Nanobody Expression and Purification

[0269]

[0270]

[0271] The purity test results of 57 nanobody strains for SEC are shown below. Figure 8 .

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

[0273] Experimental principle: hEGFR-His protein is linked to a solid-phase carrier. The antibody to be tested binds to the antigen and then to the enzyme-labeled secondary antibody, forming an antigen-antibody-enzyme-labeled secondary antibody complex. The amount of complex formed is directly proportional to the amount of antibody to be tested.

[0274] Detailed experimental procedure:

[0275] 1. Dilute the target protein hEGFR-His to 1 μg / mL, add 100 μL to each well of the microplate and coat overnight;

[0276] 2. Dilute the nanobody to be tested to a starting concentration of 4 μg / mL, and then dilute it in a 4-fold to 6-fold gradient, adding 100 μl to each well;

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

[0278] 4. Dilute the enzyme-labeled secondary antibody 1:10000, add 100 μL of the enzyme-labeled secondary antibody to each well, and incubate at 37°C for 30 minutes;

[0279] 5. Add 100 μL of TMB colorimetric solution to each well, develop the color at room temperature for 5 minutes, add 50 μL of stop solution, and take the reading after termination;

[0280] The experimental results are shown in Figure 9AAs shown in -F: Except for KY303-23, KY303-26, and KY303-27, which have relatively weak binding activity, the remaining nanobodies all have good binding activity with EGRF protein.

[0281] The results are shown in Table 4: Nanobody ELISA combined with EC50.

[0282] Table 4 Nanobody ELISA combined with 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 binding activity of 57 nanobody strains

[0285] The specific implementation steps are as follows:

[0286] 1. Pre-experiment preparation: Prepare EGFR-CHO-K1 cells in the logarithmic growth phase in advance;

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

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

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

[0290] 5. Incubation with primary antibody: Add 100 μL of antibody dilution buffer per well to the cell plate containing cell suspension. After adding, mix thoroughly by pipetting with a pipette and incubate at 4°C in the dark for 60 minutes.

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

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

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

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

[0295] 10. FACS detection: Median fluorescence intensity (Median-PE) of the expression in the test cells, and plot the fluorescence intensity curve;

[0296] The experimental results are shown in Figure 10A As shown in -F: Among them, KY303-12, KY303-16, KY303-20, KY303-23, KY303-24, KY303-25, KY303-26, KY303-27, KY303-28, KY303-29, KY303-30, KY303-31, and KY303-54 nanobodies did not bind at the FACS level, while the rest showed strong binding.

[0297] The results are shown in Table 5: nanobody FACS binding to EC50.

[0298] Table 5. Nanobody FACS binding to 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 nanobody strains

[0301] The specific implementation steps are as follows:

[0302] 1. Pre-experiment preparation: Prepare CHO-K1 cells in the logarithmic growth phase in advance;

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

[0304] 3. Cell plating: Add 100 μL of cell suspension per well 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 with primary antibody: Add 100 μL of antibody dilution buffer per well to the cell plate containing cell suspension. After adding, mix thoroughly by pipetting with a pipette and incubate at 4°C in the dark for 60 minutes.

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

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

[0309] 8. Washing: After incubation, centrifuge at 500g for 5 minutes and discard the supernatant; add 200μL / well of PBS + 1% BSA to wash the cells twice;

[0310] 9. Resuspend: Resuspend cells in 200 μL of PBS + 1% BSA in each well;

[0311] 10. FACS detection: Median fluorescence intensity of expression in the test cells (Median-PE).

[0312] The experimental results are shown in Figure 11A -B shows that KY303-25, KY303-28, KY303-29, and KY303-30 have nonspecific binding to CHO-K1.

[0313] Example 11: The specific steps for detecting the endocytic activity of EGFR-highly binding nanobodies in different tumor cell lines are as follows:

[0314] 1. Prepare the test cells (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 1.5mL EP tubes;

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

[0317] 4. Preparation of the antibody to be tested: Dilute the antibody to be tested to 10 μg / mL with complete culture medium, add 100 μL of the antibody to be tested per well, incubate at 4℃ 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) to each well to wash away unbound antibodies, centrifuge at 500g for 5 minutes, discard the supernatant, and repeat twice.

[0319] 6. Add 100 μL of complete culture medium to each well. Cells for internalization efficiency were incubated at 37°C for 2 h, while control cells were incubated at 4°C as a control.

[0320] Centrifuge at 7.500g for 5 minutes and discard the supernatant;

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

[0322] 9. Incubate at 4°C in the dark for 30 minutes, then 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. Detect the mean fluorescence intensity (MFI) expressed in the test cells using FACS.

[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: The reaction is terminated by adding L-Cysteine ​​solution;

[0340] 4. ADC purification: The coupled solution was repeatedly centrifuged and the solution was changed using a 10 kDa ultrafiltration tube;

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

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

[0343] Table 8. KY303-39, KY303-50, KY303-52 nanobodies and BMK3 antibody conjugated with MMAE

[0344]

[0345] Example 11: In vitro cytotoxic activity assay of three MMAE-conjugated nanobodies

[0346] Specific implementation steps:

[0347] 1. Experimental preparation: Prepare the test cells in the logarithmic growth phase in advance;

[0348] 2. Cell Plating: Digest and collect the cells to be tested, and prepare a cell suspension using complete culture medium. Add 100 μL of cell suspension per well to a 96-well black transparent flat-bottomed plate. Discard the edge wells of the cell plate and add 100 μL of PBS per well. Place the cell-plate in an incubator overnight to allow the cells to adhere.

[0349] 3. ADC preparation: Dilute the ADC with complete culture medium. Use 120 μg / mL for the first well, then perform 4-fold serial dilutions to obtain a total of 6 concentrations.

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

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

[0352] 6. Plate reading: The ELISA reader detects the luminescence value of the sample, calculates the cell viability based on the luminescence value, creates a curve, and calculates the IC50.

[0353] 7. Experimental results are shown in [link to experimental results]. Figure 14A -G shows that the three nanobody-conjugate drugs all have strong killing activity against seven tumor cell lines.

[0354] The results are shown in Table 9: The three ADCs showed better killing effects on seven tumor cell lines (IC50, IC ...

[0355] Table 9. IC50 of the three ADCs against the seven tumor cell lines.

[0356]

[0357] Example 12

[0358] The technical details of the in vivo efficacy evaluation of the three ADCs are as follows:

[0359] 1. Tumor cells HCT116 and NCI-H1975 (cell bank of the Chinese Academy of Sciences Type Culture Collection Committee) were inoculated into female NCG mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., 5-6 weeks old, 18-21g);

[0360] 2. The tumor grows to 100-150mm 3 ADC and control PBS were injected via the tail vein at both the left and right sides;

[0361] 3. Mice inoculated with HCT116 tumor cell line 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, respectively; mice inoculated with NCI-H1975 tumor cell line 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, respectively.

[0362] 4. Measure changes in tumor volume and calculate the tumor inhibition rate;

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

[0364] The above results indicate that KY303-39-MMAE, KY303-50-MMAE, and KY303-52-MMAE can significantly inhibit tumor growth and have good in vivo safety, showing potential for development as ADC anti-tumor drugs.

[0365] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-EGFR nanobody, characterized in that, The complementarity-determining region (CDR) of the VHH chain of the nanobody is shown below: CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:

11.

2. The anti-EGFR nanobody as described in claim 1, characterized in that, The amino acid sequence of the VHH chain of the nanobody is SEQ ID NO:

3.

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

4. The polynucleotide as described in claim 3, characterized in that, The polynucleotides include DNA, RNA, or cDNA.

5. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 3.

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

7. The host cell as described in claim 6, characterized in that, The host cells include prokaryotic cells or eukaryotic cells.

8. The host cell as described in claim 6, characterized in that, The host cell is selected from the group consisting of: Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

9. A method for generating anti-EGFR nanobodies, characterized in that, Including the following steps: (a) Culturing the host cells of claim 6 under conditions suitable for the production of nanobodies, thereby obtaining a culture containing the anti-EGFR nanobodies; and (b) Isolating or recovering the anti-EGFR nanobody from the culture; and (c) Purification and / or modification of the anti-EGFR nanobody obtained in step (b).

10. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) the anti-EGFR nanobody as described in claim 1; and (b) The coupling part selected from the group below: detectable markers, drugs, toxins, or combinations thereof.

11. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the sequence of the nanobody as described in claim 1; and (ii) Tag sequences that assist in expression and / or purification.

12. The recombinant protein as described in claim 11, characterized in that, The tag sequence includes the 6His tag and the HA tag.

13. The recombinant protein as described in claim 11, characterized in that, The recombinant protein specifically binds to the EGFR protein.

14. A pharmaceutical composition, characterized in that, 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) Pharmaceutically acceptable carriers.

15. The use of the anti-EGFR nanobody as claimed in claim 1, or the immunoconjugate as claimed in claim 10, or the recombinant protein as claimed in claim 11, or the pharmaceutical composition as claimed in claim 14, characterized in that, Used for: (a) To prepare drugs for the prevention and / or treatment of EGFR-positive tumors; and / or (b) Prepare reagents, test plates or kits for detecting EGFR; The tumors mentioned are selected from the following group: bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer, or combinations thereof.

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