EGFR (epidermal growth factor receptor) targeting nano antibody, drug conjugate and application thereof

Abstract

The invention discloses an anti-EGFR nano antibody as well as a coding sequence and application thereof. Specifically, the invention discloses an anti-human EGFR (epidermal growth factor receptor) specific nano antibody and a VHH chain of the anti-human EGFR specific nano antibody. The invention also discloses a coding sequence for coding the nano antibody or the VHH chain thereof, a corresponding expression vector and a host cell, and a method for producing the nano antibody. The nano antibody disclosed by the invention has high endocytosis activity and high specificity, and can be used for detection and targeted therapy aiming at EGFR (Epidermal Growth Factor Receptor).

Description

[0001] This is a divisional application of a Chinese invention patent application with an application date of November 30, 2023, an application number of CN202311625877.5, and an invention title of "Nanobody Targeting EGFR, Drug Conjugate and Their Uses". Technical Field

[0002] The present invention belongs to the field of biotechnology and specifically relates to nanobodies that can target EGFR and their applications. Background Art

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

[0004] Compared with traditional treatment methods, the latest methods based on targeted therapeutic drugs (such as antibodies) have smaller side effects. At the same time, after conjugating antibodies with cytotoxic drugs or toxins, the treatment effect can be greatly improved through cell internalization. These antibody-drug conjugates are also called antibody-drug conjugates (ADCs).

[0005] Nanobodies are variable regions of heavy-chain antibodies that are naturally lacking in light chains and are derived from camelid animals, discovered by scientists in 1989. Its protein crystal structure has a length of 4.0 nm and a diameter of 2.5 nm, and it is the smallest known antibody in terms of molecular mass. Therefore, it is called a nanobody. Compared with traditional antibodies, nanobodies have high stability and stronger tolerance. In addition, they also have the advantages of easy expression and easy genetic engineering modification. Based on their advantages in terms of stability, penetrability, etc., nanobodies have attracted wide attention in the fields of disease treatment, diagnosis, and substance detection.

[0006] Phage display technology inserts foreign genes into the genome of filamentous phages, enabling the proteins or polypeptides encoded by the target genes to be displayed on the surface of phages in the form of fusion proteins; compared with traditional antibodies, nanobodies, due to their natural lack of light chains, do not require heavy-light chain assembly pairing, and have a smaller molecular weight, making it easier to be expressed and displayed on the surface of phages. Combining with the huge library advantages of phage display technology, it is easier to discover nanobody molecules with sequence differentiation and functional differentiation during the development of nanobodies.

[0007] The epidermal growth factor receptor (EGFR) is a transmembrane receptor encoded by the c-erbB proto-oncogene, with a molecular weight of approximately 170 KD. It is the receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. After binding to its specific ligands, including epidermal growth factor (EGF) and transforming growth factor α (TGFα), EGFR forms dimers, further stimulating the activity of intracellular protein tyrosine kinases and triggering downstream signaling cascades, leading to DNA synthesis and cell proliferation. EGFR is also involved in the regulation of phenotypes such as cell migration, adhesion, and proliferation.

[0008] EGFR overexpression is associated with many malignancies, 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 poor prognosis of patients.

[0009] Currently, the development of EGFR-targeted drugs is mainly based on the form of function blockers, which are specifically divided into two categories: one is small molecule drugs, tyrosine kinase inhibitors (TKIs) synthesized based on intracellular signal transduction, including gefitinib, erlotinib, etc.; the other is ligand blockers, mainly monoclonal antibody drugs developed based on traditional antibodies to block extracellular ligand signal transduction, such as cetuximab, panitumumab, etc. However, for cancer treatment, there are still many deficiencies in current products, such as limited efficacy, high drug resistance, large toxic and side effects, and many other problems, and the social benefits brought are also very limited.

[0010] The mechanism of action of ADCs targeting the EGFR target is different from that of drugs that act by inhibiting the receptor signaling pathway. ADCs can directly deliver cytotoxins into tumor cells with high EGFR expression. Through the process of cellular internalization, the cytotoxins are brought into the tumor cells, enhancing the killing effect of the drugs and improving the effectiveness of targeted therapy. Therefore, theoretically speaking, ADCs have a broader range of indications and more powerful drug effects than traditional therapies. Currently, most ADCs targeting the EGFR target are in the clinical research stage. For example, AVID-100, ABT-414, and ABBV-221, etc.; and related patents such as CN106470697, CN114585391, CN111295201, etc. The antibody forms disclosed in many patents are traditional monoclonal antibodies. Traditional monoclonal antibodies usually have many disadvantages such as large molecular weight, poor stability, difficult preparation process, and high immunogenicity. Moreover, two related ADCs, IMGN-289 and AMG-595, targeting EGFR, were also forced to terminate in clinical research due to toxicity problems (such as skin toxicity, gastrointestinal toxicity, etc.). Due to the toxicity problems, AstraZeneca's AZD9592 (patent number: US20230183358A1) also made a design to weaken the affinity at the EGFR end, aiming to reduce EGFR-driven tissue toxicity. Therefore, the research on ADCs targeting the EGFR target still faces many challenges in terms of effectiveness and safety.

[0011] Nanobodies with a smaller molecular weight have many advantages over traditional antibodies, such as small size, high stability, low immunogenicity, stronger tissue permeability, and low production cost.

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

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

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

[0015] In the first aspect of the present invention, a nanobody against EGFR is provided. The complementarity-determining regions (CDRs) of the VHH chain of the nanobody are one or more selected from the following groups:

[0016] (1) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:6, 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 example, the CDR region of the nanobody VHH chain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any one of the above sequences.

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

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

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

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

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

[0025] In another preferred example, the nanobody binds to human, murine or simian EGFR.

[0026] In another preferred example, the nanobody can be endocytosed in cells expressing the EGFR antigen.

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

[0028] In another preferred example, 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 divalent body (divalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (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, SEQ ID NO:3.

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

[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 is a linker sequence;

[0037] Z3 is an immunomodulatory molecule moiety.

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

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

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

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

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

[0043] In a fifth aspect of the present invention, there is provided a host cell which contains the expression vector as described in the fourth aspect of the present invention, or the polynucleotide as described in the third aspect of the present invention is 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: Escherichia coli, yeast cells, mammalian cells, phages, or combinations thereof.

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

[0047] (a) culturing the host cell according to the fifth aspect of the present invention under conditions suitable for producing nanobodies, thereby obtaining a culture containing the anti-EGFR nanobodies; and

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

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

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

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

[0052] (b) a conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, gold nanoparticles / nanorods, magnetic nanoparticles, a virus capsid protein or VLP, or combinations thereof.

[0053] In another preferred embodiment, the conjugate 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 reagent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or combinations thereof.

[0056] In another preferred example, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines, and maytansinoids (such as DM1 and DM4), taxanes, benzodiazepines, or benzodiazepine-containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0057] In another preferred example, the toxin is selected from the group consisting of: auristatins (such as auristatin E, auristatin F, MMAE, and MMAF), calicheamicin, maytansinol, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, anthramycin, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A-chain, momordin A-chain, alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, croton toxin, calicheamicin, saponaria officinalis inhibitor, glucocorticoid, or combinations thereof.

[0058] In another preferred example, the conjugate moiety is a detectable label.

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

[0060] In another preferred example, the immunoconjugate contains: multivalent (such as divalent) anti-EGFR nanobodies as described in the first aspect of the present invention. The multivalent means that multiple repeats of the anti-EGFR nanobodies as described in the first aspect of the present invention are included in the amino acid sequence of the immunoconjugate.

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

[0062] In another preferred example, the detection includes flow cytometry detection and cellular immunofluorescence detection.

[0063] In the ninth aspect of the present invention, there is provided a use of one or more anti-EGFR nanobodies:

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

[0065] (ii) for flow cytometry detection;

[0066] (iii) for cellular immunofluorescence detection;

[0067] (iv) for treating tumors;

[0068] (v) for tumor diagnosis.

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

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

[0071] In another preferred example, the multispecific antibody further contains the Fc segment of an antibody.

[0072] In the eleventh aspect of the present invention, there is provided a recombinant protein, which has:

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

[0074] (ii) An optional Fc segment; and

[0075] (iii) An optional tag sequence for assisting expression and / or purification.

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

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

[0078] In the twelfth aspect of the present invention, there is provided the use of the nanobody as described in the first aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention, which is used for preparing a medicament, a reagent, a test plate or a kit;

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

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

[0081] In another preferred example, the tumors include: 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 the thirteenth aspect of the present invention, there is provided a pharmaceutical composition, which contains:

[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) A pharmaceutically acceptable carrier.

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

[0086] In another preferred example, the pharmaceutical composition is used for preparing a medicament for treating tumors, and the tumors include 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 a combination thereof.

[0087] In another preferred example, the pharmaceutical composition is used for preparing a medicament for treating tumors, and the tumors include cancers 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 conjugate moiety 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 a combination thereof.

[0091] In the fourteenth aspect of the present invention, there is provided the use of 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, or the pharmaceutical composition as described in the thirteenth aspect of the present invention, for:

[0092] (a) preparing a drug for preventing and / or treating EGFR-related diseases; and / or

[0093] (b) preparing a reagent, a test strip 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 the EGFR protein or a fragment thereof in a sample.

[0098] In another preferred embodiment, the detection includes flow cytometry and immunofluorescence cytochemical detection.

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

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

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

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

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

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

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

[0106] In the seventeenth aspect of the present invention, there is provided an EGFR protein detection reagent, 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 carrier acceptable in detection science.

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

[0110] In another preferred embodiment, the carrier acceptable in detection science 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 detection reagents, cellular immunofluorescence detection 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 dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, buccal tablet, aerosol).

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

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

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

[0117] In another preferred embodiment, the detection is used for the diagnosis or prognosis of diseases or disorders related to EGFR.

[0118] In the twentieth aspect of the present invention, a method for treating diseases related to EGFR is provided. The method includes administering to a subject in need 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, or the pharmaceutical composition described in the thirteenth aspect of the present invention.

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

[0120] In another preferred example, the non-human mammal includes a rodent (such as a mouse, a rabbit), a non-human primate (such as a monkey).

[0121] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 Shows the overall technical route of the present invention.

[0123] Figure 2 Shows the electrophoresis of EGFR-His protein.

[0124] Figure 3 Shows the verification of the binding activity between EGFR antigen and BMK.

[0125] Figure 4 Shows the detection of the titer of alpaca serum.

[0126] Figure 5 Shows the technical route for constructing an alpaca nanobody library.

[0127] Figure 6 Shows the panning of nanobodies targeting EGFR.

[0128] Figure 7 Shows the technical route for the expression and preparation of nanobodies.

[0129] Figure 8 Shows the SEC detection of nanobodies after purification.

[0130] Figure 9A Shows the ELISA activity detection of nanobodies KY303-01 to KY303-10.

[0131] Figure 9B Shows the ELISA activity detection of nanobodies KY303-11 to KY303-20.

[0132] Figure 9C Shows the ELISA activity detection of nanobodies KY303-21 to KY303-30.

[0133] Figure 9D Shows the ELISA activity detection of nanobodies KY303-31 to KY303-40.

[0134] Figure 9E Shows the ELISA activity detection of nanobodies KY303-41 to KY303-50.

[0135] Figure 9F Shows the ELISA activity detection of nanobodies KY303-51 to KY303-57.

[0136] Figure 10A Shows the FACS activity detection of nanobodies KY303-01 to KY303-10.

[0137] Figure 10B Shows the FACS activity detection of nanobodies KY303-11 to KY303-20.

[0138] Figure 10C Shows the FACS activity detection of nanobodies KY303-21 to KY303-30.

[0139] Figure 10D Shows the FACS activity detection of nanobodies KY303-31 to KY303-40.

[0140] Figure 10E Shows the FACS activity detection of nanobodies KY303-41 to KY303-50.

[0141] Figure 10F Shows the FACS activity detection of nanobodies KY303-51 to KY303-57.

[0142] Figure 11A Shows the specific binding detection of nanobodies KY303-01 to KY303-30.

[0143] Figure 11B Shows the specific binding detection of nanobodies KY303-31 to KY303-57.

[0144] Figure 12A Shows the endocytosis rate detection of nanobodies on HCT116 cells.

[0145] Figure 12B Shows the endocytosis rate detection of nanobodies on 769-P cells.

[0146] Figure 12CShows the detection of the endocytosis rate of the nanobody on 786-O cells.

[0147] Figure 13 Shows the technical route of nanobody conjugated with MMAE.

[0148] Figure 14A Shows the killing effect of ADC on NCI-H1975 cells.

[0149] Figure 14B Shows the killing effect of ADC on HCT116 cells.

[0150] Figure 14C Shows the killing effect of ADC on BxPC-3 cells.

[0151] Figure 14D Shows the killing effect of ADC on MDA-MB-468 cells.

[0152] Figure 14E Shows the killing effect of ADC on NCI-H1993 cells.

[0153] Figure 14F Shows the killing effect of ADC on HT29 cells.

[0154] Figure 14G Shows the killing effect of ADC on MDA-MB-231 cells.

[0155] Figure 15A Shows the change in tumor volume of the mouse HCT116 model.

[0156] Figure 15B Shows the change in body weight of the mice in the HCT116 model.

[0157] Figure 15C Shows the change in tumor volume of the mouse NCI-H1975 model.

[0158] Figure 15D Shows the change in body weight of the mice in the NCI-H1975 model. Detailed implementation manners

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

[0160] The experimental results show that the nanobody of the present invention has good binding activity and high endocytosis functional activity. In addition, after the nanobody of the present invention is conjugated with the cytotoxin (MMAE), it all has strong tumor cell killing activity, can significantly inhibit tumor growth, and has good in vivo safety. Based on this, the present invention was completed.

[0161] The present invention has developed a nanobody molecule targeting EGFR based on alpaca nanobody and phage display technology. This molecule has high binding and high endocytosis functional activities for tumor cell lines, and when developed into an antibody-drug conjugate, it has good tumor cell killing effects and has the potential to be developed into an ADC anti-tumor drug. The overall technical route of the present invention is as Figure 1 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 in which the 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 approximately 150,000 daltons with the same structural characteristics, which is composed 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, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intra-chain 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)" and "nanobody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a single-domain antibody (VHH) composed of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant region of the heavy chain (CH1), the variable region of the antibody heavy chain is then cloned to construct a single-domain antibody (VHH) composed of only one heavy chain variable region.

[0165] As used herein, the term "variable" indicates that certain portions of the variable regions in an antibody differ in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions in the variable regions are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally in a β-sheet conformation and are connected by three CDRs forming connecting loops, and in some cases can form partial β-sheet structures. The CDRs in each chain are brought closely 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, Volume I, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0166] As is known to those skilled in the art, immunoconjugates and fusion expression products include: conjugates formed by the binding of 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 anti-EGFR protein antibody or fragments thereof described above.

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

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

[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. There are currently various methods for partitioning CDRs, including the IMGT method, the Kabat method, the Chothia method, the VBASE2 method, etc. The CDR partitioning method mentioned in this patent uses the IMGT method.

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

[0171] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are interchangeable and all refer to polypeptides that specifically bind to the EGFR protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain a starting 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 at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibody of the present invention.

[0173] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the heavy chain variable region, called complementarity-determining regions (CDRs), which divide this segment into four framework 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 loop structures that are brought close to each other in spatial structure by the β-sheets formed by the intervening FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

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

[0175] The present invention includes not only intact antibodies, but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Thus, the present invention also includes fragments, derivatives, and analogs of the said antibodies.

[0176] As used herein, the terms "fragment", "derivative", and "analog" 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 conservative or non-conservative amino acid residues (preferably conservative 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 a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0177] The antibody of the present invention refers to a polypeptide having EGFR protein binding activity and including the above CDR regions. This term also includes variant forms of the polypeptide containing the above CDR regions that have the same function as the antibody of the present invention. These variant forms 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, when substituting amino acids with similar or close properties, the function of the protein is usually not changed. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. This term also includes active fragments and active derivatives of the antibody of the present invention.

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

[0179] The present invention also provides other polypeptides, such as fusion proteins containing nanobodies or their fragments. In addition to almost full-length polypeptides, the present invention also includes fragments of the nanobodies of the present invention. Generally, 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, most preferably at least about 100 consecutive amino acids.

[0180] In the present invention, the "conservative variant of the antibody of the present invention" refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids in the amino acid sequence of the antibody of the present invention with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0181] Table 1

[0182]

[0183]

[0184] The present invention also provides polynucleotide molecules encoding the above antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0185] The polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; the coding sequences of the mature polypeptides and various additional coding sequences; the coding sequences of the mature polypeptides (and optional additional coding sequences) and non-coding sequences.

[0186] The term "polynucleotide encoding a polypeptide" may be a polynucleotide comprising the polynucleotide encoding this polypeptide, or may also be a polynucleotide further comprising 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%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at a lower ionic strength and a higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0188] The full-length nucleotide sequences or fragments of the antibodies of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis methods. A feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments and then ligating them, fragments with a very long sequence can be obtained. 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 sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually done by cloning them into a vector, then transferring them into cells, and then isolating the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0190] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then, this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0191] The present invention also relates to vectors containing the above-described appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0192] The host cell can be a prokaryotic cell, such as a bacterial cell; or 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; animal cells such as CHO, COS7, 293 cells, etc.

[0193] Transformation of the host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with CaCl 2 method, and the steps used are well known in the art. Another method is to use MgCl 2 . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

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

[0195] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods utilizing 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, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0196] The antibody of the present invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or any combination of the above substances.

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

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

[0199] Drug composition

[0200] The present invention also provides a composition. Preferably, the composition is a drug composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Usually, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substances being formulated and the disease to be treated. The formulated drug composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

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

[0202] The drug composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned nanobody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The drug preparation should match the administration method. The drug composition of the present invention can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Drug compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 10 micrograms per kilogram of body weight - about 50 milligrams per kilogram of body weight per day. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0203] When using the drug composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually 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 about 10 micrograms per kilogram of body weight - about 10 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are within the scope of the skills of a skilled physician.

[0204] Labeled nanobody

[0205] In a preferred embodiment of the present invention, the nanobody is conjugated with a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels, or fluorescent labels.

[0206] Colloidal gold labeling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-EGFR nanobody is labeled with colloidal gold to obtain a colloidal gold-labeled nanobody.

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

[0208] Detection method

[0209] The present invention also relates to a method for detecting EGFR protein. The steps of the method are generally 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] This

[0211] In the detection method of the present invention, the sample used is not particularly limited. Representative examples are cell-containing samples present in cell preservation solutions.

[0212] Kit

[0213] The present invention also provides a kit containing the antibody (or its fragment) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, an instruction manual, a buffer, etc.

[0214] The present invention also provides a detection kit for detecting EGFR level. The kit includes an antibody that recognizes EGFR protein, a lysis medium for dissolving the sample, 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 nanobody of the present invention has broad biological and clinical application values. 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 clinical diagnosis and targeted therapy against EGFR.

[0217] Main advantages of the present invention:

[0218] (a). The EGFR-targeted nanobody developed based on alpaca nanobody of the present invention has high endocytosis activity.

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

[0220] (c) Compared with the traditional antibodies developed on the current market, the nanobodies developed by the present invention have a smaller molecular weight, lower immunogenicity in the human body, are easier to modify, have higher stability, are easier to engineer, and can further reduce costs during commercialization, bringing better social benefits.

[0221] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as 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 stated, percentages and parts are weight percentages and weight parts.

[0222] Example 1. Antigen Preparation

[0223] Construct an hEGFR-His protein expression vector, culture and induce expression and purification in the 293 cell line to prepare the purified hEGFR-His protein; electrophoretically verify the purified hEGFR-His protein, and the protein purity is shown in Figure 2 : Protein purity > 90%.

[0224] Verify the activity of the purified EGFR protein: Dilute the hEGFR-His protein to 1 μg / mL, coat the enzyme-linked immunosorbent assay (ELISA) plate overnight, dilute the BMK molecule to 4 μg / mL, and detect the binding activity according to an 8-fold gradient with a 4-fold ratio; where 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 Alpacas with hEGFR-His Protein

[0226] Immunize alpacas according to the alpaca immunization strategy in Table 1;

[0227] Table 1 Alpaca 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] For the first immunization, Freund's complete adjuvant is used, and for the second to fifth immunizations, Freund's incomplete adjuvant is used. A total of five immunizations are carried out. After each immunization, blood is taken to prepare antiserum for serum titer detection:

[0230] Titer detection test: Dilute the hEGFR-His protein to 1 μg / mL and the antiserum to 1:1000, and perform titer detection according to 8 gradients of 3-fold dilution. Among them, Im.0: Alpaca serum before immunization; 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. After the fifth immunization of the alpaca, blood was collected and PBMC was isolated. The technical method is as follows:

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

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

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

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

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

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

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

[0239] Extract alpaca RNA from alpaca PBMC and reverse transcribe it 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 carried out according to the kit steps.

[0241] cDNA preparation was performed using PrimeScript TM 1st Strand cDNA Synthesis Kit (Brand: Takara, Catalog Number: 6110A), and the cDNA preparation process was carried out according to the kit instructions to complete cDNA preparation.

[0242] Example 5: Nanobody library construction

[0243] According to Figure 5Construct the alpaca nanobody library according to the technical route shown below. Specifically, it includes:

[0244] 1. Design the upstream and downstream primers of alpaca nanobodies to amplify the alpaca VHH fragment and obtain the alpaca VHH fragment;

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

[0246] 3. Recover and ligate the digested VHH fragment and the vector to prepare the VHH ligation product;

[0247] 4. Prepare electrocompetent Escherichia coli cells and electrotransform the ligation product to complete the construction of the nanobody library.

[0248] Example 6: Conduct library panning and screening for anti-EGFR nanobodies;

[0249] According to Figure 6 the technical route shown below, pan and screen for anti-EGFR nanobodies.

[0250] Specific implementation steps:

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

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

[0253] 3. Enrich and pan using the EGFR protein solid-phase coating method to obtain a positive library;

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

[0255] 5. Perform gene sequencing on the positive monoclonal strains to obtain the anti-EGFR nanobody sequence.

[0256] Experimental results: The results of monoclonal ELISA are shown in Table 2. Pick the positive clones with values above 0.3 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 ELISA Results of Monoclonal Antibody B4 Plate

[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. The technical route is shown in Figure 7 .

[0267] According to the above route, 57 nanobodies were expressed and prepared. The CHO cell system was used to express a 20 mL system, and the nanobodies were obtained after purification. The information of the purified nanobodies is shown in Table 3.

[0268] Table 3 Expression and Purification of Nanobodies

[0269]

[0270]

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

[0272] Example 8: ELISA Detection of the Binding Activity of 57 Nanobodies

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

[0274] Specific Experimental Procedure:

[0275] 1. Dilute the target protein hEGFR-His to 1 μg / mL and add 100 μL per well to the wells of the enzyme-labeled plate for overnight coating;

[0276] 2. Dilute the nanobodies to be tested starting from 4 μg / mL and perform 6-fold serial dilutions at a ratio of 4. Add 100 μl per well;

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

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

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

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

[0281] The results analysis is shown in Table 4: The EC50 of nanobody ELISA binding.

[0282] Table 4 The EC50 of nanobody ELISA binding

[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: Detection of the binding activities of 57 nanobodies by FACS

[0285] The specific implementation steps are as follows:

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

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

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

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

[0290] 5. Incubate the primary antibody: Add 100 μL / well of the antibody dilution to the cell plate with the cell suspension. After adding, mix well with a multi-channel pipette and incubate at 4°C in the dark for 60 minutes;

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

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

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

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

[0295] 10. FACS detection: Median fluorescence intensity (Median-PE) expressed in the cells to be tested, and a curve was made based on the fluorescence intensity;

[0296] The experimental results are shown in Figure 10A -F: Among them, 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 had no binding at the FACS level, and the rest had strong binding.

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

[0298] Table 5 FACS binding EC50 of nanobodies

[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: Specific binding detection of 57 nanobodies by FACS

[0301] The specific implementation steps are as follows:

[0302] 1. Preparation before experiment: Prepare CHO-K1 cells in the 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 a buffer of PBS + 1% BSA. Dilute the antibody to 20 μg / mL;

[0306] 5. Incubate the primary antibody: Add 100 μL / well of antibody dilution to the cell plate with the cell suspension. After adding, pipette and mix well, and incubate at 4°C in the dark for 60 minutes;

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

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

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

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

[0311] 10. FACS detection: Median fluorescence intensity (Median-PE) expressed in the cells to be tested.

[0312] The experimental results are shown in Figure 11A -B: Among them, KY303-25, KY303-28, KY303-29, and KY303-30 have non-specific binding to CHO-K1.

[0313] Example 11: Detection of the endocytosis activity of nanobodies with high EGFR binding activity in different tumor cell lines. The specific implementation steps are as follows:

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

[0317] 4. Prepare the antibody to be detected: 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 500 g 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 the unbound antibody, centrifuge at 500 g for 5 minutes, and discard the supernatant. Repeat 2 times;

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

[0320] 7. Centrifuge at 500 g 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 500 g 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 500 g for 5 minutes, discard the supernatant, and repeat 2 times;

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

[0325] 12. Detect the mean fluorescence intensity (MFI) expressed in the cells to be tested by FACS.

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

[0327] Example 12: Detection of the binding affinity of three nanobodies with high endocytic activity to the 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; the affinity of KY303-52 is 2.72E-7.

[0329] Table 6 Detection of the affinity of nanobodies with high endocytic activity

[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 nanobodies with high endocytic activity, 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: Conjugation of three nanobodies with high endocytic activity, KY303-39, KY303-50, and KY303-52, with MMAE

[0335] The technical route for the conjugation of nanobodies with MMAE is as Figure 13 shown.

[0336] The specific implementation steps are as follows:

[0337] 1. Antibody reduction: Fix the reduction system and reduce at 200 rpm in a metal bath at 37 °C for 4 hours;

[0338] 2. Toxin conjugation: Fix the feed amount of Vc-MMAE and react at 200 rpm in a metal bath at 4 °C for 2 hours;

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

[0340] 4. ADC purification: The conjugated solution is centrifuged and exchanged with a 10 kDa ultrafiltration tube repeatedly;

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

[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 KY303-50-MMAE is 2.93; the DAR value of KY303-52-MMAE is 2.99.

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

[0344]

[0345] Example 11: Detection of in vitro killing activity of three nanobodies conjugated with MMAE

[0346] Specific implementation steps:

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

[0348] 2. Cell plating: Digest and collect the cells to be tested, and prepare a cell suspension with complete medium. Add 100 μL / well of the 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 the plated cells in the incubator and wait overnight for the cells to adhere;

[0349] 3. ADC preparation: Dilute the ADC with complete medium. The first well is 120 μg / mL, and then dilute it 4-fold gradient, with a total of 6 concentrations;

[0350] 4. Incubate the ADC: Add 20 μL / well of the ADC dilution 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 / well of the detection solution to the 96-well cell culture plate, let it stand for 15 minutes until the cells are completely lysed;

[0352] 6. Read the plate: Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the luminescence value to be measured, 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: The three nanobodies conjugated with the drug have strong killing activity against seven tumor cell lines;

[0354] The results analysis is shown in Table 9: The IC50 values of the killing effects of the three ADCs on 7 tumor cell lines. For KY303-39-MMAE, KY303-50-MMAE, and KY303-52-MMAE, their killing effects on NCI-H1975, BxPC-3, MDA-MB-468, and NCI-H1993 cells are better than those of BMK3-MMAE.

[0355] Table 9 The IC50 values of the killing effects of the three ADCs on 7 tumor cell lines

[0356]

[0357] Example 12

[0358] In vivo pharmacodynamic evaluation of the three ADCs, the technical details are as follows:

[0359] 1. Inoculate tumor cells HCT116 and NCI-H1975 (Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection) into female NCG mice (purchased from: Jiangsu Jicui Yakang Biotechnology Co., Ltd., 5-6 weeks old, 18-21 g);

[0360] 2. When the tumor grows to about 100 - 150 mm 3 inject ADC and control PBS via the tail vein;

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

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

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

[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, and have the potential to be developed into ADC anti-tumor drugs.

[0365] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

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 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 according to 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 according to claim 1.

4. An expression vector, characterized in that the expression vector contains the polynucleotide according to claim 3.

5. A host cell, characterized in that the host cell contains the expression vector according to claim 4, or the polynucleotide according to claim 3 is integrated into its genome.

6. A method for producing an anti-EGFR nanobody, characterized in that it includes the steps of: (a) culturing the host cell according to claim 5 under conditions suitable for producing nanobodies, thereby obtaining a culture containing the anti-EGFR nanobody; and (b) separating or recovering the anti-EGFR nanobody from the culture; and (c) optionally, purifying and / or modifying the anti-EGFR nanobody obtained in step (b).

7. An immunoconjugate, characterized in that the immunoconjugate contains: (a) the anti-EGFR nanobody according to claim 1; and (b) a conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, or a combination thereof.

8. A recombinant protein, characterized in that the recombinant protein has: (i) the sequence of the nanobody according to claim 1; and (ii) an optional Fc segment; and (iii) an optional tag sequence for assisting expression and / or purification.

9. A pharmaceutical composition, characterized in that the pharmaceutical composition contains: (i) the anti-EGFR nanobody according to claim 1, or the immunoconjugate according to claim 7, or the recombinant protein according to claim 8; and (ii) a pharmaceutically acceptable carrier.

10. Use of the anti-EGFR nanobody according to claim 1, or the immunoconjugate according to claim 7, or the recombinant protein according to claim 8, or the pharmaceutical composition according to claim 9, characterized in that it is used for: (a) preparing a drug for preventing and / or treating tumors; and / or (b) preparing a reagent, test plate or kit for detecting EGFR.

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