Nanobodies against HPV18 subtypes and their applications

Abstract

The present disclosure provides nanobodies or active fragments thereof against human papillomavirus (HPV) 18 subtype, as well as nucleic acid molecules encoding the nanobodies or active fragments thereof, protein conjugates containing the nanobodies or active fragments thereof, and pharmaceutical compositions. The present disclosure also provides the use of the nanobodies or active fragments thereof in drugs for diagnosing diseases associated with HPV18 subtypes, or treating or preventing diseases associated with HPV18 subtypes.

Description

Technical Field

[0001] The present disclosure belongs to the field of biotechnology, and particularly relates to nano antibodies against HPV18 subtypes and applications thereof. Background Art

[0002] Human papillomavirus (HPV) is a non-enveloped DNA virus belonging to the genus Papillomavirus of the family Papillomaviridae. HPV infects the epidermis and mucosal tissues of the human body. Currently, more than 170 types of HPV have been identified. According to the risk of tumor occurrence, HPV is divided into low-risk subtypes and high-risk subtypes. Low-risk HPV subtypes often cause benign lesions such as condyloma of the external genital tract, including low-grade cervical intraepithelial lesions. High-risk HPV subtypes are closely related to the occurrence of cervical cancer and high-grade intraepithelial neoplasia of the cervix. HPV18 is the human papillomavirus 18 subtype, which is a high-risk HPV subtype. HPV18 and HPV16 subtypes are the culprits of cervical cancer in women. Compared with other subtypes of HPV, cervical cancer caused by HPV18 is the most serious, and the risk of death in patients is even higher than that of HPV16 subtypes. Cervical cancer is the only cancer with a clear cause and can be completely prevented and treated through early screening and early detection. Therefore, HPV18 screening is still one of the keys to preventing cervical cancer.

[0003] In the process of studying HPV18, antibodies are a very important research tool, which has great value and significance for patient diagnosis, virus analysis and research. Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present invention relies on antibodies produced by the immune system of alpacas, which are called "nanoantibodies". Nanoantibodies are tiny antibody fragments separated from immunoglobulins in animals such as camels and sharks. They have the same antigen binding ability and structural stability as complete antibodies. They are the smallest units that can bind to target antigens, and their relative molecular mass is only 15kD. Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune systems in animals such as alpacas can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanoantibodies. Summary of the invention

[0004] In order to solve one of the above-mentioned technical problems existing in the prior art, the present invention provides a nano antibody or its active fragment against human papillomavirus (HPV) 18 subtype, and the use of the nano antibody or its active fragment in the treatment or diagnosis of diseases related to HPV18 subtype.

[0005] According to one aspect of the present disclosure, a nanobody or an active fragment thereof against human papillomavirus (HPV) 18 subtype is provided. In some embodiments, the Nanobody or its active fragment comprises at least one heavy chain variable region, which comprises: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86 or 91; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, 87 or 92; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, 73, 78, 83, 88 or 93.

[0006] In some embodiments, the Nanobody or its active fragment can specifically bind to the L1 protein of the HPV18 subtype.

[0007] In some embodiments, the active fragment of the Nanobody is a fragment of the Nanobody that can specifically bind to the HPV18 subtype, in particular to the L1 protein of the HPV18 subtype.

[0008] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 3.

[0009] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO:6; a CDR2 having an amino acid sequence as shown in SEQ ID NO:7; and a CDR3 having an amino acid sequence as shown in SEQ ID NO:8.

[0010] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 11; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 12; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 13.

[0011] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 16; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 17; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 18.

[0012] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23.

[0013] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 26; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 27; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 28.

[0014] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 31; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 32; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 33.

[0015] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 36; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 37; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 38.

[0016] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO:41; a CDR2 having an amino acid sequence as shown in SEQ ID NO:42; and a CDR3 having an amino acid sequence as shown in SEQ ID NO:43.

[0017] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 46; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 47; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 48.

[0018] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 51; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 52; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 53.

[0019] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 56; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 57; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 58.

[0020] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO:61; a CDR2 having an amino acid sequence as shown in SEQ ID NO:62; and a CDR3 having an amino acid sequence as shown in SEQ ID NO:63.

[0021] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 66; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 67; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 68.

[0022] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO:71; a CDR2 having an amino acid sequence as shown in SEQ ID NO:72; and a CDR3 having an amino acid sequence as shown in SEQ ID NO:73.

[0023] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 76; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 77; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 78.

[0024] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO:81; a CDR2 having an amino acid sequence as shown in SEQ ID NO:82; and a CDR3 having an amino acid sequence as shown in SEQ ID NO:83.

[0025] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 86; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 87; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 88.

[0026] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may include: a CDR1 having an amino acid sequence as shown in SEQ ID NO:91; a CDR2 having an amino acid sequence as shown in SEQ ID NO:92; and a CDR3 having an amino acid sequence as shown in SEQ ID NO:93.

[0027] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 4.

[0028] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 9.

[0029] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 14.

[0030] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 19, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 19.

[0031] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 24, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 24.

[0032] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 29, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0033] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 34, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 24.

[0034] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 39, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0035] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 44, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 24.

[0036] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 49, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0037] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 54, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 24.

[0038] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 59, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0039] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 64, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 24.

[0040] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 69, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0041] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 74, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 24.

[0042] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 79, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0043] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 84, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 24.

[0044] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 89, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0045] In some embodiments, at least one heavy chain variable region of the Nanobody or its active fragment may have an amino acid sequence as shown in SEQ ID NO: 94, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 24.

[0046] According to another aspect of the present disclosure, a nucleic acid molecule encoding the Nanobody or an active fragment thereof of the present disclosure is provided.

[0047] In some embodiments, the nucleic acid molecule can have a nucleotide sequence as shown in SEQ ID NO:5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.8% sequence identity thereto.

[0048] According to another aspect of the present disclosure, an expression vector is provided, wherein the expression vector comprises the above-mentioned nucleic acid molecule of the present disclosure.

[0049] According to another aspect of the present disclosure, a host cell is provided, the host cell comprising the above-mentioned expression vector of the present disclosure. In some embodiments, the host cell can express the nano antibody or its active fragment of the present disclosure. In some embodiments, the host cell can be a mammalian cell, an insect cell, a yeast cell or a prokaryotic cell. In a specific embodiment, the host cell can be Escherichia coli (E.coli).

[0050] According to yet another aspect of the present disclosure, there is provided a humanized antibody or an active fragment thereof, which is obtained from the Nanobody or an active fragment thereof of the present disclosure.

[0051] According to another aspect of the present disclosure, a protein conjugate is provided, the protein conjugate comprising the nanobody or active fragment thereof of the present disclosure, and a ligand. In some embodiments, the ligand is selected from a radioisotope, a fluorescent group and / or a delivery vector.

[0052] According to yet another aspect of the present disclosure, there is provided a pharmaceutical composition comprising the Nanobody or an active fragment thereof of the present disclosure, and a pharmaceutically acceptable carrier.

[0053] According to another aspect of the present disclosure, a chimeric antigen receptor (CAR) is provided, which comprises the Nanobody or an active fragment thereof of the present disclosure.

[0054] According to another aspect of the present disclosure, there is provided the use of the Nanobodies or active fragments thereof of the present disclosure in the diagnosis or treatment of diseases associated with HPV, particularly HPV18 subtype.

[0055] According to another aspect of the present disclosure, there is provided use of the Nanobodies or active fragments thereof of the present disclosure in a kit for diagnosing diseases associated with HPV, particularly HPV18 subtype.

[0056] According to another aspect of the present disclosure, there is provided use of the Nanobodies or active fragments thereof of the present disclosure in drugs for treating or preventing diseases associated with HPV, particularly HPV18 subtype.

[0057] In some embodiments, the HPV-related diseases may include cervical cancer, anal canal cancer, tonsil cancer, oral cancer, laryngeal cancer, nasal cancer, esophageal cancer, Bowen disease, basal cell carcinoma, Paget's disease, squamous cell carcinoma, flat warts, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The amino acid sequence of the Nanobody is shown.

[0059] Figure 2 The results of the detection of the binding affinity of the nanobody to the antigen are shown. Figure 2 A to 2S show the affinity test results of Nanobodies 1A1, 1B4, 1B11, 1E10, 1D5, 1F3, 3D11, 2A4, 2A5, 2A7, 2H3, 2F5, 3C1, 2G11, 3B8, 3G6, 4B4, 4F5 and 4G7, respectively. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0061] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0062] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0063] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0064] Human papillomavirus (HPV) belongs to the genus Papillomavirus A of the family Papovaviridae. It is a spherical DNA virus that can cause squamous epithelial proliferation of human skin and mucous membranes. More than 130 types have been isolated so far, and different types cause different clinical manifestations. They can be divided into different subtypes according to the different tissue sites invaded. In some embodiments of the present disclosure, HPV18 subtype is used, which belongs to the mucosal high-risk type and is associated with diseases such as cervical cancer, anal and anal canal cancer, tonsil cancer, oral cancer, laryngeal cancer, nasal cancer, and esophageal cancer.

[0065] The term "virus-like particle" or "VLP" as used herein refers to a hollow particle containing one or more structural proteins of a virus, without viral nucleic acid, unable to replicate autonomously, and morphologically identical or similar to a true virus particle. In some embodiments of the present disclosure, the VLP used has the same surface structure as HPV18, but without the viral nucleic acid of HPV18 and unable to replicate autonomously. Using VLP as an antigen for animal immunization will be more conducive to obtaining antibodies that recognize the natural state of HPV18.

[0066] HPV L1 protein (major capsid protein) plays an important role in HPV infection and various stages of its life cycle, especially in the early stage of viral infection and inducing protective humoral immunity. L1 protein is scattered on the surface of HPV viral capsid. It is known that both linear epitopes and conformational epitopes of L1 protein are the main antigenic epitopes that induce protective immune response in the body.

[0067] The term "kd" value used in this article refers to the dissociation constant (kd), which is a specific type of equilibrium constant used to measure the tendency of a larger object to separate (dissociate) from another smaller component. It is the reciprocal of the association constant and is expressed in mol / L (M) or nmol / L (nM). The smaller the kd value, the stronger the binding ability of the two substances.

[0068] The terms "nanoantibody" or "Nanobody" used herein can be used interchangeably and refer to antibodies that naturally lack light chains and exist in the peripheral blood of camelids. Nanoantibodies contain only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but they are not as easy to stick to each other as artificially modified single-chain antibody fragments, or even aggregate into lumps. The VHH structure cloned and expressed separately has a structural stability comparable to that of the original heavy chain antibody, as well as binding activity with the antigen, and is the smallest unit known to bind to the target antigen. VHH crystals are 2.5nm, 4nm long, and have a molecular weight of only 15KDa, so they are also called nanoantibodies (Nanobody, Nb). Compared to traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune system in camelids can recognize the complex spatial structure of the surface of antigens and can produce highly specific and high-affinity nanoantibodies.

[0069] "Percent (%) sequence identity" relative to a reference amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference amino acid sequence, after aligning the sequences and (as needed) introducing gaps to obtain maximum percentage sequence identity, but without considering any conservative substitutions as part of the sequence identity. To determine the amino acid sequence identity percentage, alignment can be performed in various ways within the scope of the art, such as using BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0070] The term "pharmaceutically acceptable carrier" as used herein refers to ingredients in a pharmaceutical preparation other than the active ingredient that are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0071] As used herein, the term "treat" refers to alleviating and / or ameliorating a disorder and / or a disease or symptom associated therewith, as well as preventing the deterioration of the disorder's symptoms. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating symptoms, alleviating or improving prognosis. However, it should be understood that treating a disease or symptom does not require complete elimination of the disease or symptoms associated therewith.

[0072] The term "chimeric antigen receptor" or "CAR" as used herein is a receptor protein that gives immune cells new abilities to target specific antigenic proteins. Conventional CAR molecules are composed of the antigen binding region of an antibody, an extracellular hinge region, a transmembrane region, and an intracellular immunoreceptor tyrosine activation motif.

[0073] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. It should be understood by those skilled in the art that some experimental steps described in the examples can be performed with reference to the conventional operations in the art. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0074] Example

[0075] Example 1. Preparation of antigens and immunization of alpacas

[0076] In this embodiment, the present invention uses HPV18 VLP L1 (purchased from Creative Biostructure, product number CBS-V644) as an antigen for alpaca immunization. In brief, the experimental steps are as follows:

[0077] (1) The alpacas were immunized 4 times in total by subcutaneously injecting the antigen into the animals. The first immunization was counted as the first day, and the subsequent immunizations were on the 10th day, the 19th day, and the 28th day respectively;

[0078] (2) On day 28, before the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca;

[0079] (3) On day 42, i.e., 14 days after the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca.

[0080] Compared with traditional immunization technology solutions for animal antibodies such as mice and rabbits, the advantage of this technology is that it collects a large amount of alpaca venous peripheral blood, which is conducive to subsequent screening to obtain highly diverse nano-antibodies.

[0081] Example 2. Construction of Alpaca Nanobody Library

[0082] The two batches of alpaca venous peripheral blood collected in Example 1 were used as raw materials to construct a highly diverse nanobody library. The two batches of alpaca venous peripheral blood were processed by the following steps:

[0083] (1) Lymphocytes were isolated from alpaca venous peripheral blood using density gradient centrifugation;

[0084] (2) extracting total mRNA from lymphocytes and reverse transcribed into cDNA;

[0085] (3) using appropriate DNA primers and the above cDNA as a template, amplifying the VHH fragments of alpaca immunoglobulins IgG2 and IgG3, i.e., the DNA fragments of the nanobody, by polymerase chain reaction (PCR);

[0086] (4) Connecting the VHH DNA fragment to the phage surface display screening vector to form a VHH-pIII fusion protein expression vector plasmid library. Among them, pIII is a protein present on the flagella on the surface of the phage;

[0087] (5) The screening vector connected to the VHH DNA fragment obtained in step (4) is transformed into TG1 competent bacteria by electroporation, and all colonies are collected after appropriate cultivation, which is the alpaca nanoantibody library.

[0088] Compared with the traditional method of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, this embodiment can preserve all the nano-antibody fragments (nano-antibody library) of alpacas for a long time, and can continuously screen and develop nano-antibodies.

[0089] Example 3. Screening of specific nanobodies by phage surface display

[0090] Using the nanobody library obtained in Example 2 as a source, antigen-specific nanobodies were obtained by phage surface display screening. The specific steps are as follows:

[0091] (1) Take an appropriate amount of frozen nanoantibody library, inoculate it into bacterial culture medium, add an appropriate amount of helper phage after appropriate cultivation, and continue to cultivate under appropriate conditions.

[0092] (2) The phages amplified in the bacterial culture supernatant were extracted using the PEG-NaC method.

[0093] (3) The phages were appropriately incubated with antigens, and the HPV18 VLP antigens were pre-fixed in immunotubes (Maxisorp immunotubes, ThermoFisher Scientific).

[0094] (4) Panning: discard the phages, rinse the antigen several times with PBS buffer, pan and remove the phages that non-specifically bind to the antigen, and retain the phages that specifically bind to the antigen.

[0095] (5) Elution: using an acidic glycine solution to treat the phages that specifically bind to the antigen, so that the phages and the antigen are dissociated and retained. Thus, phages expressing specific nanobodies are obtained.

[0096] (6) The obtained phage is infected and cultured again to E. coli, but no helper phage is added. After the phage infection is complete, the specific nano-antibody exists in the E. coli in the form of a DNA plasmid. Collect all the E. coli to form an antigen-specific nano-antibody library. This library is used as a raw material and can be returned to step (1) for the next round of phage surface display screening.

[0097] (7) Transformation into monoclonal nanobody colonies. Take a small amount of the phage obtained in step (5), dilute it and infect E. coli again, but no helper phage is added. After the phage infection is complete, the E. coli are evenly spread on a bacterial culture dish, and monoclonal colonies containing nanobody DNA plasmids are obtained through cultivation.

[0098] Example 4. Identification of positive monoclonal nanobodies

[0099] The monoclonal colonies obtained in step (7) of Example 3 were subjected to identification of positive monoclonal nanobodies. The specific steps are as follows:

[0100] (1) Pick a single clone and culture it in a microplate;

[0101] (2) adding IPTG to induce the expression of the fusion protein VHH-pIII containing the nanobody;

[0102] (3) The bacterial culture supernatant containing nanobodies was collected and incubated with the antigen HPV18 VLP, which was pre-immobilized in a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific);

[0103] (4) using enzyme-linked immunosorbent assay (ELISA) to detect whether each monoclonal nanobody binds to the HPV18 VLP antigen;

[0104] (5) Screening out monoclonal colonies of monoclonal nanoantibodies that can bind to antigens.

[0105] Through the above method, 19 monoclonal colonies that can specifically recognize and bind to HPV18 L1 were screened.

[0106] Example 5. Recombinant expression and purification of small batches of monoclonal nanobodies

[0107] The 19 monoclonal colonies that can specifically recognize and bind to the antigen obtained by screening in Encoding Example 4 were expanded and cultured, DNA plasmids were extracted, and DNA sequencing was performed to obtain the nucleic acid sequence of the nanobody, and the amino acid sequence of the nanobody was obtained after translation. The sequences of the nanobodies obtained from these 19 monoclonal colonies are as follows: Figure 1 As shown in Tables 1 to 19.

[0108] Table 1. Amino acid sequence and nucleic acid sequence of antibody 1A1

[0109]

[0110]

[0111] Table 2. Amino acid sequence and nucleic acid sequence of antibody 1B4

[0112]

[0113] Table 3. Amino acid sequence and nucleic acid sequence of antibody 1B11

[0114]

[0115] Table 4. Amino acid sequence and nucleic acid sequence of antibody 1E10

[0116]

[0117]

[0118] Table 5. Amino acid sequence and nucleic acid sequence of antibody 1D5

[0119]

[0120] Table 6. Amino acid sequence and nucleic acid sequence of antibody 1F3

[0121]

[0122] Table 7. Amino acid sequence and nucleic acid sequence of antibody 3D11

[0123]

[0124] Table 8. Amino acid sequence and nucleic acid sequence of antibody 2A4

[0125]

[0126]

[0127] Table 9. Amino acid sequence and nucleic acid sequence of antibody 2A5

[0128]

[0129] Table 10. Amino acid sequence and nucleic acid sequence of antibody 2A7

[0130]

[0131] Table 11. Amino acid sequence and nucleic acid sequence of antibody 2H3

[0132]

[0133] Table 12. Amino acid sequence and nucleic acid sequence of antibody 2F5

[0134]

[0135] Table 13. Amino acid sequence and nucleic acid sequence of antibody 3C1

[0136]

[0137] Table 14. Amino acid sequence and nucleic acid sequence of antibody 2G11

[0138]

[0139] Table 15. Amino acid sequence and nucleic acid sequence of antibody 3B8

[0140]

[0141]

[0142] Table 16. Amino acid sequence and nucleic acid sequence of antibody 3G6

[0143]

[0144] Table 17. Amino acid sequence and nucleic acid sequence of antibody 4B4

[0145]

[0146] Table 18. Amino acid sequence and nucleic acid sequence of antibody 4F5

[0147]

[0148] Table 19. Amino acid sequence and nucleic acid sequence of antibody 4G7

[0149]

[0150]

[0151] In addition, the extracted DNA plasmid was transformed into BL21 (DE3) competent cells to express and purify monoclonal nanobodies in small batches. Then, the ELISA method was used to incubate different concentrations of nanobodies, and the affinity of the nanobodies to the antigen was measured based on the binding ability of the nanobodies to HPV18. The test results are shown in Figure 2 A~S.

[0152] from Figure 2 A~ Figure 2 From the results of S, it can be seen that the Kd values ​​of the 19 screened nanoantibodies binding to HPV18 are all below 100nM.

[0153] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A nanobody or an active fragment thereof against human papillomavirus (HPV) 18 subtype L1 protein, wherein the nanobody or the active fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises: CDR1 of the amino acid sequence shown in SEQ ID NO: 1; CDR2 of the amino acid sequence shown in SEQ ID NO: 2; and, a CDR3 having an amino acid sequence as shown in SEQ ID NO:3; CDR1 of the amino acid sequence shown in SEQ ID NO:11; CDR2 of the amino acid sequence shown in SEQ ID NO:12; and CDR3 of the amino acid sequence shown in SEQ ID NO:

13.

2. The nanobody or active fragment thereof according to claim 1, characterized in that The Nanobody or its active fragment comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 4 or 14.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Nanobody or its active fragment as described in claim 1 or 2.

4. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 3.

5. A host cell, characterized in that The host cell comprises the expression vector according to claim 4.

6. The host cell according to claim 5, characterized in that The host cell is selected from mammalian cells, insect cells, yeast cells or prokaryotic cells.

7. The host cell according to claim 5, characterized in that The host cell is Escherichia coli.

8. A protein conjugate, characterized in that The protein conjugate is composed of the nanobody or active fragment thereof as claimed in claim 1 or 2, and a ligand, and the ligand is selected from a radioactive isotope, a fluorescent group and / or a delivery vector.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody or its active fragment as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

10. Use of the nanobody or active fragment thereof as described in claim 1 or 2 in the preparation of a kit for diagnosing diseases infected with HPV18 subtype.

11. The use according to claim 10, characterized in that: The diseases include cervical cancer, anal canal cancer, tonsil cancer, oral cancer, laryngeal cancer, nasal cancer, esophageal cancer, Bowen's disease and flat warts.

12. The use according to claim 10, characterized in that: Such diseases include squamous cell carcinoma.

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