Single-domain antibody specifically binding to ganglioside glycan GD2

Abstract

The invention discloses a single-domain antibody capable of being specifically combined with ganglioside glycan GD2. The single-domain antibody can be specifically combined with GD2 glycan. The invention provides a single domain antibody which can be used for targeting and / or treating several cancers (such as neuroblastoma, glioma and the like) related to overexpressed GD2 cells. The invention also relates to a recombinant nucleic acid sequence for coding the single-domain antibody, an expression vector and a host cell.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a single domain antibody that specifically binds to ganglioside glycan GD2. The single domain antibody can specifically bind to GD2 glycan and can be used to target and / or treat several cancers associated with cells that overexpress GD2. Background Art

[0002] Tumor-associated carbohydrate antigens (TACAs) are abnormal sugar chains overexpressed on the surface of tumor cells and can be used as markers to distinguish normal cells from tumor cells. These abnormal sugar chains originate from the combined action of glycosidases and glycosyltransferases abnormally expressed in tumor cells. Ganglioside TACAs (GD2, GD3, GM2, GM3) are glycosphingolipids modified with sialic acid. Their unique structure makes them widely used in the field of medicine and health care. Their sugar chain parts are highly expressed on the surface of tumors. As the terminal antigenic determinant of gangliosides, they have a high degree of chemical structure conservation and tumor specificity, so they are regarded as tumor markers. Among them, disialyl ganglioside GD2 is closely related to the occurrence and development of tumors and malignant phenotypes by enhancing cell proliferation, motility, migration, adhesion and invasion. This provides a theoretical basis for the development of anti-GD2 monoclonal antibodies and other therapeutic methods for tumor treatment with disialyl ganglioside GD2 as a target. However, the treatment of GD2 monoclonal antibodies for tumors with high GD2 expression is still hindered by pharmacological factors, such as the large size of monoclonal antibodies, which leads to insufficient penetration into the tumor microenvironment.

[0003] Unlike traditional mammalian IgG antibodies, single-domain antibodies contain only heavy chains and no light chains. Single-domain antibodies with antigen-binding function are also called VHH or nanobodies. VHH have several general advantages over traditional antibodies. First, they are only 13-15kDa in size, which is about 10 times smaller than traditional IgG antibodies (150KDa). Even in crowded cellular environments, they have better epitope binding ability and can better penetrate into tissues, organs and animals. Compared with traditional antibodies, they have higher stability and longer shelf life, and are easy to produce and modify. Therefore, the development of single-domain antibodies that can specifically target the tumor-associated carbohydrate antigen GD2 has good application prospects as research tool carriers, disease diagnostic tools and disease treatment strategies. Summary of the invention

[0004] Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides a single domain antibody that specifically binds to ganglioside GD2, and provides a single domain antibody that can be used to target and / or treat several cancers associated with cells overexpressing GD2.

[0005] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a single domain antibody or a polypeptide comprising the single domain antibody, wherein the single domain antibody has binding specificity to ganglioside glycan GD2 and comprises complementary determining domains: CDR1, CDR2 and CDR3, wherein:

[0007] The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 21; or

[0008] The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; or

[0009] The CDR1 comprises the amino acid sequence shown in SEQ ID NO:25, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:27.

[0010] Optionally, in some embodiments of the present invention, the single domain antibody further comprises framework regions FR1, FR2, FR3 and FR4, and has the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein:

[0011] The FR1 is selected from the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:11 or SEQ ID NO:15;

[0012] The FR2 is selected from the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 16;

[0013] The FR3 is selected from the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:17;

[0014] The FR4 is selected from the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18.

[0015] Optionally, in some embodiments of the present invention, the amino acid sequence of the single-domain antibody is selected from any one of the following: SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.

[0016] Optionally, in some embodiments of the invention, at least one single domain antibody has at least 80% sequence identity with the sequences of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.

[0017] Further optionally, in some embodiments of the present invention, at least one single domain antibody has at least 90% sequence identity with the sequences of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.

[0018] In a second aspect, the present invention provides a recombinant nucleic acid molecule encoding a single domain antibody or a polypeptide comprising the single domain antibody as described in any one of the above items.

[0019] In a third aspect, the present invention provides a vector comprising the above recombinant nucleic acid molecule.

[0020] In a fourth aspect, the present invention provides a host cell comprising the above-mentioned recombinant nucleic acid molecule or the vector as claimed in claim 6.

[0021] In a fifth aspect, the present invention provides a composition comprising the single domain antibody as described in any one of the above items or a polypeptide comprising the single domain antibody and a pharmaceutically acceptable carrier.

[0022] In a sixth aspect, the present invention provides a method for treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of the single domain antibody as described in any one of the above or a polypeptide comprising the single domain antibody.

[0023] In a seventh aspect, the present invention provides use of any of the above single domain antibodies or polypeptides comprising the single domain antibodies in the preparation of drugs for cancer treatment and / or diagnosis.

[0024] Optionally, in some embodiments of the present invention, the cancer includes several cancers associated with cells that overexpress GD2, such as neuroblastoma, glioma, etc. Beneficial effect: The advantage of the present invention is that the present invention provides a method for obtaining a GD2-specific single-domain antibody, a high-titer single-domain antibody phage display library is constructed using KLH-GD2 vaccine, and HSA-GD2 is used for solid phase panning to obtain the GD2 single-domain antibody gene sequence. The method is simple to operate and multiple GD2-specific single-domain antibody sequences can be obtained at the same time. Another advantage of the present invention is that the GD2 single-domain antibody provided by the present invention can be used to target and / or treat several cancers associated with cells that overexpress GD2 (such as neuroblastoma and glioma), which can be stably, soluble, and expressed in large quantities in a prokaryotic expression system, which is convenient for batch acquisition of high-purity antibodies; in addition, the GD2 single-domain antibody obtained by the present invention has strong antigen specificity, high affinity, and high activity both in vivo and in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the vaccine synthesis route.

[0026] Figure 2 This is a schematic diagram of the GD2 serum titer results of alpacas 7 weeks after immunization in Example 2.

[0027] Figure 3 Schematic diagram of the positive rate detection results of the phage library in Example 3, wherein 1-16 represent randomly selected monoclonal phages, and M represents DL5000 marker.

[0028] Figure 4 This is a schematic diagram of the results of phage ELISA determination of GD2 positive monoclonal clones in Example 5.

[0029] Figure 5 This is an SDS-page diagram of the expression and purification of the GD2 single domain antibody in Example 6, wherein 1 represents the supernatant of the single domain antibody fermentation broth, 2-7 represent protein eluates with different concentrations of imidazole, and M represents protein Marker.

[0030] Figure 6 This is the binding kinetics diagram of the GD2 single domain antibody detected by SPR in Example 7.

[0031] Figure 7 This is a flow cytometry analysis diagram of detecting the recognition of GD2 single domain antibody on HEK293 cells artificially coated with GD2 glycolipid in Example 8. Figure 8 This is the therapeutic ability of the GD2 single domain antibody in Example 9 on EL4 tumor-bearing mice. DETAILED DESCRIPTION

[0032] definition

[0033] It should be noted that the term "a" or "an" entity refers to one or more of the entity; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0034] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence, which means that when compared, the percentage of bases (or amino acids) is the same in the two sequences being compared. Such comparisons and percentage homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, the comparison is performed using default parameters. One comparison program is BLAST, using default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort by = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Bioequivalent polynucleotides are those having the above specified percentage homology and encoding polypeptides having the same or similar biological activity.

[0035] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a certain degree of homology or sequence identity with the nucleotide sequence of the nucleic acid or its complement. The homologue of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence with a certain degree of homology with it or its complement. On the one hand, the homologue of a nucleic acid can hybridize with a nucleic acid or its complement. Similarly, "equivalent polypeptide" refers to a polypeptide with a certain degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some aspects, compared with a reference polypeptide or polynucleotide, an equivalent polypeptide or polynucleotide has one, two, three, four or five additions, deletions, substitutions and combinations thereof. In some aspects, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of a reference sequence.

[0036] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule with antigen-binding biological activity. Such examples include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or its ligand-binding portion, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least one portion of a binding protein.

[0037] As used herein, the term "antibody fragment" or "antigen binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment can bind to the antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror images, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0038] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, these regions are connected to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine to increase flexibility, or rich in serine or threonine to increase solubility, and it can connect the N-terminus of VH and the C-terminus of VL, or it can be connected in reverse. This protein retains the specificity of the original immunoglobulin, despite the removal of the constant region and the introduction of a linker. ScFv molecules are known in the art, as described in U.S. Patent 5,892,019.

[0039] The term "antibody" includes various major classes of polypeptides that can be distinguished by biochemical methods. It will be appreciated by those skilled in the art that heavy chains can be divided into γ, μ, α, δ or ε, with some subclasses (e.g., γl-γ4) therein. The nature of the chain determines the "class" of the antibody, which is IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized, and it is known that they have functional specificity. In view of the present disclosure, modified versions of each of these classes and isotypes are easily distinguishable to those skilled in the art, and are therefore within the scope of the present disclosure. All immunoglobulin classes are obviously within the scope of the present disclosure, and the following discussion is generally directed to IgG class immunoglobulin molecules. For IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 daltons. These four chains are usually connected by disulfide bonds to form a "Y"-shaped structure, in which the light chain starts from the mouth of the "Y"-shaped structure and extends to the variable region, surrounding the heavy chain.

[0040] The antibodies, antigen-binding polypeptides, variants or derivatives thereof disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising VK or VH domains, expression libraries from Fab, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies of LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

[0041] "Specific binding" or "having specificity" generally refers to the binding of an antibody to an epitope through its antigen binding domain, and such binding requires a certain complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" an epitope when it binds to the epitope more easily through its antigen binding domain than to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B", or it may be said that antibody "A" binds to epitope "C" with higher specificity than it binds to the related epitope "D".

[0042] As used herein, the term "treatment" refers to therapeutic and prophylactic treatment or preventive measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or total relief), whether detectable or undetectable. "Treatment" can also refer to prolonging survival compared to the expected survival without treatment. Those in need of treatment include those who already have the disease or disorder and those who are prone to the disease or disorder or those in which the disease or disorder is to be prevented.

[0043] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo, sport or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0044] As used herein, phrases such as "in a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from the administration of an antibody or composition used in the present disclosure, e.g., for detection, for diagnostic procedures, and / or for therapy.

[0045] Example Single domain antibody specifically binding to ganglioside GD2

[0046] The present invention discloses a single domain antibody that specifically binds to ganglioside glycan GD2. The single domain antibody can specifically bind to GD2 glycan, wherein the single domain antibody is a variable domain of a heavy chain antibody, and the heavy chain antibody naturally lacks a light chain and a constant region 1.

[0047] Based on this, specifically, the present invention proposes a single domain antibody or a polypeptide comprising the single domain antibody, wherein the single domain antibody has binding specificity to ganglioside glycan GD2 and comprises complementary determining domains: CDR1, CDR2 and CDR3, wherein:

[0048] The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 21; or

[0049] The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; or

[0050] The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0051] In some examples of the present invention, the present invention discloses a single domain antibody that specifically binds to ganglioside glycan GD2, wherein the single domain antibody can specifically bind to GD2 glycan, and the single domain antibody comprises a framework region (FR) and a complementarity determining region (CDR). Specifically, the single domain antibody comprises framework regions FR1, FR2, FR3 and FR4, and has the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein:

[0052] The FR1 is selected from the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:11 or SEQ ID NO:15;

[0053] The FR2 is selected from the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 16;

[0054] The FR3 is selected from the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:17;

[0055] The FR4 is selected from the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18.

[0056] Preferably, in some examples of the present invention, the single-domain antibody comprises the following regions in sequence:

[0057] Framework region 1 (FR1) selected from SEQ ID NO:7, SEQ ID NO:11 or SEQ ID NO:15;

[0058] a complementarity determining domain 1 (CDR1) selected from SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO: 25;

[0059] Framework region 2 (FR2) selected from SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 16;

[0060] a complementarity determining domain 2 (CDR2) selected from SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 26;

[0061] a framework region 3 (FR3) selected from SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17;

[0062] a complementarity determining domain 3 (CDR3) selected from SEQ ID NO:21, SEQ ID NO:24 or SEQ ID NO:27;

[0063] Framework region 4 (FR4) selected from SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18.

[0064] In some examples of the present invention, the optional instances of each framework region 1 (FR1), each complementary determining region 1 (CDR1), each framework region 2 (FR2), each complementary determining region 2 (CDR2), each framework region 3 (FR3), each complementary determining region 3 (CDR3) and each framework region 4 (FR4) in the present invention have a high sequence homology, for example, a sequence homology of more than 80%.

[0065] More preferably, in some examples of the present invention, the single-domain antibody comprises the following regions in sequence:

[0066] a framework region 1 (FR1) selected from the group consisting of SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:15;

[0067] a complementarity determining domain 1 (CDR1) as shown in SEQ ID NO: 19;

[0068] a framework region 2 (FR2) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16;

[0069] a complementarity determining domain 2 (CDR2), which is shown in SEQ ID NO: 20;

[0070] a framework region 3 (FR3) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 17;

[0071] a complementarity determining domain 3 (CDR3), which is shown in SEQ ID NO: 21;

[0072] A framework region 4 (FR4) selected from the group consisting of SEQ ID NO:10, SEQ ID NO:14 and SEQ ID NO:18.

[0073] More preferably, in some examples of the present invention, the single-domain antibody comprises the following regions in sequence:

[0074] a framework region 1 (FR1) selected from the group consisting of SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:15;

[0075] a complementarity determining domain 1 (CDR1) as shown in SEQ ID NO: 22;

[0076] a framework region 2 (FR2) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16;

[0077] a complementarity determining domain 2 (CDR2), which is shown in SEQ ID NO: 23;

[0078] a framework region 3 (FR3) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 17;

[0079] a complementarity determining domain 3 (CDR3) as shown in SEQ ID NO: 24;

[0080] A framework region 4 (FR4) selected from the group consisting of SEQ ID NO:10, SEQ ID NO:14 and SEQ ID NO:18.

[0081] More preferably, in some examples of the present invention, the single-domain antibody comprises the following regions in sequence:

[0082] a framework region 1 (FR1) selected from the group consisting of SEQ ID NO: 7, SEQ ID NO 11 and SEQ ID NO: 15;

[0083] a complementarity determining domain 1 (CDR1) as shown in SEQ ID NO: 25;

[0084] a framework region 2 (FR2) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16;

[0085] a complementarity determining domain 2 (CDR2), which is set forth in SEQ ID NO: 26;

[0086] a framework region 3 (FR3) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 17;

[0087] a complementarity determining domain 3 (CDR3), which is set forth in SEQ ID NO: 27;

[0088] A framework region 4 (FR4) selected from the group consisting of SEQ ID NO:10, SEQ ID NO:14 and SEQ ID NO:18.

[0089] More preferably, in some examples of the present invention, the amino acid sequence of the single-domain antibody is selected from the following: SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.

[0090] More preferably, in some examples of the present invention, at least one single domain antibody has at least 80% sequence identity with the sequences of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.

[0091] More preferably, in some examples of the present invention, at least one single domain antibody has at least 90% sequence identity with the sequences of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.

[0092] On the other hand, the present invention also provides a recombinant nucleic acid molecule encoding a single domain antibody or a polypeptide comprising the single domain antibody as described in any one of the above.

[0093] In another aspect, the present invention also provides a vector comprising the above-mentioned recombinant nucleic acid molecule.

[0094] In another aspect, the present invention also provides a host cell comprising the above-mentioned recombinant nucleic acid molecule or the above-mentioned vector.

[0095] On the other hand, the present invention also provides a composition comprising the single domain antibody as described in any one of the above items or a polypeptide comprising the single domain antibody and a pharmaceutically acceptable carrier.

[0096] It will also be appreciated by those of ordinary skill in the art that the antibodies disclosed herein can be modified so that their amino acid sequences are different from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein may be similar to the starting sequence, for example, having a certain percentage of identity, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to the starting sequence. In some embodiments, the modified antibody or fragment retains the specified CDR sequence.

[0097] In another aspect, the present invention also provides a method for treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of any of the above single domain antibodies or polypeptides comprising the single domain antibodies.

[0098] On the other hand, the present invention also provides use of any of the above single domain antibodies or polypeptides comprising the single domain antibodies in the preparation of drugs for cancer treatment and / or diagnosis.

[0099] In another aspect, the present invention provides a method for preparing the above-mentioned single domain antibody that specifically binds to ganglioside glycan GD2 or a polypeptide comprising the single domain antibody, comprising the following steps:

[0100] (1) preparing KLH-GD2 vaccine to immunize alpacas;

[0101] (2) ELISA was used to detect the titer of serum after immunization of alpacas;

[0102] (3) Construction of phage display library;

[0103] (4) solid phase panning of GD2-specific phage;

[0104] (5) Indirect phage ELISA to detect positive monoclones;

[0105] (6) Using the E. coli expression system for soluble expression of single domain antibodies.

[0106] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. According to the following embodiments, the present invention can be better understood. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0107] Example 1: Preparation of KLH-GD2 vaccine

[0108] This embodiment is intended to Figure 1 As shown, KLH-GD2 was prepared according to the synthetic route in reference (Chinese Chemical Letters 32(2021)4041–4044).

[0109] Preparation of GD2 tumor sugar vaccine: Take 300ug of KLH-GD2, completely dissolve it with 1mL of PBS, and mix it thoroughly with 1mL of complete Freund's adjuvant to prepare KLH-GD2 tumor sugar vaccine.

[0110] Alpaca immunization: Before immunization, blood was collected from the alpaca's neck vein using a vacuum blood collection tube as a non-immunized serum control (D0); multiple immunizations were performed in the alpaca's neck by subcutaneous injection, with an immunization dose of 0.3 mg glycoprotein vaccine per time, and boosted once a week for 7 consecutive immunizations; after multiple immunizations, peripheral venous blood was collected from the alpaca, and serum was separated for antibody titer determination. After the last immunization, 90 mL of peripheral venous blood was collected from the alpaca for the separation of peripheral blood lymphocytes.

[0111] Example 2: ELISA detection of serum titer after alpaca immunization

[0112] This example is intended to detect the serum titer of alpaca immunization by ELISA

[0113] Take 10mL of the alpaca peripheral blood obtained in Example 1 and let it stand for 30 minutes at 4°C and 37°C respectively, then centrifuge at 5000rpm for 10 minutes. The supernatant after centrifugation is the alpaca serum. Dissolve HSA-GD2 to 2μg / mL with coating buffer, 100μL per well, coat the antigen at 4°C overnight and transfer to a 37°C incubator for incubation for 1h. After coating, wash the wells 3 times with PBST. Add 200μL ELISA blocking buffer to each well and block at room temperature for 2h. After blocking, wash the wells 3 times with PBST. Add gradient diluted immune serum or D0 serum to the corresponding wells and incubate at 37°C for 2h. After incubation, wash the wells 3 times with PBST. According to the secondary antibody (here refers to MonoRab TM Rabbit Anti-Camelid VHH Cocktail [HRP]) instructions, dilute the secondary antibody with PBS at the dilution multiple (1:5000) recommended in the instructions, and incubate at 37°C for 1 hour. After incubation, wash the wells 3 times with PBST. According to the instructions of the TMB kit, the absorbance value of each well was detected at 450nm. The maximum dilution multiple corresponding to the well with (Px / Nx)>2 was regarded as the titer of the alpaca serum. P is the absorbance value corresponding to the immune serum well, N is the absorbance value corresponding to the D0 serum well, and x is the dilution multiple.

[0114] The results of ELISA testing the titer of serum after alpaca immunization are as follows Figure 2 As shown, after immunizing alpacas three times, the serum titer of GD2-specific heavy chain antibodies (35804) increased to 1.75 times that of non-immunized alpacas (20391), and after immunizing alpacas seven times (53892) increased to 2.64 times that of non-immunized alpacas.

[0115] Example 3: Construction of phage display library

[0116] This example aims to construct a phage display library.

[0117] Using the cDNA of alpaca peripheral blood lymphocytes obtained in Example 1 as a template, nested PCR was performed using the leader peptide conserved region primer CALL001 and the CH2 region primer CALL002 to obtain the VHH gene sequence. The PCR-amplified VHH product was connected to the vector by restriction enzyme ligation and then transformed into TG1 competent cells. The bacterial solution after transformation and recovery was mixed, and the initial library bacterial library capacity obtained by gradient dilution plate counting method was about 1.1×10 5 cfu, 24 clones were randomly selected for colony PCR, and the positive rate was about 96%.

[0118] The titer of the helper phage after amplification was determined by the gradient dilution plate counting method to be approximately 1.23×10 10 cfu / mL, and the library was amplified at a multiplicity of infection of 100:1 to obtain a titer of 6×10 14 cfu / mL of the nanobody phage library, 16 clones were randomly selected for colony PCR, such as Figure 3 As shown, the positive rate was 100%.

[0119] Example 4: Solid phase panning of GD2-specific phage

[0120] This example aims at solid phase panning of GD2-specific phage.

[0121] HSA-GD2 (1 mg / mL) was diluted to 200 μg / mL with coating buffer, 100 μL was added to each well of the plate, and incubated at 4°C overnight. Washed three times with PBS, 3% BSA was added, 300 μL per well, and blocked at 37°C for 1 hour. Washed three times with PBS, phage library was added, and incubated at 37°C for 1 hour. Washed 5 times with PBS, 100 μL of phage elution buffer was added, and incubated at 37°C for 10 minutes. Take the eluate in a 1.5 mL centrifuge tube, immediately add 15 μL of phage neutralization buffer to neutralize, mix well, and take 10 μL of the eluate to measure the elution amount.

[0122] After mixing the magnetic beads, take 100uL to a 1mL centrifuge tube, remove the supernatant by magnetic separation, wash twice with 200uL MEST solution, and remove the supernatant. Quickly add freshly prepared 100uL EDC solution and 100uL NHS solution to the magnetic beads, and activate on a rotating mixer for 30min. Remove the supernatant by magnetic separation of the activated magnetic beads, add 100ug sugar antigen (1mg / mL), and couple at room temperature for 2h, keeping the magnetic beads suspended during coupling. Remove the supernatant by magnetic separation, resuspend the magnetic beads with 200uL PBST (1% BSA), react at room temperature for 1h, and block the unreacted carboxyl groups on the surface of the magnetic beads. Remove the supernatant by magnetic separation, wash 3 times with 200uL PBS, and store at 4°C.

[0123] The magnetic beads were allowed to stand at room temperature for 20 minutes, and the supernatant was removed by magnetic separation. The beads were washed three times with 200 μL of TBST, and the supernatant was removed by magnetic separation. 3% BSA (PBS) was added and blocked at 4°C for 1 hour. The blocking solution was removed by magnetic separation, and the beads were washed three times with 200 μL of TBST. The supernatant was removed by magnetic separation, and the phage library was added. The beads were incubated at room temperature for 1 hour, and the magnetic beads were kept suspended during incubation. Unbound phages were removed by magnetic separation, and the magnetic beads were washed ten times with 200 μL of TBST. The supernatant was removed by magnetic separation. 500 μL of elution solution was added to the magnetic beads, and the magnetic beads were suspended by gentle pipetting. The beads were rotated and incubated for 5 minutes, and the elution solution was removed by magnetic separation. The magnetic beads were washed five times with PBS. The eluate was transferred to a 1.5 mL centrifuge tube, and 100 μL of phage neutralization buffer was added immediately for neutralization. The mixture was gently mixed, and 10 μL of the eluate was taken to measure the elution amount.

[0124] Example 5: Indirect phage ELISA detection of positive monoclonal

[0125] This example is intended to detect positive single clones by indirect phage ELISA.

[0126] Pick 48 single colonies from the plate for titer detection and inoculate them in 1mL 2YT-A / Glu, and culture until OD600 = 0.4. Add 10μL helper phage, let stand at 37℃ for 15min, and then culture at 220rpm for 2h. Centrifuge at 3000g for 10min, discard the supernatant, resuspend the precipitate with an equal volume of 2YT-AK / Glu, and culture overnight at 30℃ and 250rpm. Centrifuge at 8000rpm, 4℃ for 10min, and take the supernatant for ELISA identification.

[0127] Dilute the glycoprotein to 8 μg / mL with coating buffer, add 100 μL per well to the plate, and incubate at 4°C overnight. Wash 3 times with PBST, add 4% BSA, 300 μL per well, and block at 37°C for 1 hour. Wash 3 times with PBST, add phage supernatant, 100 μL per well, and incubate at 37°C for 2 hours. Wash 3 times with PBST, dilute HRP / anti-M13 secondary antibody with blocking buffer (1:5000), 100 μL per well, and incubate at 37°C for 1 hour. Wash 6 times with PBST, add TMB colorimetric solution, 100 μL per well, and incubate at 37°C for 15 minutes. Add 2M H2SO4 stop solution, 50 μL per well to stop the reaction, and measure the OD450 value.

[0128] The results of indirect phage ELISA detection of positive monoclonal Figure 4 As shown, among the 48 randomly selected monoclonal clones, the single domain antibodies displayed by most phages had weak recognition of GD2 antigen (OD450 in the ELISA test was lower than 0.3), so the positive monoclonal clones were selected based on OD450=0.3.

[0129] Example 6: Soluble expression of single domain antibodies using the E. coli expression system

[0130] This example aims to express single domain antibodies in a soluble form in an E. coli expression system.

[0131] Take 100 μL of glycerol bacteria and transfer to 10 mL of LB-A medium, 200 rpm, 37 ° C overnight culture to obtain seed liquid. Transfer the seed liquid to TB medium at a 2% inoculation ratio, 200 rpm, 37 ° C to OD 600 When the pH was between 0.6 and 0.8, IPTG was added to a final concentration of 0.5 mM for induction, and the temperature was switched to 16°C for inducible expression of nanobodies.

[0132] The bacterial liquid after 48h of fermentation was collected, centrifuged at 9000rpm for 3min, the bacterial precipitate was collected, and the bacterial cells were resuspended with 50mL buffer A. The bacterial cells were broken in an ice bath, and the working mode of the ultrasonic crusher was set to break for 3s and stop for 2s, with a maximum power of 30%, and an effective breaking time of 30min. The suspension after breaking was centrifuged at 9000rpm for multiple times, each time for 10min, and all the broken supernatants were collected and filtered with a 0.22μm filter membrane for standby use. The nickel ion-coupled agar gravity column for protein purification was rinsed with ultrapure water for 10 column volumes, and buffer A was rinsed for 10 column volumes for column balance, and the filtered broken supernatant was added for loading, all the flow-through liquid was collected, and the filtered buffer A was rinsed for 2 column volumes. After Coomassie Brilliant Blue G250 detected that there was no protein outflow, several column volumes were then rinsed with 5%, 10%, 20%, 50%, and 100% buffer B, and all the eluents were collected for SDS-PAGE detection.

[0133] The results of soluble expression of single domain antibodies in E. coli expression system are as follows Figure 5 As shown, the fermentation broth of the single domain antibody is centrifuged, the bacteria are collected, and the supernatant after crushing is purified by a nickel ion-coupled agar gravity column. Lane 1 is the unpurified crushed supernatant, and lanes 2-7 are protein samples eluted with different imidazole concentrations. SDS-PAGE analysis shows that the expression form of the single domain antibody in the E. coli system is intracellular soluble expression, and high-purity single domain antibodies can be obtained by nickel column purification, and the molecular weight is between 17-18 kDa, which is consistent with the size of single domain antibodies.

[0134] By the above method, the present invention obtains three GD2 single domain antibodies: 2D24; 2D26; 2D29, the amino acid sequences of each single variable domain are listed in Table 1 below, and the nucleotide sequences are shown in SEQ ID NO:4, SEQ ID NO:5SEQ ID NO:6 respectively. The CDR and FR sequences are summarized in Tables 1A-1F. Some CDR sequences include dipeptides, such as NG and DG, which may be susceptible to post-translational modifications. The table also provides certain "de-risked" versions of these sequences, which are expected to retain antibody activity while reducing the risk of such post-translational modifications.

[0135] Table 1. Variable region sequences

[0136]

[0137] Table 1A. CDR sequences of 2D24

[0138] name Amino acid sequence SEQ ID NO 2D24-CDR1 FTLGYYT 19 2D24-CDR2 SDGST 20 2D24-CDR3 ADLERIDADSCMRYAYGAL 21

[0139] Table 1B. FR sequence of 2D24

[0140]

[0141]

[0142] Table 1C. CDR sequences of 2D26

[0143] name Amino acid sequence SEQ ID NO 2D26-CDR1 TGFSVYA 22 2D26-CDR2 RGGIT 23 2D26-CDR3 TDQFFTLTTISTDPD 24

[0144] Table 1D. FR sequence of 2D26

[0145]

[0146] Table 1E. CDR sequences of 2D29

[0147] name Amino acid sequence SEQ ID NO 2D29-CDR1 FTFSSYA 25 2D29-CDR2 TGGDIT 26 2D29-CDR3 RWGTRGWVDGMD 27

[0148] Table 1F. FR sequence of 2D29

[0149]

[0150]

[0151] Example 7: SPR detection of specificity and affinity of GD2 single domain antibody

[0152] This example aims to study the specificity and affinity of SPR detection of GD2 single domain antibodies. SPR can show the binding mode and binding constant of antibodies and antigens. In addition, we detected a total of 4 structurally similar carbohydrate antigens, proving that single domain antibodies have specificity and no cross-binding activity.

[0153] The CM5 chip was activated with equal volumes of NHS and EDC for 500s, and the single domain antibody was diluted to a concentration of 30-50μg / mL with sodium acetate buffer at pH 5.0 for coupling, and the coupling was performed at a flow rate of 10μL / min. The flow channel recorded the background response value until the response value no longer increased. PBS-P+ solution was used as the running buffer, and the sugar antigen (15.6-4000nM) was flowed through the chip at a flow rate of 30μL / min, with a contact time of 180s and a dissociation time of 300s. The experimental results were analyzed according to the 1:1 binding model for kinetic data and calculated KD values.

[0154] The results of SPR detection of the specificity and affinity of GD2 single domain antibody in this example are as follows Figure 6 As shown, the single-domain antibody 2D29 has GD2 binding specificity, with an affinity of 24.6μM and a binding mode of fast binding and slow dissociation, which is consistent with the antigen-antibody binding mode; the single-domain antibody 2D29 has an affinity for the GD3 antigen greater than 1mM, that is, the affinity is very weak, and has no binding ability to GM2 and GM3 with similar structures, proving that 2D29 has specificity for the GD2 antigen. In addition, the affinity data of 2D24 and 2D26 are shown in the following table.

[0155] Table 2. Affinity data of GD2 single domain antibody

[0156] 2D24 2D26 2D29 GD2 311μM 102μM 24.6μM GD3 197μM No binding 1.5mM GM2 No binding No binding No binding GM3 No binding No binding No binding

[0157] Example 8: Recognition of HEK293 cells artificially coated with GD2 glycolipid by GD2 single domain antibody

[0158] This example aims to study the recognition of GD2 single domain antibody on HEK293 cells artificially coated with GD2 glycolipid.

[0159] HEK293 cells were resuspended in PBS buffer to 8 × 10 5 / mL, GD2 glycolipid (concentration of 200μM) was mixed with 100μL cell suspension, incubated at 37℃ for 20min, washed with PBS buffer, added with single domain antibodies of different concentrations (2.44-150μM), incubated on ice for 30min, washed twice with FACS buffer, added with Myc antibody diluent diluted 1:1000, incubated on ice for 30min, washed twice with FACS buffer, added with Alexa Fluor 647 labeled goat anti-mouse IgG antibody diluted 1:2000, incubated on ice for 30min, washed twice with FACS buffer, resuspended to 100μL, detected the samples by flow cytometer, and analyzed the results with FlowJo software.

[0160] The results of GD2 single domain antibody recognizing HEK293 cells artificially coated with GD2 glycolipid are shown in Figure 7 As shown, GD2 has no recognition ability for HEK293 cells alone, but has significant recognition ability for HEK293 cells artificially coated with GD2 glycolipids, and its recognition is concentration gradient dependent, with an affinity of 20.87 μM.

[0161] Example 9: The therapeutic ability of GD2 single domain antibody on EL4 tumor-bearing mice

[0162] This example aims to study the therapeutic ability of GD2 single domain antibody on EL4 tumor-bearing mice.

[0163] Mouse lymphoma EL4 cells were resuspended in PBS buffer to 5 × 10 7 100 μL of cell suspension was injected subcutaneously into the left flank of Balb / c mice. One week later, the tumor volume was between 100-300 mm 3 The mice were randomly divided into 4 groups (PBS group, 2D29 group, 2D26 group and 2D24 group), with 5 mice in each group. Intratumoral administration was performed three times a week for two weeks. The PBS group was injected with 50 μL PBS each time; the other groups were injected with 50 μL single domain antibody (100 μg) each time. After the treatment, the mice were dissected and the tumor mass was detected.

[0164] The therapeutic ability of GD2 single domain antibody on EL4 tumor-bearing mice Figure 8 As shown, after two weeks of continuous administration, the tumor mass of mice in the single-domain antibody group was significantly lower than that in the PBS control group, which preliminarily demonstrated the in vivo therapeutic potential of the GD2 single-domain antibody.

[0165] The above in vivo and in vitro experiments demonstrate that the GD2 single domain antibody prepared in the embodiments of the present invention can be used to target and / or treat several cancers associated with cells overexpressing GD2 (such as neuroblastoma, glioma, etc.).

[0166] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A single domain antibody or a polypeptide comprising the single domain antibody, characterized in that: The single domain antibody has binding specificity to ganglioside GD2 and comprises complementary determining domains: CDR1, CDR2 and CDR3, wherein: The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 21; or The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; or The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:

27.

2. The single domain antibody or a polypeptide comprising the single domain antibody according to claim 1, characterized in that: The single domain antibody further comprises framework regions FR1, FR2, FR3 and FR4, and has the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein: The FR1 is selected from the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:11 or SEQ ID NO:15; The FR2 is selected from the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 16; The FR3 is selected from the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:17; The FR4 is selected from the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:

18.

3. The single domain antibody or the polypeptide comprising the single domain antibody according to claim 1, characterized in that: The amino acid sequence of the single domain antibody is selected from any one of the following: SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:

5.

4. The single domain antibody or a polypeptide comprising the single domain antibody according to claim 1, characterized in that: at least one single domain antibody has at least 80% sequence identity to the sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5; Preferably, at least one single domain antibody has at least 90% sequence identity to the sequences of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:

5.

5. A recombinant nucleic acid molecule encoding a single domain antibody or a polypeptide comprising the single domain antibody according to any one of claims 1 to 4. A vector comprising the recombinant nucleic acid molecule of claim 5 .

7. A host cell comprising the recombinant nucleic acid molecule according to claim 5 or the vector according to claim 6.

8. A composition comprising the single domain antibody according to any one of claims 1 to 4 or a polypeptide comprising the single domain antibody and a pharmaceutically acceptable carrier.

9. A method for treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of the single domain antibody or a polypeptide comprising the single domain antibody according to any one of claims 1 to 4.

10. Use of the single domain antibody or a polypeptide comprising the single domain antibody according to any one of claims 1 to 4 in the preparation of a medicament for cancer treatment and / or diagnosis.

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