A single-domain antibody that specifically binds to the ganglioside glycan gd2

Abstract

Disclosed is a single-domain antibody specifically binding to ganglioside GD2, which can specifically bind to GD2 glycan. The present application provides a single-domain antibody that can be used for targeting and / or treating several cancers (such as neuroblastoma, glioma, etc.) associated with GD2-overexpressing cells. The present application also relates to a recombinant nucleic acid sequence encoding the above-mentioned single-domain antibody, as well as an expression vector and a host cell.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and specifically relates to a single-domain antibody specifically binding to ganglioside GD2, which can specifically bind to GD2 glycan and can be used for targeting and / or treating several cancers related to GD2-overexpressing cells. BACKGROUND

[0002] Tumor-associated carbohydrate antigens (TACAs) are abnormal sugar chains overexpressed on the surface of tumor cells, which can be used as markers to distinguish normal cells and tumor cells. These abnormal sugar chains are derived from the combined action of glycosidase and glycosyltransferase abnormally expressed in tumor cells. Ganglioside TACAs (GD2, GD3, GM2, GM3) are sialic acid-modified glycosphingolipids, and their unique structure makes them have a wide range of uses in the field of medicine and health care. The sugar chain part thereof is highly expressed on the surface of tumors, and as the terminal antigenic determinant of gangliosides, it has high chemical structure conservation and tumor specificity, and is therefore regarded as a tumor marker. Among them, bisialyl ganglioside GD2 is closely related to tumor occurrence, development and malignant phenotype by enhancing cell proliferation, movement, migration, adhesion and invasion. This provides a theoretical basis for developing anti-GD2 monoclonal antibodies and other therapeutic methods for tumor treatment by targeting bisialyl ganglioside GD2. However, the treatment of GD2-overexpressing tumors by GD2 mAb is still hindered by pharmacological factors, such as the insufficient penetration of the tumor microenvironment caused by the large size of the monoclonal antibody.

[0003] Unlike traditional mammalian IgG antibodies, single-domain antibodies only contain heavy chains and do not contain light chains. Single-domain antibodies with antigen binding function are also called VHH or nanobodies. VHH has several general advantages compared to traditional antibodies. First, their size is only 13-15 kDa, which is about 10 times smaller than traditional IgG antibodies (150 KDa). Even in a crowded cellular environment, they can have better epitope binding capacity and better penetration into tissues, organs and animal bodies. 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 tumor-associated carbohydrate antigens GD2 has good application prospects as research tool carriers, disease diagnosis tools and disease treatment strategies. SUMMARY

[0004] The present application provides a single-domain antibody specifically binding to ganglioside GD2, and provides a single-domain antibody that can be used for targeting and / or treating several cancers related to GD2-overexpressing cells.

[0005] Technical solutions: To achieve the above-mentioned purposes, the technical solutions adopted by the present application are:

[0006] In a first aspect, the present application provides a single-domain antibody or a polypeptide comprising the single-domain antibody, the single-domain antibody having binding specificity to ganglioside GD2 and comprising complementarity determining regions: CDR1, CDR2 and CDR3, wherein,

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

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

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

[0010] Optionally, in some embodiments of the present application, 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 an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 15;

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

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

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

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

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

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

[0018] In a second aspect, the present application provides a recombinant nucleic acid molecule encoding a single-domain antibody or a polypeptide comprising the same according to any one of the above.

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

[0020] In a fourth aspect, the present application provides a host cell comprising the recombinant nucleic acid molecule according to the above or the vector according to claim 6.

[0021] In a fifth aspect, the present application provides a composition comprising the single-domain antibody or the polypeptide comprising the same according to any one of the above and a pharmaceutically acceptable carrier.

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

[0023] In a seventh aspect, the present application provides use of the single-domain antibody or the polypeptide comprising the same according to any one of the above in the manufacture of a medicament for the treatment and / or diagnosis of cancer.

[0024] Optionally, in some embodiments of the present invention, the cancer includes several cancers associated with GD2-overexpressing cells, such as neuroblastoma and glioma. Beneficial effects: The advantages of the present invention are that it provides a method for obtaining GD2-specific single-domain antibodies. A high-titer single-domain antibody phage display library is constructed using a KLH-GD2 vaccine, and GD2 single-domain antibody gene sequences are obtained through solid-phase panning using HSA-GD2. This method is simple to operate and can simultaneously obtain multiple GD2-specific single-domain antibody sequences. Another advantage of the present invention is that the GD2 single-domain antibodies provided by the present invention, which can be used to target and / or treat several cancers associated with GD2-overexpressing cells (such as neuroblastoma and glioma), can be stably, soluble, and expressed in large quantities in prokaryotic expression systems, facilitating the batch acquisition of high-purity antibodies. Furthermore, the GD2 single-domain antibodies obtained by the present invention have strong antigen specificity, high affinity, and high activity both in vivo and in vitro. Attached Figure Description

[0025] Figure 1 This is a 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 This is a schematic diagram of the phage library positivity rate detection results in Example 3, where 1-16 represent randomly selected monoclonal phages, and M represents the DL5000 marker.

[0028] Figure 4 This is a schematic diagram of the results of phage ELISA assay for GD2-positive monoclonal antibodies 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. In the diagram, 1 represents the supernatant of the single-domain antibody fermentation broth, 2-7 represent protein elution buffers with different concentrations of imidazole, and M represents the protein marker.

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

[0031] Figure 7 This is a flow cytometry diagram showing the recognition of GD2 single-domain antibody on HEK293 cells artificially coated with GD2 glycolipid in Example 8.

[0032] Figure 8 This demonstrates the therapeutic efficacy of the GD2 single-domain antibody in Example 9 against EL4 tumor-bearing mice. Detailed Implementation

[0033] definition

[0034] It should be noted that the terms "a" or "an" entity refer to one or more of that entity; for example, "an antibody" is understood to represent one or more antibodies. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0035] A polynucleotide or a region of a polynucleotide (or a polypeptide or a region of a polypeptide) 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 the percentage of bases (or amino acids) that are identical in the compared sequences is the same in the two sequences when aligned. Such alignment and percent 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 alignment is performed using default parameters. One alignment 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; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Biologically equivalent polynucleotides are those polynucleotides that have a specified percentage homology as described above and encode a polypeptide having the same or similar biological activity.

[0036] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a certain degree of homology or sequence identity to the nucleotide sequence of the nucleic acid or its complement. Homologs of a double-stranded nucleic acid are intended to include nucleic acids having a nucleotide sequence that has a certain degree of homology to it or its complement. In one aspect, a homolog of a nucleic acid is capable of hybridizing to the nucleic acid or its complement. Likewise, an "equivalent polypeptide" refers to a polypeptide having a certain degree of homology or sequence identity to 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, an equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof, compared to a reference polypeptide or polynucleotide. In some aspects, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0037] As used herein, an "antibody" or "antigen binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be an intact antibody and any antigen binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule that has the ability to bind an antigen. Examples of such include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0038] 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, and the like. Regardless of structure, an antibody fragment is capable of binding to an antigen that the intact antibody recognizes. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by forming a complex with a specific antigen.

[0039] A "single chain variable fragment" or "scFv" refers to a fusion protein of immunoglobulin heavy (VH) and light (VL) chain variable regions. In some aspects, these regions are connected with a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for increased flexibility, as well as serine or threonine for increased solubility, either N- or C-terminally to VH and C- or N-terminally to VL. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and introduction of the linker. ScFv molecules are known in the art, as described in U.S. Patent 5,892,019.

[0040] The term "antibody" includes various large classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that the heavy chains can be of the gamma, mu, alpha, delta, or epsilon types, with some subtypes (e.g., gamma l-gamma 4). The class of an antibody (IgG, IgM, IgA, IgG, or IgE) is determined by the type of heavy chain it possesses. Immunoglobulin subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2, are well characterized and known to have functional specificities. Modified versions of each of these classes and isotypes are readily discernible to those skilled in the art in light of the present disclosure and are therefore within the scope of the present disclosure. All immunoglobulin classes are expressly 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 polypeptides of approximately 23,000 daltons in molecular weight and two identical heavy polypeptides of 53,000-70,000 daltons in molecular weight. These four chains are typically linked together by disulfide bonds into a "Y" shape, with the light chains extending from the mouth of the "Y" shape, all the way to the variable region, encircling the heavy chains.

[0041] Antibodies, antigen-binding polypeptides, variants or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized 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 a VK or VH domain, expressed from a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to the LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0042] "Specifically binds" or "has specificity for" generally refers to the binding of an antibody by its antigen binding domain to an epitope, and such binding requires a certain degree of complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope with greater affinity than to a random, unrelated epitope by its antigen binding domain. The term "specificity" is used herein to qualify the relative affinity of a particular antibody to a particular epitope. For example, an antibody "A" can be said to have a higher specificity for a given epitope than antibody "B", or it can be said that antibody "A" binds to epitope "C" with a higher specificity than it binds to related epitope "D".

[0043] As used herein, the term "treatment" refers to therapeutic and prophylactic treatment or prophylactic measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or condition, e.g., the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those in which the condition or disorder is to be prevented.

[0044] A "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, in need of diagnosis, prognosis, or treatment. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.

[0045] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, e.g., mammalian subjects, who would benefit from administration of an antibody or composition used in the present disclosure, e.g., for detection, for a diagnostic procedure, and / or for treatment.

[0046] Embodiment Single-domain antibody specifically binding to the ganglioside-like glycan GD2

[0047] The present disclosure discloses a single-domain antibody specifically binding to the ganglioside-like glycan GD2, which can specifically bind to the GD2 glycan, wherein the single-domain antibody is a variable domain of a heavy chain antibody, which naturally lacks a light chain and a constant region 1.

[0048] Based on this, in particular, the present disclosure provides a single-domain antibody or a polypeptide comprising the same, which has binding specificity to the ganglioside-like glycan GD2 and comprises complementarity determining domains: CDR1, CDR2 and CDR3, wherein,

[0049] the CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 19, the CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 20, and the CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 21; or

[0050] the CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 22, the CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 23, and the CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 24; or

[0051] the CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 25, the CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 26, and the CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 27.

[0052] In some examples of the present application, a single-domain antibody specifically binding to ganglioside GD2 is disclosed, 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 a framework region FR1, FR2, FR3 and FR4, and has the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein,

[0053] the FR1 is selected from an amino acid sequence as set forth in SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 15;

[0054] the FR2 is selected from an amino acid sequence as set forth in SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 16;

[0055] the FR3 is selected from an amino acid sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17;

[0056] the FR4 is selected from an amino acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 18.

[0057] Preferably, in some examples of the present application, the single-domain antibody comprises the following regions in sequence:

[0058] a framework region 1 (FR1) selected from SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 15;

[0059] a complementarity determining region 1 (CDR1) selected from SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO: 25;

[0060] a framework region 2 (FR2) selected from SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 16;

[0061] a Complementarity Determining Region 2 (CDR2) selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26;

[0062] a Framework Region 3 (FR3) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 17;

[0063] a Complementarity Determining Region 3 (CDR3) selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27;

[0064] a Framework Region 4 (FR4) selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 18.

[0065] In some examples of the present application, there is a high sequence homology, for example, more than 80% sequence homology, between the optional examples of each of the Framework Region 1 (FR1), each of the Complementarity Determining Region 1 (CDR1), each of the Framework Region 2 (FR2), each of the Complementarity Determining Region 2 (CDR2), each of the Framework Region 3 (FR3), each of the Complementarity Determining Region 3 (CDR3), and each of the Framework Region 4 (FR4) in the present application.

[0066] More preferably, in some examples of the present application, the single-domain antibody comprises the following regions in sequence:

[0067] a Framework Region 1 (FR1) selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15;

[0068] a Complementarity Determining Region 1 (CDR1) as shown in SEQ ID NO: 19;

[0069] a Framework Region 2 (FR2) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16;

[0070] a Complementarity Determining Region 2 (CDR2) as shown in SEQ ID NO: 20;

[0071] a Framework Region 3 (FR3) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 17;

[0072] a Complementarity Determining Region 3 (CDR3) as shown in SEQ ID NO: 21;

[0073] a framework region 4 (FR4) selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 18.

[0074] More preferably, in some examples of the application, the single-domain antibody comprises, in order, the following regions:

[0075] a framework region 1 (FR1) selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15;

[0076] a complementarity determining domain 1 (CDR1) as set forth in SEQ ID NO: 22;

[0077] a framework region 2 (FR2) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16;

[0078] a complementarity determining domain 2 (CDR2) as set forth in SEQ ID NO: 23;

[0079] a framework region 3 (FR3) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 17;

[0080] a complementarity determining domain 3 (CDR3) as set forth in SEQ ID NO: 24;

[0081] a framework region 4 (FR4) selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 18.

[0082] More preferably, in some examples of the application, the single-domain antibody comprises, in order, the following regions:

[0083] a framework region 1 (FR1) selected from the group consisting of SEQ ID NO: 7, SEQ ID NO 11, and SEQ ID NO: 15;

[0084] a complementarity determining domain 1 (CDR1) as set forth in SEQ ID NO: 25;

[0085] a framework region 2 (FR2) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16;

[0086] a complementarity determining domain 2 (CDR2) as set forth in SEQ ID NO: 26;

[0087] a framework region 3 (FR3) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 17;

[0088] a Complementarity Determining Region 3 (CDR3) set forth in SEQ ID NO: 27;

[0089] a Framework Region 4 (FR4) selected from the group consisting of SEQ ID NO: 10, SEQ ID NO 14, and SEQ ID NO: 18.

[0090] More preferably, in some examples of the present application, the amino acid sequence of the single domain antibody is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.

[0091] More preferably, in some examples of the present application, the at least one single domain antibody has at least 80% sequence identity to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.

[0092] More preferably, in some examples of the present application, the at least one single domain antibody has at least 90% sequence identity to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.

[0093] In another aspect, the present application also provides a recombinant nucleic acid molecule encoding a single domain antibody or a polypeptide comprising the same as described in any of the above.

[0094] In another aspect, the present application also provides a vector comprising the recombinant nucleic acid molecule as described above.

[0095] In another aspect, the present application also provides a host cell comprising the recombinant nucleic acid molecule as described above or the vector as described above.

[0096] In another aspect, the present application also provides a composition comprising the single domain antibody or the polypeptide comprising the same as described in any of the above and a pharmaceutically acceptable carrier.

[0097] It will also be appreciated by one of ordinary skill in the art that the antibodies disclosed herein can be modified such that their amino acid sequences are not identical to the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein can be similar to the starting sequence, e.g., have a certain percentage of identity, e.g., can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence. In some embodiments, the modified antibody or fragment retains the specified CDR sequences.

[0098] In another aspect, the present application also provides a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of the single domain antibody or the polypeptide comprising the same as described in any of the above.

[0099] On the other hand, the present invention also provides the use of any of the single-domain antibodies described above or peptides containing said single-domain antibodies in the preparation of medicaments for cancer treatment and / or diagnosis.

[0100] In another aspect, the present invention provides a method for preparing the above-mentioned single-domain antibody that specifically binds to ganglioside GD2 or a polypeptide containing said single-domain antibody, comprising the following steps:

[0101] (1) Preparation of KLH-GD2 vaccine for immunization of alpacas;

[0102] (2) ELISA was used to detect serum titers of alpacas after immunization;

[0103] (3) Construct a phage display library;

[0104] (4) Solid-phase panning of GD2-specific phages;

[0105] (5) Positive monoclonal antibodies detected by indirect phage ELISA;

[0106] (6) Soluble expression of single-domain antibodies using the Escherichia coli expression system.

[0107] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the present invention as described in detail in the claims.

[0108] Example 1: Preparation of KLH-GD2 vaccine

[0109] This embodiment is intended to be as follows: Figure 1 As shown, KLH-GD2 was prepared using the synthetic route in the reference (Chinese Chemical Letters 32(2021)4041–4044).

[0110] Preparation of GD2 tumor glycovaccine: Take 300ug of KLH-GD2, dissolve it completely in 1mL of PBS, and mix it thoroughly with 1mL of complete Freund's adjuvant to prepare KLH-GD2 tumor glycovaccine.

[0111] Camel immunization: Before immunization, the blood of the camel was collected from the jugular vein with a vacuum blood collection tube, which was used as the control serum before immunization (D0); the camel was immunized by subcutaneous injection at multiple points on the neck, and the immunization dose was 0.3 mg of glycoprotein vaccine per time, which was strengthened once a week for 7 times; after multiple immunizations, the peripheral venous blood of the camel was collected, and the serum was separated for determination of the antibody titer. After the last immunization, 90 mL of peripheral venous blood of the camel was collected for isolation of peripheral blood lymphocytes.

[0112] Example 2: ELISA detection of serum titer of camel after immunization

[0113] This example is intended to detect the serum titer of the camel after immunization by ELISA

[0114] Take 10 mL of the camel peripheral blood obtained in Example 1 and place it at 4°C and 37°C for 30 min, then centrifuge at 5000 rpm for 10 min. The supernatant after centrifugation is the camel serum. Dissolve HSA-GD2 to 2 μg / mL with coating buffer, 100 μL per well, and coat the antigen at 4°C overnight, then transfer to a 37°C incubator for 1 h. After coating, wash the wells with PBST for 3 times. Add 200 μL of ELISA blocking buffer to each well and incubate at room temperature for 2 h. After blocking, wash the wells with PBST for 3 times. Add gradient-diluted immune serum or D0 serum to the corresponding wells, and incubate at 37°C for 2 h. After incubation, wash the wells with PBST for 3 times. Dilute the secondary antibody (here referring to MonoRab TM Rabbit Anti-Camelid VHH Cocktail[HRP]) according to the recommended dilution ratio (1:5000) in the instruction, and incubate at 37°C for 1 h. After incubation, wash the wells with PBST for 3 times. Detect the absorbance value of each well at 450 nm according to the instruction of the TMB kit, and the maximum dilution ratio corresponding to the wells with (Px / Nx)>2 is regarded as the titer of the camel serum. Wherein 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 ratio.

[0115] The results of ELISA detection of serum titer of the camel after immunization are shown in Figure 2 After 3 times of immunization, the serum titer of the GD2-specific heavy chain antibody of the immunized camel (35804) increased by 1.75 times of that of the non-immunized camel (20391), and after 7 times of immunization (53892), it increased by 2.64 times of that of the non-immunized camel.

[0116] Example 3: Construction of phage display library

[0117] This example is intended to construct a phage display library.

[0118] The llama peripheral blood lymphocyte cDNA obtained in Example 1 was used as a template, and the VHH gene sequence was obtained by using the primer CALL001 for the conserved region of the leader peptide and the primer CALL002 for the CH2 region to perform nested PCR. The VHH product amplified by PCR was ligated to a vector by enzyme digestion, and the TG1 competent cells were transformed. The bacteria liquid after resuscitation was mixed, and the initial library capacity of the bacteria was about 1.1 x 10 5 cfu was obtained by gradient dilution plate counting method. Twenty-four clones were randomly picked for colony PCR, and the positive rate was about 96%.

[0119] The titer of the amplified helper phage was about 1.23 x 10 10 cfu / mL, and the nanobody phage library with a titer of 6 x 10 14 cfu / mL was obtained after amplification by infecting the library bacteria at an infection multiplicity of 100:1. Sixteen clones were randomly picked for colony PCR, and the positive rate was 100% as shown in the following table. Figure 3

[0120] Example 4: Solid-phase panning of GD2-specific phage

[0121] This example is intended to solid-phase panning of GD2-specific phage.

[0122] HSA-GD2 (1 mg / mL) was diluted to 200 μg / mL with coating buffer, 100 μL per well was added to the plate, and 4°C overnight. PBS was washed three times, 3% BSA was added, 300 μL per well, 37°C blocking for 1 h. PBS was washed three times, the phage library was added, and 37°C incubation for 1 h. PBS was washed 5 times, 100 μL phage elution buffer was added, and 37°C incubation for 10 min. The eluate was taken into a 1.5 mL centrifuge tube, and 15 μL phage neutralization buffer was immediately added for neutralization. After mixing, 10 μL of the eluate was taken for elution.

[0123] After mixing the magnetic beads, 100 uL was taken into a 1 mL centrifuge tube, and the supernatant was removed by magnetic separation. The magnetic beads were washed twice with 200 uL MEST solution, and the supernatant was removed. Freshly prepared 100 uL EDC solution and 100 uL NHS solution were quickly added to the magnetic beads, and the magnetic beads were activated on a rotating mixer for 30 min. The activated magnetic beads were magnetically separated to remove the supernatant, 100 ug of sugar antigen (1 mg / mL) was added, and the magnetic beads were coupled at room temperature for 2 h, and the magnetic beads were kept suspended during coupling. The supernatant was removed by magnetic separation, the magnetic beads were resuspended with 200 uL PBST (1% BSA), and the magnetic beads were reacted at room temperature for 1 h to block the unreacted carboxyl groups on the surface of the magnetic beads. The supernatant was removed by magnetic separation, the magnetic beads were washed with 200 uL PBS for 3 times, and the magnetic beads were stored at 4°C.

[0124] ​The magnetic beads were allowed to stand at room temperature for 20 min, and the supernatant was removed by magnetic separation. The magnetic beads were washed with 200 μL of TBST three times, and the supernatant was removed by magnetic separation. 3% BSA (PBS) was added, and the magnetic beads were allowed to stand at 4°C for 1 h. The supernatant was removed by magnetic separation, and the magnetic beads were washed with 200 μL of TBST three times. The supernatant was removed by magnetic separation, and the phage library was added. The mixture was allowed to stand at room temperature for 1 h, and the magnetic beads were kept in suspension during the incubation. The unbound phage was removed by magnetic separation, and the magnetic beads were washed with 200 μL of TBST ten times. 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 blowing and sucking. The mixture was allowed to stand at room temperature for 5 min, and the elution solution was removed by magnetic separation. The magnetic beads were washed with PBS five times. The elution solution was transferred to a 1.5 mL centrifuge tube, and 100 μL of phage neutralization buffer was immediately added to neutralize the elution solution. The mixture was mixed gently, and 10 μL of the elution solution was taken for determination of the elution amount.

[0125] Example 5: Indirect phage ELISA detection of positive monoclonal antibodies

[0126] This example is intended to detect positive monoclonal antibodies by indirect phage ELISA.

[0127] Forty-eight single colonies from the plate for determination of the titer were inoculated into 1 mL of 2YT-A / Glu, and the mixture was allowed to stand at 37°C for 15 min and then incubated at 220 rpm for 2 h. The mixture was centrifuged at 3000 g for 10 min, and the supernatant was discarded. The precipitate was resuspended with an equal volume of 2YT-A-K / Glu, and the mixture was incubated at 30°C at 250 rpm overnight. The mixture was centrifuged at 8000 rpm at 4°C for 10 min, and the supernatant was taken for ELISA identification.

[0128] The glycoprotein was diluted with coating buffer to 8 μg / mL, and 100 μL of the solution was added to each well of a plate. The plate was allowed to stand at 4°C overnight. The plate was washed with PBST three times, and 300 μL of 4% BSA was added to each well. The plate was allowed to stand at 37°C for 1 h. The plate was washed with PBST three times, and 100 μL of the phage supernatant was added to each well. The plate was allowed to stand at 37°C for 2 h. The plate was washed with PBST three times, and 100 μL of HRP / anti-M13 secondary antibody diluted with blocking buffer (1:5000) was added to each well. The plate was allowed to stand at 37°C for 1 h. The plate was washed with PBST six times, and 100 μL of TMB color developing solution was added to each well. The plate was allowed to stand at 37°C for 15 min. The reaction was terminated by adding 50 μL of 2M H2SO4 to each well, and the OD450 value was determined.

[0129] The results of the indirect phage ELISA detection of positive monoclonal antibodies are shown in Table 1. Among the 48 randomly selected monoclonal antibodies, most of the phage displayed single-domain antibodies that weakly recognized the GD2 antigen (the OD450 value in the ELISA test was lower than 0.3). Therefore, the positive monoclonal antibodies were selected according to the criterion of OD450 = 0.3. Figure 4

[0130] ​Example 6: Soluble expression of single domain antibody by E. coli expression system

[0131] This example is intended to express the single domain antibody by E. coli expression system.

[0132] Take 100 μL of glycerol bacteria and transfer it to 10 mL of LB-A medium, 200 rpm, 37°C overnight culture to obtain seed liquid. The seed liquid is transferred to TB medium at a 2% inoculation ratio, 200 rpm, 37°C culture to OD 600 When the OD is between 0.6 and 0.8, add IPTG to a final concentration of 0.5 mM for induction, and at the same time, change the temperature to 16°C for induced expression of nanobody.

[0133] Collect the bacteria liquid after 48 hours of fermentation, centrifuge at 9000 rpm for 3 min, collect the bacterial precipitate, resuspend the bacteria with 50 mL of buffer A, and perform ice bath crushing of the bacteria. The ultrasonic crushing instrument is set to crushing for 3 s and stopping for 2 s, the maximum power is 30%, and the effective crushing time is 30 min. The suspension after crushing is centrifuged multiple times at 9000 rpm for 10 min each time, and all the broken wall supernatant is collected and filtered with a 0.22 μm filter for standby use. For protein purification, the nickel ion coupled agarose gravity column is washed with 10 column volumes of ultrapure water, 10 column volumes of buffer A for column equilibration, and the filtered broken wall supernatant is loaded for loading, and all the flow-through is collected. After detecting that no protein flows out with Coomassie Brilliant Blue G250, 5%, 10%, 20%, 50%, and 100% buffer B are used to wash the column for several column volumes, and all the eluate is collected for SDS-PAGE detection.

[0134] The results of soluble expression of single domain antibody by E. coli expression system are shown in Figure 5 The fermentation broth of single domain antibody is centrifuged, the bacterial precipitate is collected, and the supernatant after crushing is purified by nickel ion coupled agarose gravity column. Lane 1 is the un-purified broken supernatant, lanes 2-7 are protein samples eluted with different imidazole concentrations, SDS-PAGE analysis shows that the expression form of single domain antibody in E. coli system is intracellular soluble expression, and high-purity single domain antibody can be obtained by nickel column purification, with a molecular weight of 17-18 kDa, which is consistent with the size of single domain antibody.

[0135] By the above method, the present application obtains three GD2 single domain antibodies: 2D24; 2D26; 2D29, the amino acid sequences of each single variable domain of which are listed in Table 1 below, the nucleotide sequences of which are respectively shown as SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6. The CDR and FR sequences are summarized in Tables 1A-1F. Some CDR sequences include dipeptides, such as NG and DG, which can be susceptible to post-translational modifications. Certain "de-risked" versions of these sequences are also provided in the table, which are expected to retain antibody activity while reducing the risk of such post-translational modifications.

[0136] Table 1. Variable region sequences

[0137]

[0138] Table 1A. CDR sequences for 2D24

[0139] Name Amino acid sequence SEQ ID NO 2D24-CDR1 FTLGYYT 19 2D24-CDR2 SDGST 20 2D24-CDR3 ADLERIDADSCMRYAYGAL 21

[0140] Table 1B. FR sequences for 2D24

[0141]

[0142]

[0143] Table 1C. CDR sequences for 2D26

[0144] Name Amino acid sequence SEQ ID NO 2D26-CDR1 TGFSVYA 22 2D26-CDR2 RGGIT 23 2D26-CDR3 TDQFFTLTTISTDPD 24

[0145] Table 1D. FR sequences for 2D26

[0146]

[0147] Table 1E. CDR sequences for 2D29

[0148] Name Amino acid sequence SEQ ID NO 2D29-CDR1 FTFSSYA 25 2D29-CDR2 TGGDIT 26 2D29-CDR3 RWGTRGWVDGMD 27

[0149] Table 1F. FR sequences for 2D29

[0150]

[0151]

[0152] Example 7: SPR detection of specificity and affinity of GD2 single domain antibodies

[0153] This example aims to study the specificity and affinity of GD2 single domain antibodies detected by SPR, which can exhibit the binding mode and binding constant of the antibody and antigen; in addition, we have detected a total of four structurally similar sugar antigens, proving that the single domain antibody has specificity and does not have cross-binding activity.

[0154] The CM5 chip was activated with equal volumes of NHS and EDC for 500 s. Single-domain antibodies were diluted to a concentration of 30-50 μg / mL with pH 5.0 sodium acetate buffer and coupled at a flow rate of 10 μL / min. Background response values ​​were recorded in the flow channel until the response value no longer increased. Using PBS-P+ solution as the run buffer, glycoantigens (15.6-4000 nM) were flowed through the chip at a flow rate of 30 μL / min, with a contact time of 180 s and a dissociation time of 300 s. Experimental results were analyzed kineticly using a 1:1 model, and KD values ​​were calculated.

[0155] The results of SPR detection of the specificity and affinity of the GD2 single-domain antibody in this embodiment are as follows: Figure 6 As shown, the single-domain antibody 2D29 exhibits GD2 binding specificity with an affinity of 24.6 μM and a fast binding-slow dissociation mode, consistent with antigen-antibody binding patterns. However, 2D29 shows a weak affinity for the GD3 antigen (greater than 1 mM) and no binding ability for structurally similar antigens GM2 and GM3, demonstrating its specificity for the GD2 antigen. Furthermore, the affinity data for 2D24 and 2D26 are shown in the table below.

[0156] Table 2. Affinity data for GD2 single-domain antibodies

[0157] 2D24 2D26 2D29 GD2 311 μΜ 102 μΜ 24.6 μΜ GD3 197 μΜ No binding 1.5 mM GM2 No binding No binding No binding GM3 No binding No binding No binding

[0158] Example 8: Recognition of HEK293 cells artificially coated with GD2 glycolipids by GD2 single-domain antibody

[0159] This embodiment aims to investigate the recognition of HEK293 cells artificially coated with GD2 glycolipids by a GD2 single-domain antibody.

[0160] HEK293 cells were resuspended in PBS buffer to a concentration of 8 × 10⁸. 5 GD2 glycolipid (200 μM) was mixed with 100 μL of cell suspension and incubated at 37 °C for 20 min. Cells were washed with PBS buffer, and single-domain antibodies of different concentrations (2.44-150 μM) were added. Cells were incubated on ice for 30 min, washed twice with FACS buffer, and Myc antibody diluted 1:1000 was added. Cells were incubated on ice for 30 min, washed twice with FACS buffer, and Alexa Fluor 647-labeled goat anti-mouse IgG antibody diluted 1:2000 was added. Cells were incubated on ice for 30 min, washed twice with FACS buffer, and resuspended in 100 μL. The sample was analyzed by flow cytometry, and the results were analyzed using FlowJo software.

[0161] The recognition results of the GD2 single-domain antibody to HEK293 cells artificially coated with GD2 glycolipid are shown in Figure 7 GD2 has no recognition ability to the single HEK293 cells, but has significant recognition ability to the HEK293 cells artificially coated with GD2 glycolipid, and the recognition presents a concentration gradient dependence, and the affinity is 20.87 μM.

[0162] Example 9: Therapeutic ability of the GD2 single-domain antibody to EL4 tumor-bearing mice

[0163] This example is intended to study the therapeutic ability of the GD2 single-domain antibody to EL4 tumor-bearing mice.

[0164] The mouse lymphoma cells EL4 were resuspended to 5×10 7 / mL with PBS buffer, and 100 μL of the cell suspension was subcutaneously injected into the left flank of Balb / c mice, one week later, the mice with tumor volume between 100-300 mm 3 were randomly divided into 4 groups (PBS group, 2D29 group, 2D26 group and 2D24 group), 5 mice in each group. Intratumoral administration was performed three times a week for two weeks. The PBS group was intratumorally injected with 50 μL of PBS each time, and the other groups were intratumorally injected with 50 μL of single-domain antibody (100 μg) each time. After the treatment, the mice were dissected, and the tumor weight of the mice was detected.

[0165] The therapeutic ability of the GD2 single-domain antibody to EL4 tumor-bearing mice is shown in Figure 8 After two weeks of administration, the tumor weight of the mice in the single-domain antibody group was significantly lower than that of the PBS control group, which preliminarily proved the in vivo therapeutic potential of the GD2 single-domain antibody.

[0166] The above in vivo and in vitro experiments prove that the GD2 single-domain antibody prepared in the embodiments of the present application can be used for targeting and / or treating several cancers (such as neuroblastoma, glioma, etc.) related to GD2-overexpressing cells.

[0167] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A single domain antibody or a polypeptide comprising said single domain antibody, characterized in that, The single-domain antibody has binding specificity for ganglioside glycan GD2 and comprises complementarity determining domains: CDR1, CDR2 and CDR3, wherein, the amino acid sequences of the CDR1, CDR2, CDR3 are as shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, respectively; or the amino acid sequences of the CDR1, CDR2, CDR3 are as shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, respectively; or the amino acid sequences of the CDR1, CDR2, CDR3 are as shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, respectively.

2. The single-domain antibody or polypeptide comprising the same of claim 1, characterized in that, the amino acid sequences of the CDR1, CDR2, CDR3 are as shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, respectively.

3. The single-domain antibody or polypeptide comprising the same of claim 1, wherein, 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 polypeptide comprising the same of claim 3, wherein, The at least one single-domain antibody has at least 80% sequence identity to the sequence of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO:

5. The at least one single-domain antibody has at least 90% sequence identity to the sequence of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO:

5.

5. A recombinant nucleic acid molecule encoding the single-domain antibody or the polypeptide comprising the single-domain antibody of any one of claims 1-4.

6. A vector comprising the recombinant nucleic acid molecule of claim 5.

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

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

9. Use of the single-domain antibody or the polypeptide comprising the single-domain antibody of any one of claims 1-4 in the manufacture of a medicament for treating lymphoma.

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