CN114539412A

CN114539412A - Single-domain antibody of anti-HLA-A2/WT 1 complex and preparation method and application thereof

Info

Publication number: CN114539412A
Application number: CN202210174458.3A
Authority: CN
Inventors: 卢小玲; 杨晓梅; 李海霞; 尹时华; 姜晓兵
Original assignee: Guangxi Medical University
Current assignee: Guangxi Medical University
Priority date: 2022-02-24
Filing date: 2022-02-24
Publication date: 2022-05-27
Anticipated expiration: 2042-02-24
Also published as: CN114539412B

Abstract

The invention discloses a single domain antibody of an anti-HLA-A2/WT 1 complex, a preparation method and application thereof. The single domain antibody comprises an epitope-complementary region comprising CDR1, CDR2, and CDR3 having at least 75% or greater identity to the amino acid sequences set forth in SEQ ID Nos. 2, 4, and 6, respectively, and a framework region. The invention also relates to a derivative antibody derived from the single domain antibody, biological materials related to the antibody, a method for preparing the antibody and application of the antibody in preparing products for binding T cells expressing HLA-A2/WT1 complex. The antibody can be more efficiently expressed in escherichia coli, has higher binding rate with the compound, and is more suitable for preparing a detection reagent for detecting the compound, a tumor inhibitor or a tumor cell inhibitor, and a medicament for inhibiting the activity of an HLA-A2/WT1 compound and promoting the proliferation of T cells.

Description

Single-domain antibody of anti-HLA-A2/WT 1 complex and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a single domain antibody of an anti-HLA-A2/WT 1 compound, and a preparation method and application thereof.

Background

The wilms tumor protein (WT1) antigen is an ideal target for targeting tumor intracellular antigens for immunotherapy. In recent years, many studies show that the WT1 antigen is highly expressed or abnormally expressed (including over-expression, deletion, mutation and translocation) in cancer tissues such as leukemia, breast cancer and thyroid cancer, and plays an important role in the occurrence and development of different tumors. The WT1 antigen polypeptide can induce specific Cytotoxic T Lymphocyte (CTL) reaction, and has good anti-tumor effect in vivo and in vitro experiments. The complex formed by the WT1 peptide and HLA-A2 molecule (named as HLA-A2/WT1) is often expressed in WT1 positive tumor cells. At present, researchers report that a single-chain antibody is successfully screened against an HLA-A2/WT1 complex, an immunotherapy targeting the HLA-A2/WT1 complex is developed by adopting the single-chain antibody, and a good anti-tumor effect is generated in preclinical research. However, single chain antibodies are formed by linking the variable regions of the antibody heavy chain, the hinge region and the variable region of the light chain. The activity and application of conventional single chain antibodies are limited due to the susceptibility of heavy and light chain variable regions to mismatch and the complexity of hinge region optimization processes. Therefore, it is of great interest to explore novel antibodies that overcome the disadvantages of conventional antibodies and to develop immunotherapies based on the novel antibodies.

However, the application of the existing antibody drugs has a plurality of problems, such as long development period and overhigh production cost; difficult to mass produce; poor stability, easy degradation and high storage cost; is easy to be polluted, and the maintenance cost is high; and has immunogenicity and the like, thereby limiting the application range of the medicine in clinic.

Single domain antibody technology is a revolution of antibody engineering by biomedical scientists based on traditional antibodies, using molecular biology technology combined with the concept of nanoparticle science, to develop the latest and smallest antibody molecules. In 1993, ham et al reported that a camelid has a heavy chain antibody with a naturally deleted light chain and heavy chain constant region 1(CH1) in it, and the variable region was cloned to obtain a single domain antibody consisting of only one heavy chain variable region, called VHH (variable domain of heavy chain of heavy-chain antibody) or Nanobody (Nb). Single domain antibodies have the smallest antigen-binding fragment with full function, with an elliptical crystal structure, a diameter of 2.5nm and a length of 4 nm. The single domain antibody has a plurality of unique properties, is very suitable for genetic modification, and shows wide application prospects in the aspects of accurate diagnosis, targeted therapy and the like of diseases. Single domain antibodies are much simpler in chemical composition and shape than antibodies, are not chemically hydrophobic, are more heat and acid and base resistant, bind more readily to each other or to other compounds, can be encoded by a single gene, and are readily synthesized by microorganisms. The single domain antibody has good tolerance to the environment, high conformational stability, smaller molecular weight and better clinical treatment effect, and meanwhile, the small protein molecules are easier to synthesize and have lower price. Due to the unique properties of the single-domain antibody, the single-domain antibody has wider application prospects in the aspects of accurate diagnosis of diseases, immune targeted therapy and the like.

At present, the preparation of WT1 Nb-CAR T cells targeting HLA-A2/WT1 complex and the in vitro and in vivo experimental verification of the anti-tumor effect are reported. However, no screening process of the single-domain antibody targeting the HLA-A2/WT1 complex exists, and the binding determination of the single-domain antibody and the HLA-A2/WT1 complex and the sequence report of the single-domain antibody exist.

Disclosure of Invention

The object of the present invention is to prepare a single domain antibody capable of effectively treating tumors, which overcomes the above-mentioned problems of the prior art, and to develop an antibody drug or therapy based on the single domain antibody.

The present invention provides in a first aspect a single domain antibody comprising an epitope-complementing region and a framework region, wherein:

the epitope-complementary regions include CDR1, CDR2, and CDR 3;

the amino acid sequence of the CDR1 has at least 75 percent of identity with the amino acid sequence shown in SEQ ID NO. 2;

the amino acid sequence of the CDR2 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 4;

the amino acid sequence of the CDR3 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 6.

In some preferred embodiments, the framework region may comprise four domains FR1, FR2, FR3 and FR 4. Wherein the amino acid sequence of FR1 has at least 75% or more identity with the amino acid sequence shown in SEQ ID NO.1, and enables the single-domain antibody to have a biologically acceptable binding rate with HLA-A2/WT1 complex.

It is further preferred or additionally preferred that the amino acid sequence of FR2 has at least 75% or more identity with the amino acid sequence shown in SEQ ID No.3 and that the single domain antibody has a biologically acceptable binding rate to the HLA-a2/WT1 complex.

It is further preferred or additionally preferred that the amino acid sequence of FR3 has at least 75% or more identity with the amino acid sequence shown in SEQ ID No.5 and that the single domain antibody has a biologically acceptable binding rate to the HLA-a2/WT1 complex.

It is further preferred or additionally preferred that the amino acid sequence of FR4 has at least 75% or more identity with the amino acid sequence shown in SEQ ID No.7 and that the single domain antibody has a biologically acceptable binding rate to the HLA-a2/WT1 complex.

In some preferred embodiments, the amino acid sequence of the CDR1 comprises the amino acid sequence set forth in SEQ ID No. 2; more preferably, the amino acid sequence of the CDR1 consists of the amino acid sequence shown in SEQ ID NO. 2. The amino acid sequence of the CDR2 comprises an amino acid sequence shown in SEQ ID NO. 4; more preferably, the amino acid sequence of the CDR2 consists of the amino acid sequence shown in SEQ ID No. 4. The amino acid sequence of the CDR3 comprises the amino acid sequence shown in SEQ ID NO.6, and more preferably, the amino acid sequence of the CDR3 consists of the amino acid sequence shown in SEQ ID NO. 6.

In some preferred embodiments, the amino acid sequence of FR1 comprises the amino acid sequence shown in SEQ ID No.1, more preferably the amino acid sequence of FR1 consists of the amino acid sequence shown in SEQ ID No. 1.

In some preferred embodiments, the amino acid sequence of FR2 comprises the amino acid sequence set forth in SEQ ID No. 3; more preferably, the amino acid sequence of FR2 consists of the amino acid sequence shown in SEQ ID NO. 3.

In some preferred embodiments, the amino acid sequence of FR3 comprises the amino acid sequence set forth in SEQ ID No. 5; more preferably, the amino acid sequence of FR3 consists of the amino acid sequence shown in SEQ ID NO. 5.

In some preferred embodiments, the amino acid sequence of FR4 comprises the amino acid sequence set forth in SEQ ID No. 7; more preferably, the amino acid sequence of FR4 consists of the amino acid sequence shown in SEQ ID NO. 7.

In some preferred embodiments, the amino acid sequence of CDR1 is amino acids 26 to 35 of SEQ ID No.9 of the sequence Listing; the amino acid sequence of the CDR2 is the 51 st to 59 th amino acids of SEQ ID No.9 in the sequence table; the amino acid sequence of the CDR3 is the amino acid at position 100-113 of SEQ ID No.8 in the sequence table.

Further preferably or additionally preferably, the amino acid sequence of FR1 is amino acids 1-25 of SEQ ID No.9 of the sequence Listing; the amino acid sequence of FR2 is the 36 th-50 th amino acid of SEQ ID No.9 in the sequence table; the amino acid sequence of FR3 is the 60 th-99 th amino acid of SEQ ID No.9 in the sequence table; the amino acid sequence of the FR4 is the amino acid at the 114 th and 124 th positions of SEQ ID No.9 in the sequence table.

In some more preferred embodiments, the single domain antibody is encoded by a nucleic acid sequence set forth in SEQ ID No.8 of the sequence Listing; and/or the amino acid sequence of the single domain antibody is shown as SEQ ID NO.9 in the sequence table.

In a second aspect, the invention provides a derivative antibody derived from a single domain antibody according to the first aspect of the invention, and the derivative antibody is a), b), c), d), or e):

a) a single chain antibody comprising the single domain antibody of any one of claims 1 to 6;

b) a fusion antibody comprising a) said single chain antibody;

c) a fusion antibody comprising the single domain antibody of any one of claims 1 to 6;

d) a Fab comprising the single domain antibody of any one of claims 1 to 6;

e) an intact antibody comprising a single domain antibody according to any one of claims 1 to 6.

The present invention provides in a third aspect a biomaterial that is associated with a single domain antibody according to the first aspect of the invention or with a derivatized antibody according to the second aspect of the invention, and is any one of B1) to B12) as follows:

B1) a nucleic acid molecule encoding a single domain antibody according to the first aspect of the invention or a derivative antibody according to the second aspect of the invention;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line containing the recombinant vector of B4);

preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.8 in the sequence table; more preferably, the nucleic acid molecule is a cDNA molecule or a DNA molecule.

In a fourth aspect, the invention provides a method of producing a single domain antibody according to the first aspect of the invention or a derivative antibody according to the second aspect of the invention, said method comprising the steps of:

(1) introducing a nucleic acid molecule encoding a single domain antibody according to the first aspect of the invention or a derivative antibody according to the second aspect of the invention into a recipient cell, resulting in a transgenic cell expressing said single domain antibody or said derivative antibody;

(2) culturing the transgenic cell, and separating the single-domain antibody from the cultured transgenic cell;

preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.8 in the sequence table, or the nucleic acid molecule codes the amino acid sequence shown as SEQ ID No.9 in the sequence table;

more preferably, the recipient cell is a microbial cell.

In a fifth aspect, the invention provides the use of any one of the following A1-A4:

use of a1, a single domain antibody according to the first aspect of the invention, in the manufacture of a product for binding to a T cell expressing HLA-a2/WT1 complex;

use of a2, a derivative antibody according to the invention in the second aspect, in the manufacture of a product for binding to a T cell expressing HLA-a2/WT1 complex;

use of A3, a biomaterial according to the third aspect of the invention in the manufacture of a product for binding to T cells expressing HLA-a2/WT1 complex;

a4 use of a method according to the fourth aspect of the invention in the manufacture of a product for binding to T cells expressing the HLA-A2/WT1 complex.

Compared with the prior art, the single domain antibody or derivative antibody can be more efficiently expressed in escherichia coli, has higher binding rate with an HLA-A2/WT1 compound, is more suitable for being applied to the development of molecular detection reagents of the HLA-A2/WT1 compound, and is used for preparing tumor inhibitors or tumor cell inhibitors and preparing medicines for inhibiting the activity of the HLA-A2/WT1 compound and promoting the proliferation of T cells.

Drawings

FIG. 1 is a DNA electrophoresis of a single domain antibody, with DNA bands from left to right gel wells: the first path is a molecular marker of 2000bp, the second path is a PCR product, and the PCR product band is about 400 bp;

FIG. 2 is an electrophoretogram of SDS-PAGE of a single domain antibody WT1/VHH1-25 of the anti-HLA-A2/WT 1 complex purified by nickel column resin gel affinity chromatography; lane Marker indicates protein molecular weight in KDa;

FIG. 3A is the experimental results of the single domain antibody WT1/VHH1-25 with T2 cells, respectively, which were not loaded with peptide; FIG. 3B is the experimental results of the blank control and single domain antibody WT1/VHH1-25 with T2 cells loaded with irrelevant peptide; FIG. 3C is the experimental results of the blank control and single domain antibody WT1/VHH1-25 with T2 cells loaded with WT1 peptide.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described more clearly and completely in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

As described above, the present invention provides, in a first aspect, a single domain antibody (sometimes referred to herein as WT1/VHH1-25) comprising epitope-complementary regions CDRs and framework region FRs; the epitope-complementary region CDRs of the single domain antibody consist of CDR1, CDR2, and CDR 3. The amino acid sequence of the CDR1 has at least 75 percent of identity with the amino acid sequence shown in SEQ ID NO. 2; the amino acid sequence of the CDR2 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 4; the amino acid sequence of the CDR3 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 6.

Preferably, the amino acid sequence of the CDR1 has at least 75% or more identity to the amino acid sequence shown in SEQ ID No.2 and such that the single domain antibody has a biologically acceptable binding rate to the HLA-a2/WT1 complex; the amino acid sequence of the CDR2 has at least 75% or more identity with the amino acid sequence shown in SEQ ID No.4, and enables the single domain antibody to have a biologically acceptable binding rate with the HLA-A2/WT1 complex; the amino acid sequence of the CDR3 has at least 75% identity with the amino acid sequence shown in SEQ ID NO.6, and the single domain antibody has a biologically acceptable binding rate with the HLA-A2/WT1 complex.

In some preferred embodiments, the framework region FR of the single domain antibody is comprised of four domains FR1, FR2, FR3 and FR 4. Wherein, the amino acid sequence of FR1 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 1. Preferably, the amino acid sequence of FR1 is such that the single domain antibody has a biologically acceptable binding rate to the HLA-A2/WT1 complex.

It is further preferred or additionally preferred that the amino acid sequence of FR2 has at least 75% or more identity with the amino acid sequence shown in SEQ ID No. 3. Preferably, the amino acid sequence of FR2 is such that the single domain antibody has a biologically acceptable binding rate to the HLA-A2/WT1 complex.

It is further preferred or additionally preferred that the amino acid sequence of FR3 has at least 75% or more identity with the amino acid sequence shown in SEQ ID No. 5. Preferably, the amino acid sequence of FR3 is such that the single domain antibody has a biologically acceptable binding rate to the HLA-A2/WT1 complex.

It is further preferred or additionally preferred that the amino acid sequence of FR4 has at least 75% or more identity with the amino acid sequence shown in SEQ ID No. 7. Preferably, the amino acid sequence of FR4 is such that the single domain antibody has a biologically acceptable binding rate to the HLA-A2/WT1 complex.

Herein, the term "biologically acceptable binding rate" has the meaning generally understood by a person skilled in the art, e.g. in respect of binding rate to the HLA-a2/WT1 complex, "biologically acceptable binding rate" means that the binding rate of the single domain antibody or derivative antibody to the HLA-a2/WT1 complex has a biologically acceptable binding rate (e.g. a detectable binding rate) such that the single domain antibody has technical progress or utility.

In some preferred embodiments, in order to facilitate purification of the single domain antibody WT1/VHH1-25, a tag as depicted in Table 1 may be attached to the amino-terminal or carboxy-terminal end of the protein as depicted in amino acids 1-124 of SEQ ID No.9 of the sequence Listing (i.e., the single domain antibody).

TABLE 1 sequences of tags

Label (R)	Residue(s) of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL
HA	9	YPYDVPDYA

b) a fusion antibody comprising a) said single chain antibody;

d) a Fab comprising the single domain antibody of any one of claims 1 to 6;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line containing the recombinant vector of B4);

preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.8 in the sequence table.

In the above biological material, the nucleic acid molecule may be DNA (e.g., cDNA), genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above-mentioned biological materials, the expression cassette containing a nucleic acid molecule encoding the single domain antibody or the derivative antibody described in B2), which is sometimes referred to as WT1/VHH1-25 gene expression cassette, refers to DNA capable of expressing the single domain antibody or the derivative antibody in a host cell, and the DNA may include not only a promoter for initiating transcription of the single domain antibody gene or the derivative antibody gene, but also a terminator for terminating transcription of the single domain antibody gene or the derivative antibody gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the expression cassette of the single domain antibody gene or the derived antibody gene can be constructed using an existing expression vector.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above-mentioned biomaterial, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into pMECS. In one embodiment of the invention, B3) the recombinant vector is a recombinant vector pMECS-WT1/VHH1-25 obtained by introducing the gene (nucleotide sequence can be, for example, nucleotides 1-372 of SEQ ID No.8 in a sequence table) encoding the single domain antibody or the derivative antibody into pMECS, and the recombinant vector pMECS-WT1/VHH1-25 expresses the single domain antibody WT1/VHH1-25 shown in SEQ ID No. 9.

In the above biomaterial, the microorganism may be yeast, bacteria, algae, or fungi.

In the above biological material, the transgenic animal cell line does not include propagation material; the recombinant microorganism may be a recombinant microorganism obtained by introducing the nucleic acid molecule of B1) into Escherichia coli WK 6.

The nucleotide sequence of the single domain antibody or derivative antibody of B1) of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides that are artificially modified to have 75% or more than 75% identity to the nucleotide sequence of the single domain antibody or the derivative antibody of B1) of the present invention are within the scope of the present invention as long as they encode the single domain antibody or the derivative antibody and have single domain antibody or derivative antibody activity (e.g., biologically acceptable binding to the HLA-a2/WT1 complex), are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

In some embodiments, the biological material B1) the nucleic acid molecule is 1) or 2) or 3) below:

1) the nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No.8 in a sequence table;

2) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding said single domain antibody WT1/VHH 1-25;

3) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in 1) and codes for the single-domain antibody WT1/VHH 1-25.

In a fourth aspect, the present invention provides a method of producing a single domain antibody according to the first aspect of the invention or a derivative antibody according to the second aspect of the invention, said method comprising the steps of:

(2) culturing the transgenic cells, and separating the single-domain antibody from the cultured transgenic cells.

In some preferred embodiments, the method may further comprise the steps of: a step of synthesizing a gene encoding the single domain antibody or derivative antibody.

In some preferred embodiments, the method for preparing a single domain antibody according to the first aspect of the present invention or a derivative antibody according to the second aspect of the present invention may comprise: (1) synthesizing a gene encoding the single domain antibody or derivative antibody; (2) expressing the encoding gene by a biological method, thereby obtaining the single domain antibody or derivative antibody.

In some preferred embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.8 of the sequence Listing, or is a nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.9 of the sequence Listing.

In some more preferred embodiments, the recipient cell is a microbial cell, such as e.coli, and in particular e.coli WK 6.

In some preferred embodiments, the gene encoding the single domain antibody or the derivative antibody may be obtained by deleting one or more amino acid residues of a codon in the DNA sequence (e.g., the sequence shown in SEQ ID No.8 of the sequence listing) of the single domain antibody or the derivative antibody, and/or by performing a missense mutation of one or more base pairs, and/or by linking the coding sequence of the tag shown in table 1 above at its 5 'end and/or 3' end.

As used herein, the term "identity" characterizes sequence similarity between sequences (e.g., nucleic acid sequences or amino acid sequences) for comparison. The "at least 75% identity" means no less than 75% identity, for example, 75, 80, 85, 90, 95, or 99% identity or more. Identity can be assessed by visual statistics or computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above biological material, the nucleic acid molecule of B1) is 1) or 2) or 3) below:

3) a cDNA molecule or a genomic DNA molecule which hybridizes with the nucleotide sequence defined in 1) under stringent conditions and codes for the single domain antibody WT1/VHH 1-25.

In some preferred embodiments, the product may be a medicament, a tumor suppressor or a tumor cell suppressor or a product that inhibits the activity of the HLA-a2/WT1 complex or promotes T cell proliferation.

In some more specific embodiments, the use is any one of a 1-A8:

a1, the application of the single domain antibody WT1/VHH1-25 in preparing tumor inhibitors or tumor cell inhibitors;

a2, the application of the biological material in preparing tumor inhibitors or tumor cell inhibitors;

a3, and the application of the derivative antibody of the single domain antibody in preparing tumor inhibitors or tumor cell inhibitors;

a4, and the application of the preparation method of the single domain antibody WT1/VHH1-25 in the preparation of tumor inhibitors or tumor cell inhibitors;

a5, the application of the single domain antibody WT1/VHH1-25 in preparing products for inhibiting the activity of HLA-A2/WT1 complex or promoting the proliferation of T cells;

a6, and application of the biomaterial in preparing products for inhibiting activity of HLA-A2/WT1 complex or promoting proliferation of T cells;

a7, and the application of the derivative antibody in preparing products for inhibiting the activity of HLA-A2/WT1 complex or promoting T cell proliferation;

a8, and application of the preparation method of the single domain antibody WT1/VHH1-25 in preparation of products for inhibiting activity of HLA-A2/WT1 complex or promoting proliferation of T cells.

The product may be a medicament.

The primer pair for amplifying the nucleic acid molecule of the amino acid sequence shown in SEQ ID No.9 of the sequence list or any fragment of the amino acid sequence also belongs to the protection scope of the invention, and the primer pair can be designed by the technicians in the field according to the sequence disclosed in the application.

In summary, the present invention provides a single domain antibody of anti-HLA-a 2/WT1 complex, a derivative antibody thereof, or a nucleic acid molecule encoding the antibody, a biological material (e.g., a host cell comprising the nucleic acid molecule) related to the antibody, and methods for preparing and using the single domain antibody, the derivative antibody, and the biological material. The single domain antibody or derivative antibody of the invention can be efficiently expressed in escherichia coli, and can be applied to the development of HLA-A2/WT1 compound molecule detection reagents, the preparation of tumor inhibitors or tumor cell inhibitors and the preparation of drugs for inhibiting the activity of HLA-A2/WT1 compounds and promoting the proliferation of T cells.

The present invention is further described in detail below with reference to specific examples, which are given only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

The experimental procedures in the following examples are all conventional ones unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Coli WK6 used in the examples described below was obtained from the university of cantonese medical science.

Example 1 preparation of Single Domain antibodies

The amino acid sequence of the single domain antibody WT1/VHH1-25 prepared in this example is shown as SEQ ID No.9 in the sequence table, and is encoded by the nucleotide sequence of SEQ ID No. 8.

The nucleotide electrophoresis diagram of the single domain antibody WT1/VHH1-25 is shown in FIG. 1, wherein the first channel is a molecular marker of 2000bp, the second channel is a PCR product, and the PCR product band is about 400 bp.

Wherein the DNA fragment between PstI and NotI recognition sequences of the vector pMECS is replaced with the DNA molecule represented by SEQ ID No.8 having a nucleotide sequence encoding a HHHHHHHHHH tag (Poly-His) at the C-terminus and a nucleotide sequence encoding a YPYDVPDYA tag (HA) at the N-terminus, and none of the other sequences is changed, resulting in the recombinant vector pMECS-WT1/VHH1-25, pMECS-WT1/VHH1-25 differing from pMECS only in that the DNA fragment between PstI and NotI recognition sequences of pMECS is replaced with the DNA molecule represented by SEQ ID No. 8. The recombinant vector pMECS-WT1/VHH1-25 expresses the single domain antibody WT1/VHH1-25 shown in SEQ ID No. 9. pMECS-WT1/VHH1-25 is introduced into escherichia coli WK6 to obtain recombinant bacteria WK6-pMECS-WT1/VHH 1-25.

The specific preparation steps of the single domain antibody are as follows:

(1) WK6-pMECS-WT1/VHH1-25 was spread on LB plates containing ampicillin and glucose (in LB plates, concentrations of ampicillin and glucose were 100. mu.g/mL and 20mg/mL, respectively), and cultured overnight (culture time may be 10-14 hours, and time used in this example is 12 hours) while controlling the temperature in the range of 25-37 ℃;

(2) selecting single colony, inoculating in 5mL LB culture solution containing ampicillin (in LB culture solution, concentration of ampicillin is 100 μ g/mL), and shake culturing overnight (culture time can be 10-14 hr, 10 hr in this example, and shaking table speed is 200rpm) at 25-37 deg.C;

(3) inoculating the overnight cultured culture solution obtained in the step (2) into a fresh TB culture solution according to the ratio of (1: 300) - (1: 350), performing shake culture at 25-37 ℃ until OD reaches 0.6-1.0 (0.8 in the example and the shaking table speed is 200rpm), adding IPTG (isopropyl-beta-D-1-25), culturing the culture solution at 20-30 ℃ until the concentration of IPTG in the culture solution WK6-pMECS-WT1/VHH1-25 is 1mM, and culturing the culture solution at 20-30 ℃ on a shaking table (the shaking table rotation speed is controlled to 220 and 250rpm) overnight (10-14 hours in the example and 14 hours in the example) to obtain an induced solution WK6-pMECS-WT1/VHH 4-25;

(4) centrifuging the induction liquid WK6-pMECS-WT1/VHH1-25 obtained in the step (3) at 4 ℃, and collecting thalli;

(5) obtaining antibody crude extract by using a conventional osmosis method;

(6) single domain antibody WT1/VHH1-25 was prepared using nickel column ion affinity chromatography. SDA-PAGE electrophoretograms of single domain antibody WT1/VHH1-25 are shown in FIG. 2, respectively, and the size of single domain antibody WT1/VHH1-25 is about 15 kDa. The purity of the single domain antibody WT1/VHH1-25 obtained by the method can reach more than 90 percent through SDA-PAGE electrophoresis detection.

Example 2 Single domain antibodies WT1/VHH1-25 and HLA-A2/WT1_126-134Determination of Complex binding Rate

By expressing HLA-A2/WT1_126-134Loading of the Complex WT1_126-134Peptide T2 cells (purchased from Shanghai Reinforcement Biotech Co., Ltd.) detection of Single domain antibodies WT1/VHH1-25 and HLA-A2/WT1_126-134The binding rate of the complex. Specifically, the single domain antibody WT1/VHH1-25 (1. mu.g) prepared in example 1 was added to 1-6X 10⁶The above T2 cells were incubated at 4 ℃ for 30 minutes in the absence of light (20-40 min is acceptable), washed with PBS 2 times, then 5. mu.l of Alexa Fluor 647 anti-HA tag antibody (from Cell signaling) was added, incubated at 4 ℃ for 30 minutes (20-40 min is acceptable), washed with PBS 2 times, and then the samples were subjected to BACKMAN flow cytometer for detection, the results of which are shown in FIG. 3B. T2 cells not loaded with peptide were used as a control as shown in fig. 3A. FIG. 3A is the percentage of binding of the blank control and single domain antibody WT1/VHH1-25, respectively, to T2 cells that were not loaded with peptide; FIG. 3B is the percent binding of the blank control and single domain antibody WT1/VHH1-25 to T2 cells loaded with an unrelated peptide that has no binding activity to WT 1; FIG. 3C is a blank control and single domain antibody WT1/VHH1-25 with loading WT1_126-134Percent binding of peptide T2 cells; in FIG. 3, the horizontal axis represents fluorescence intensity (Alexa Fluor 647), the vertical axis represents percentage by number (% of Max), S2 represents a blank control, and S1 represents the single-domain antibody WT1/VHH 1-25. As can be seen from the results shown in the figure, the single domain antibody WT1/VHH1-25 prepared in example 1 was well compatible with the loading of WT1_126-134T2 cell binding of the peptide.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Guangxi university of medical science

<120> single domain antibody of anti-HLA-A2/WT 1 complex, preparation method and application thereof

<130> GY22100096

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> PRT

<213> Artificial sequence

<400> 1

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 2

<211> 10

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<213> Artificial sequence

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Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly

1 5 10

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<212> PRT

<213> Artificial sequence

<400> 3

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala

1 5 10 15

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<211> 9

<212> PRT

<213> Artificial sequence

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Ile Asp Ser Asp Gly Ser Thr Arg Tyr

1 5

<210> 5

<211> 40

<212> PRT

<213> Artificial sequence

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Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys

1 5 10 15

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

20 25 30

Met Tyr Tyr Cys Ala Ala Asp Leu

35 40

<210> 6

<211> 14

<212> PRT

<213> Artificial sequence

<400> 6

Ile Ala Thr Met Cys Arg Gly Leu Ser Ile Gly Ala Gly Tyr

1 5 10

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<213> Artificial sequence

<400> 7

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

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<211> 372

<212> DNA

<213> Artificial sequence

<400> 8

caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60

tcctgtgcag cctctggata cacctacagt agctattgca tgggctggtt ccgccaggct 120

ccagggaagg agcgcgaggg ggtcgcagct attgatagtg atggtagcac aaggtacgca 180

gactccgtga agggccgatt caccatctcc aaagacaacg ccaagaacac tctgtatctg 240

caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcggc agaccttata 300

gcgactatgt gtcggggact atcgatcggg gcgggttact ggggccaggg gacccaggtc 360

accgtctcct ca 372

<210> 9

<211> 124

<212> PRT

<213> Artificial sequence

<400> 9

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Asp Ser Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Leu Ile Ala Thr Met Cys Arg Gly Leu Ser Ile Gly Ala Gly

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

Claims

1. A single domain antibody comprising an epitope-complementing region and a framework region, wherein:

the epitope-complementary regions include CDR1, CDR2, and CDR 3;

2. The single domain antibody of claim 1, characterized in that:

the framework regions comprise FR1, FR2, FR3 and FR 4;

the amino acid sequence of FR1 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 1;

the amino acid sequence of FR2 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 3;

the amino acid sequence of FR3 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 5; and/or

The amino acid sequence of FR4 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 7.

3. The single domain antibody of claim 1 or 2, characterized in that:

the amino acid sequence of the CDR1 comprises the amino acid sequence shown in SEQ ID NO.2, and preferably, the amino acid sequence of the CDR1 consists of the amino acid sequence shown in SEQ ID NO. 2;

the amino acid sequence of the CDR2 comprises the amino acid sequence shown in SEQ ID NO.4, and preferably, the amino acid sequence of the CDR2 consists of the amino acid sequence shown in SEQ ID NO. 4;

the amino acid sequence of the CDR3 comprises the amino acid sequence shown in SEQ ID NO.6, and preferably, the amino acid sequence of the CDR3 consists of the amino acid sequence shown in SEQ ID NO. 6.

4. The single domain antibody of any one of claims 1 to 3, characterized in that:

the amino acid sequence of FR1 comprises the amino acid sequence shown in SEQ ID NO.1, and preferably, the amino acid sequence of FR1 consists of the amino acid sequence shown in SEQ ID NO. 1;

the amino acid sequence of FR2 comprises the amino acid sequence shown in SEQ ID NO.3, preferably, the amino acid sequence of FR2 consists of the amino acid sequence shown in SEQ ID NO. 3;

the amino acid sequence of FR3 comprises the amino acid sequence shown in SEQ ID NO.5, and preferably, the amino acid sequence of FR3 consists of the amino acid sequence shown in SEQ ID NO. 5; and/or

The amino acid sequence of FR4 comprises the amino acid sequence shown in SEQ ID NO.7, and preferably, the amino acid sequence of FR4 consists of the amino acid sequence shown in SEQ ID NO. 7.

5. The single domain antibody of any one of claims 1 to 4, characterized in that:

the amino acid sequence of the CDR1 is the 26 th to 35 th amino acids of SEQ ID No.9 in the sequence table; the amino acid sequence of the CDR2 is the 51 st to 59 th amino acids of SEQ ID No.9 in the sequence table; the amino acid sequence of the CDR3 is the amino acid at position 100-113 of SEQ ID No.8 in the sequence table; and/or

The amino acid sequence of FR1 is the 1 st-25 th amino acid of SEQ ID No.9 in the sequence table; the amino acid sequence of FR2 is the 36 th-50 th amino acid of SEQ ID No.9 in the sequence table; the amino acid sequence of FR3 is the 60 th-99 th amino acid of SEQ ID No.9 in the sequence table; the amino acid sequence of the FR4 is the amino acid at the 114 th and 124 th positions of SEQ ID No.9 in the sequence table.

6. The single domain antibody of any one of claims 1 to 5, characterized in that:

the single domain antibody is coded by a nucleic acid sequence shown as SEQ ID NO.8 in a sequence table; and/or

The amino acid sequence of the single domain antibody is shown as SEQ ID NO.9 in the sequence table.

7. A derivative antibody derived from the single domain antibody of any one of claims 1 to 6, and which is a), b), c), d), or e) as follows:

b) a fusion antibody comprising a) said single chain antibody;

d) a Fab comprising the single domain antibody of any one of claims 1 to 6;

8. A biomaterial associated with the single domain antibody of any one of claims 1 to 6 or with the derivatized antibody of claim 7, and being any one of the following B1) to B12):

B1) a nucleic acid molecule encoding the single domain antibody of any one of claims 1 to 6 or encoding the derivatized antibody of claim 7;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line containing the recombinant vector of B4);

9. A method for producing a single domain antibody according to claim 1, comprising the steps of:

(1) introducing a nucleic acid molecule encoding a single domain antibody according to any one of claims 1 to 6 or encoding a derivative antibody according to claim 7 into a recipient cell, resulting in a transgenic cell expressing said single domain antibody or said derivative antibody;

more preferably, the recipient cell is a microbial cell.

10. Any one of the following A1-A4:

use of a1, a single domain antibody of any one of claims 1 to 6, in the manufacture of a product for binding to a T cell expressing HLA-a2/WT1 complex;

use of a2, the biomaterial of claim 2, in the manufacture of a product for binding to T cells expressing the HLA-a2/WT1 complex;

use of A3 or a derivatized antibody of claim 3 in the preparation of a product for binding to a T cell expressing HLA-a2/WT1 complex;

use of A4 or the method of claim 4 for the preparation of a product for binding to T cells expressing the HLA-A2/WT1 complex.