CN114539412B

CN114539412B - Single domain antibody of anti-HLA-A 2/WT1 complex, preparation method and application thereof

Info

Publication number: CN114539412B
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: 2023-09-29
Anticipated expiration: 2042-02-24
Also published as: CN114539412A

Abstract

The application discloses a single domain antibody of an anti-HLA-A 2/WT1 complex, a preparation method and application thereof. The single domain antibody comprises an epitope complementary region and a framework region, the epitope complementary region comprising CDR1, CDR2 and CDR3 having at least 75% identity to the amino acid sequences shown in SEQ ID nos. 2, 4 and 6, respectively. The application also relates to derived antibodies derived from said single domain antibodies, biological materials related to said antibodies, methods of making said antibodies and the use of said antibodies in the preparation of products for binding T cells expressing HLA-A2/WT1 complexes. The antibody can be more efficiently expressed in escherichia coli, has higher binding rate with the complex, and is more suitable for preparing detection reagents, tumor inhibitors or tumor cell inhibitors for detecting the complex, and medicaments for inhibiting the activity of the HLA-A2/WT1 complex and promoting the proliferation of T cells.

Description

Single domain antibody of anti-HLA-A 2/WT1 complex, preparation method and application thereof

Technical Field

The application relates to the biomedical field, in particular to a single domain antibody of an anti-HLA-A 2/WT1 complex, a preparation method and application thereof.

Background

The wilms tumor protein (WT 1) antigen is an ideal target for immunotherapy targeting antigens within tumor cells. In recent years, many studies show that the WT1 antigen has high expression or abnormal expression (including over-expression, deletion, mutation and translocation) in cancer tissues such as leukemia, breast cancer, thyroid cancer and the like, 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. WT1 peptide and HLA-A2 molecule form a complex (designated HLA-A2/WT 1) that is often expressed in WT1 positive tumor cells. Successful screening of single chain antibodies against the HLA-A2/WT1 complex has been reported by researchers, and immunotherapy targeting the HLA-A2/WT1 complex has been developed using the single chain antibodies, and has produced good anti-tumor effects in preclinical studies. However, single chain antibodies are made by joining the antibody heavy chain variable region, hinge region and light chain variable region. The activity and utility of conventional single chain antibodies is limited by the ease of mismatch of the heavy and light chain variable regions and the complexity of the hinge region optimization process. Therefore, it is important to find new antibodies that overcome the shortcomings of conventional antibodies and to develop new antibody-based immunotherapies.

However, the current antibody drug application has a plurality of problems, such as long research and development period and high production cost; difficult to mass produce; the stability is poor, the degradation is easy, and the storage cost is high; the pollution is easy, and the maintenance cost is high; and has immunogenicity and the like, thereby limiting the application range in clinic.

Single domain antibody technology is the latest and smallest antibody molecules developed by biomedical scientists based on traditional antibodies, utilizing molecular biology techniques in combination with the concepts of nanoparticle science. In 1993, hamers et al reported that there was a heavy chain antibody naturally deleted in the light chain and heavy chain constant region 1 (CH 1) in the camelid body, and cloning the variable region thereof resulted in a single domain antibody consisting of only one heavy chain variable region, called VHH (variable domain of heavy chain of heavy-chain antibodies) or nanobody (Nb). The single domain antibody has a fully functional minimal antigen binding fragment with an oval crystal structure, 2.5nm in diameter and 4nm in length. The single domain antibody has a plurality of unique properties, is very suitable for genetic modification, and has wide application prospect in the aspects of accurate diagnosis, targeted treatment and the like of diseases. The single domain antibody is much simpler in chemical composition and shape than the antibody, has no chemical hydrophobicity, is stronger in heat resistance and acid and alkali resistance, is easier to combine with each other or other compounds, can be encoded by a single gene, and is easy to synthesize by microorganisms. The single domain antibody has good tolerance to environment, high conformational stability, smaller molecular mass and better clinical treatment effect, and simultaneously, the small protein molecules are easier to synthesize and lower in price. The unique property of the single-domain antibody enables the single-domain antibody to have wider application prospect in the aspects of accurate diagnosis, immune targeting treatment and the like of diseases.

At present, although there are reports of the preparation of WT1 Nb-CAR T cells targeting HLA-A2/WT1 complex and the in vitro and in vivo experimental verification of the antitumor effect thereof. However, there is no screening procedure for single domain antibodies targeting the HLA-A2/WT1 complex, binding assays of single domain antibodies to the HLA-A2/WT1 complex, and sequence reports of single domain antibodies.

Disclosure of Invention

The object of the present application is to prepare a single domain antibody capable of effectively treating tumor, which can overcome the above problems in the prior art, and to develop an antibody drug or therapy based on the single domain antibody.

In a first aspect, the application provides a single domain antibody comprising an epitope-complementary region and a framework region, wherein:

the epitope complementarity regions include CDR1, CDR2, and CDR3;

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

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

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

In some preferred embodiments, the framework regions can include four domains, FR1, FR2, FR3, and FR 4. Wherein the amino acid sequence of FR1 has at least 75% or more identity to the amino acid sequence shown in SEQ ID NO.1 and 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 more than 75% 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 more than 75% identity to 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 to 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 CDR1 comprises the amino acid sequence shown in SEQ ID No. 2; more preferably, the amino acid sequence of CDR1 consists of the amino acid sequence shown in SEQ ID NO. 2. The amino acid sequence of CDR2 comprises the amino acid sequence shown in SEQ ID NO. 4; more preferably, the amino acid sequence of CDR2 consists of the amino acid sequence shown in SEQ ID NO. 4. The amino acid sequence of CDR3 comprises the amino acid sequence shown in SEQ ID No.6, more preferably, the amino acid sequence of 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 shown 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 shown 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 shown 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 26-35 of SEQ ID No.9 of the sequence listing; the amino acid sequence of the CDR2 is the 51 st to 59 th amino acid of SEQ ID No.9 in a sequence table; the amino acid sequence of the CDR3 is 100 th-113 th amino acid of SEQ ID No.8 in a sequence table.

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

In some more preferred embodiments, the single domain antibody is encoded by the 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.

The present application provides in a second aspect a derivatized antibody derived from a single domain antibody of the first aspect of the application, and the derivatized antibody is a), b), c), d), or e) as follows:

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

e) An intact antibody comprising the single domain antibody of any one of claims 1 to 6.

The present application provides in a third aspect a biomaterial which is a biomaterial associated with a single domain antibody according to the first aspect of the application or with a derived antibody according to the second aspect of the application, and which 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 application or a derived antibody according to the second aspect of the application;

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 comprising 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 comprising the recombinant vector of B3);

b12 A transgenic animal cell line comprising the recombinant vector of B4);

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

In a fourth aspect, the present application provides a method of preparing a single domain antibody according to the first aspect of the application or a derived antibody according to the second aspect of the application, the method comprising the steps of:

(1) Introducing a nucleic acid molecule encoding a single domain antibody according to the first aspect of the application or a derived antibody according to the second aspect of the application into a recipient cell to obtain a transgenic cell expressing said single domain antibody or said derived antibody;

(2) Culturing the transgenic cells, and separating the single domain antibodies from the cultured transgenic cells;

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

more preferably, the recipient cell is a microbial cell.

The present application provides in a fifth aspect any one of the applications A1 to A4 as follows:

use of A1, a single domain antibody according to the first aspect of the application for the preparation of a product for binding to a T cell expressing an HLA-A2/WT1 complex;

a2, use of a derivative antibody according to the application in a second aspect for the preparation of a product for binding to T cells expressing the HLa-A2/WT1 complex;

use of A3, a biomaterial according to the third aspect of the application in the preparation of a product for binding to a T cell expressing an HLA-A2/WT1 complex;

use of A4, a method according to the fourth aspect of the application, for the preparation of a product for binding to T cells expressing HLA-A2/WT1 complex.

Compared with the prior art, the single-domain antibody or the derivative antibody can be expressed in escherichia coli more efficiently, has higher binding rate with the HLA-A2/WT1 complex, and is more suitable for the research and development of molecular detection reagents of the HLA-A2/WT1 complex, the preparation of tumor inhibitors or tumor cell inhibitors and the preparation of medicaments for inhibiting the activity of the HLA-A2/WT1 complex and promoting the proliferation of T cells.

Drawings

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

FIG. 2 is an electrophoretogram of SDS-PAGE of anti-HLA-A 2/WT1 complex single domain antibody WT1/VHH1-25 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 that were not loaded with peptide, respectively; FIG. 3B is the experimental results of the blank and single domain antibody WT1/VHH1-25 with T2 cells loaded with unrelated peptides; FIG. 3C is the experimental results of the blank and single domain antibodies WT1/VHH1-25 with WT1 peptide loaded T2 cells.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described more clearly and completely below in conjunction with the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

As described above, the present application provides in a first aspect a single domain antibody (sometimes referred to herein as WT1/VHH 1-25) comprising an epitope-complementary region CDR and a framework region FR; the epitope complementarity region CDRs of the single domain antibody consist of CDR1, CDR2 and CDR3. The amino acid sequence of the CDR1 has at least 75% of identity with the amino acid sequence shown in SEQ ID NO. 2; the amino acid sequence of CDR2 has at least 75% identity with the amino acid sequence shown in SEQ ID NO. 4; the amino acid sequence of CDR3 has at least 75% identity with the amino acid sequence shown in SEQ ID NO. 6.

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

In some preferred embodiments, the framework region FR of the single domain antibody consists of four domains, FR1, FR2, FR3 and FR 4. Wherein the amino acid sequence of FR1 has at least 75% 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 a meaning generally understood by those skilled in the art, e.g., for binding to the HLA-A2/WT1 complex, "biologically acceptable binding rate" means that the binding rate of the single domain antibody or derived 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 is of technical advancement or utility.

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

TABLE 1 sequence of tags

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 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) 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 comprising 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 comprising the recombinant vector of B3);

b12 A transgenic animal cell line comprising 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 biological material, the expression cassette of B2) containing the nucleic acid molecule encoding the single domain antibody or the derivative antibody, sometimes referred to as a 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 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.

Recombinant vectors containing expression cassettes for the single domain antibody genes or derived antibody genes can be constructed using existing expression vectors.

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

In the above biological material, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into pMECS. In one embodiment of the present application, B3) the recombinant vector is a recombinant vector pMECS-WT1/VHH1-25 obtained by introducing the coding gene (nucleotide sequence may be, for example, nucleotide 1-372 of SEQ ID No.8 in the sequence Listing) of the single domain antibody or the derived 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 biological material, the microorganism may be yeast, bacteria, algae or fungi.

In the above biological materials, 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 E.coli WK6.

The nucleotide sequence of the single domain antibody or derived antibody of B1) of the present application can be easily mutated by a person skilled in the art using known methods, such as directed evolution and point mutation. Those artificially modified, which have 75% or more identity to the nucleotide sequence of the single domain antibody or the derivative antibody of B1) of the present application, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application, insofar as they encode the single domain antibody or the derivative antibody and have the activity of the single domain antibody or the derivative antibody (e.g., have a biologically acceptable binding rate with the HLA-A2/WT1 complex), and are within the scope of the present application.

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 the 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) Hybridizing under stringent conditions to the nucleotide sequence defined in 1) and encoding the cDNA molecule or genomic DNA molecule of the single domain antibody WT1/VHH1-25.

(2) Culturing the transgenic cells, and isolating the single domain antibodies from the cultured transgenic cells.

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

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

In some preferred embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID No.8 of the sequence Listing or is a nucleic acid molecule encoding an amino acid sequence as shown in 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 WK6.

In some preferred embodiments, the coding gene of the single domain antibody or the derivative antibody may be obtained by deleting one or several amino acid residues from the DNA sequence of the single domain antibody or the derivative antibody (e.g., the sequence shown as SEQ ID NO.8 of the sequence Listing), and/or performing missense mutation of one or several base pairs, and/or ligating the coding sequence of the tag shown in Table 1 at its 5 '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 term "at least 75% identity or more" means an identity of not less than 75%, for example 75, 80, 85, 90, 95 or 99% or more. Identity can be assessed using 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 evaluate the identity between related sequences.

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

In some preferred embodiments, the product may be a drug, a tumor suppressor or a tumor cell suppressor or a product that inhibits HLA-A2/WT1 complex activity or promotes T cell proliferation.

In some more specific embodiments, the application is any one of applications A1-A8:

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

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

a3, application of the derivative antibody of the single domain antibody in preparing a tumor inhibitor or a tumor cell inhibitor;

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

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

a6, application of the biological material in preparing products for inhibiting activity of HLA-A2/WT1 complex or promoting T cell proliferation;

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

application of A8, the preparation method of the single domain antibody WT1/VHH1-25 in preparing products for inhibiting HLA-A2/WT1 complex activity or promoting T cell proliferation.

The above product can be a medicament.

Primer pairs for amplifying nucleic acid molecules encoding the amino acid sequence shown in SEQ ID No.9 of the sequence Listing or any fragment thereof are also within the scope of the present application, and those skilled in the art are fully able to design such primer pairs based on the sequences disclosed herein.

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

The application is described in further detail below in connection with specific examples which are given solely for the purpose of illustration and are not intended to limit the scope of the application.

The experimental methods in the following examples are conventional methods unless otherwise specified.

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

Coli WK6 used in the following examples was obtained from university of medical department of guangxi.

Example 1 preparation of Single-Domain antibodies

The amino acid sequence of the single domain antibody WT1/VHH1-25 prepared in the embodiment 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 2000bp molecular marker, the second channel is a PCR product, and the PCR product band is about 400bp.

Wherein the DNA fragment between PstI and NotI recognition sequences of the vector pMECS is replaced with the DNA molecule shown in SEQ ID No.8 with the nucleotide sequence encoding HHHHH tag (Poly-His) at the C terminal and the nucleotide sequence encoding YPYDVPDYA tag (HA) at the N terminal, and the other sequences are unchanged, thus obtaining the recombinant vector pMECS-WT1/VHH1-25, and the difference between pMECS-WT1/VHH1-25 and pMECS is only that the DNA fragment between PstI and NotI recognition sequences of pMECS is replaced with the DNA molecule shown in 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. The pMECS-WT1/VHH1-25 was introduced into E.coli WK6 to obtain recombinant strain WK6-pMECS-WT1/VHH1-25.

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

(1) WK6-pMECS-WT1/VHH1-25 was coated on LB plates containing ampicillin and glucose (in which concentrations of ampicillin and glucose were 100. Mu.g/mL and 20mg/mL, respectively), and incubated overnight at 25-37℃for a period of 10-14 hours (the incubation time was 12 hours in this example);

(2) The individual colonies were selected and inoculated into 5mL of an ampicillin-containing LB medium (in which ampicillin was present at a concentration of 100. Mu.g/mL) and subjected to shaking culture at a temperature controlled in the range of 25 to 37℃overnight (the culture time may be 10 to 14 hours, the time employed in this example was 10 hours, and the shaking culture speed was 200 rpm);

(3) Inoculating the culture solution cultured overnight in the step (2) into fresh TB culture solution according to (1:300) - (1:350), shaking the culture solution at a temperature controlled within a range of 25-37 ℃ until the OD value reaches 0.6-1.0 (the shaking speed is 200rpm in the embodiment, 0.8), adding IPTG to obtain WK6-pMECS-WT1/VHH1-25 culture solution, enabling the concentration of IPTG in the WK6-pMECS-WT1/VHH1-25 culture solution to be 1mM, and culturing the WK6-pMECS-WT1/VHH1-25 culture solution on a shaking table (the rotating speed of the shaking table is controlled within a range of 220-250 rpm) at 20-30 ℃ overnight (which can be 10-14 hours, 14 hours in the embodiment), thereby obtaining WK6-pMECS-WT 1/H1-25 induction solution;

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

(5) Obtaining an antibody crude extract by a conventional osmosis method;

(6) The single domain antibody WT1/VHH1-25 was prepared by nickel column ion affinity chromatography. The SDA-PAGE patterns of the single domain antibodies WT1/VHH1-25 are shown in FIG. 2, respectively, and the size of the single domain antibodies WT1/VHH1-25 is about 15kDa. 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 _126-134 Determination of Complex binding Rate

By expression of HLA-A2/WT1 _126-134 Loading of complexes WT1 _126-134 Peptide T2 cells (purchased from Shanghai Fuxiang Biotechnology Co., ltd.) detection of Single domain antibodies WT1/VHH1-25 and HLA-A2/WT1 _126-134 Binding rate of the complex. Specifically, the single domain antibody WT1/VHH1-25 (1. Mu.g) prepared in example 1 was added to 1-6X10 ⁶ After incubation at 4deg.C for 30 min (20-40 min) in the above T2 cells in the absence of light, PBS was used for 2 times, 5. Mu.l Alexa Fluor 647 anti-HA tag anti-body (purchased from Cell signaling) was added, incubation at 4deg.C for 30 min (20-40 min) was performed, PBS was used for 2 times, and the samples were examined by BACKMAN flow cytometer, as shown in FIG. 3B. Peptide-unloaded T2 cells are shown as a control in figure 3A. FIG. 3A is a blank pairPercent binding to T2 cells not loaded with peptide by the single domain antibody WT1/VHH1-25, respectively; FIG. 3B is the percent binding of the blank and single domain antibody WT1/VHH1-25 to T2 cells loaded with unrelated peptide that have no binding activity to WT 1; FIG. 3C is a blank and single domain antibody WT1/VHH1-25 and loading WT1 _126-134 Percentage of binding of T2 cells of the peptide; in FIG. 3, the horizontal axis shows fluorescence intensity (Alexa Fluor 647), the vertical axis shows the number percentage (% of Max), S2 represents the blank, and S1 represents the single domain antibody WT1/VHH1-25. As can be seen from the results of the drawing, the single domain antibody WT1/VHH1-25 prepared in example 1 can be well matched with the loaded WT1 _126-134 Peptide T2 cell binding.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and are not limiting; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application.

SEQUENCE LISTING

<110> university of medical department of Guangxi

<120> anti-HLA-A 2/WT1 complex single domain antibody, 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

<212> PRT

<213> artificial sequence

<400> 2

Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly

1 5 10

<210> 3

<211> 15

<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

<210> 4

<211> 9

<212> PRT

<213> artificial sequence

<400> 4

Ile Asp Ser Asp Gly Ser Thr Arg Tyr

1 5

<210> 5

<211> 40

<212> PRT

<213> artificial sequence

<400> 5

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

<210> 7

<211> 11

<212> PRT

<213> artificial sequence

<400> 7

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 8

<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 against the HLA-A2/WT1 complex comprising an epitope complementary region and a framework region, characterized in that:

the epitope complementarity region consists of CDR1, CDR2, and CDR3;

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 consists of the amino acid sequence shown in SEQ ID NO. 4;

the amino acid sequence of CDR3 consists of the amino acid sequence shown in SEQ ID NO. 6.

2. The single domain antibody of claim 1, wherein:

the framework regions consist of FR1, FR2, FR3 and FR 4;

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

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

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

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

3. The single domain antibody of claim 2, wherein:

the amino acid sequence of the FR1 comprises the amino acid sequence shown in SEQ ID NO. 1;

the amino acid sequence of the FR2 comprises 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/or

The amino acid sequence of FR4 comprises the amino acid sequence shown in SEQ ID NO. 7.

4. A single domain antibody according to claim 3, characterized in that:

the amino acid sequence of the FR1 consists of the amino acid sequence shown in SEQ ID NO. 1;

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

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

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, wherein:

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

6. The single domain antibody of any one of claims 2 to 4, wherein:

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

7. The single domain antibody of any one of claims 1 to 4, wherein:

the single domain antibody is encoded 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.

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

a) A single chain antibody comprising the single domain antibody of any one of claims 1 to 7;

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 7;

d) Fab comprising the single domain antibody of any one of claims 1 to 7;

e) An intact antibody comprising the single domain antibody of any one of claims 1 to 7.

9. A biomaterial, characterized in that it is a biomaterial related to the single domain antibody of any one of claims 1 to 7 or to the derived antibody of claim 8, and is 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 7 or encoding the derivatized antibody of claim 8;

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 comprising 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 comprising the recombinant vector of B3);

b12 A transgenic animal cell line comprising the recombinant vector of B4).

10. The biomaterial according to claim 9, characterized in that:

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

11. The biomaterial according to claim 9 or 10, characterized in that:

the nucleic acid molecule is a cDNA molecule or a DNA molecule.

12. The method of preparing a single domain antibody according to any one of claims 1 to 7, characterized in that the method comprises the steps of:

(1) Introducing a nucleic acid molecule encoding the single domain antibody of any one of claims 1 to 7 into a recipient cell to obtain a transgenic cell expressing the single domain antibody;

13. The method according to claim 12, wherein the nucleic acid molecule is a nucleic acid molecule encoding an amino acid sequence shown in SEQ ID NO.9 of the sequence Listing.

14. The method of claim 12, wherein the nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID No.8 of the sequence Listing.

15. The method of any one of claims 12 to 14, wherein the recipient cell is a microbial cell.

16. The method of producing a derivatized antibody of claim 8, comprising the steps of:

(1) Introducing a nucleic acid molecule encoding the derivatized antibody of claim 8 into a recipient cell to obtain a transgenic cell expressing the derivatized antibody;

(2) Culturing the transgenic cells, and isolating the derived antibodies from the cultured transgenic cells.

17. The method of claim 16, wherein the recipient cell is a microbial cell.

18. Any of the following applications A1-A4:

use of A1, a single domain antibody according to any one of claims 1 to 7 in the manufacture of a product for binding to a T cell expressing an HLA-A2/WT1 complex;

use of A2, a biological material according to any one of claims 9 to 11 for the preparation of a product for binding to T cells expressing HLa-A2/WT1 complex;

use of A3, a derivative antibody according to claim 8 in the preparation of a product for binding to a T cell expressing HLA-A2/WT1 complex;

use of A4, the method of any one of claims 12 to 17 for the preparation of a product for binding to a T cell expressing an HLA-A2/WT1 complex.