WO2016188449A1

WO2016188449A1 - Single-domain antibody targeting cd47

Info

Publication number: WO2016188449A1
Application number: PCT/CN2016/083469
Authority: WO
Inventors: 万亚坤; 孟红; 卞忠华
Original assignee: 江苏春申堂药业有限公司
Priority date: 2015-05-27
Filing date: 2016-05-26
Publication date: 2016-12-01
Also published as: CN104804093A

Abstract

Provided is a single-domain antibody targeting the extracellular region of CD47, the amino acid sequence of the single-domain antibody being as shown in SEQ ID NO: 9. Also provided are a nucleic acid coding the antibody, an expression vector containing the nucleic acid, and a host cell containing the expression vector.

Description

Single domain antibody against CD47

Technical field

The invention relates to a single domain antibody against CD47, and specifically designs a camel source single domain antibody against the extracellular segment of CD47 molecule, and belongs to the technical field of nanobodies.

Background technique

CD47 is a membrane glycoprotein widely expressed between multiple species and tissues, which interacts with the inhibitory receptor signal-modulating protein a as a receptor and ligand, and can form a CD47-SIRPa signal complex. The complex has the function of mediating two-way signal regulation and regulating a variety of immune response processes. On normal hematopoietic stem cells (HSCs), the significance of CD47 expression is to maintain its relative stability in the body. In the study of malignant diseases such as leukemia, non-Hodgkin's lymphoma, bladder cancer and breast cancer, it is found that tumor cells have elevated CD47, and high expression of CD47 indicates poor clinical prognosis; some types of cancer stem cells (cancer stem cells) Overexpression of CD47 is present in CDCs). Tumor cells evade tumor immunity by taking the "Don't Eat Me" signal. Blocking the interaction of CD47 and SIRPa by using an anti-CD47 antibody has the effect of targeted therapy with minimal effect on normal cells. In the treatment of leukemia, combined with CD47 antibody on the basis of the existing classical chemotherapy, targeted killing of leukemia tumor stem cells can increase the anti-tumor effect to the maximum extent, and provide a new exploration direction for the treatment of leukemia. The importance of individualization and further improvement of efficacy.

Nano-antibody technology is the latest and smallest antibody molecule developed by biomedical scientists based on traditional antibodies and using the molecular biology technology combined with the concept of nanoparticle science to develop the latest and smallest antibody molecules, originally by the Belgian scientist Hamers, R. It is isolated from a single heavy chain antibody in camel blood and is therefore also called a camel single-domain antibody. The single domain nature of this antibody gives it some unique properties compared to common antibodies. (1) The molecular weight is small, the structure is stable, the solubility is high, and it can be fixed in order. Nanobodies are the least known antibodies of the molecular weight and are one tenth (15 KD) of common antibodies. Since the hydrophobic residue in the FR2 (frame-work 2) of the Nanobody is substituted with a hydrophilic residue, the water solubility of the Nanobody is increased, and the polymerizability is reduced. The study found that the Nanobody still retains 80% of the binding activity after 1 week of incubation at 37 ° C, indicating that it is easier to use and long-term storage at room temperature. Antigen binding ability can be regained after prolonged placement in an environment above 90 °C. At the same time, Nanobodies also exhibit high stability in strong denaturing agents and extreme pH environments, while ordinary antibodies undergo irreversible polymerization. Because nano-antibodies have a small volume and a regular structure, they are easier to fix and can be ordered and fixed. (2) High affinity and strong specificity. It has a longer CDR3 (complementarity-determining region 3) relative to the concave or planar antigen-binding surface of a normal antibody, and forms a binding surface of a stable, exposed convex structure. This convex structure can penetrate deep into the antigen and bind more effectively to the concave topology formed by the epitope. Only the antigen specificity and affinity of the Nanobody are increased, while many antigens that are not recognized by common antibodies are recognized. Even when the protein tightly packed hides the common antibody recognition site, the Nanobody can also perform epitope recognition. (3) weak immunogenicity and strong penetrability. Nanobodies are weakly immunogenic to humans and have good biocompatibility with humans. The immunogenicity is related to the size and chemical structure of the molecule, and the smaller the relative molecular weight, the smaller the immunogenicity. Studies have shown that nanobodies in animal experiments do not cause any humoral and cellular immune responses. At the same time, nano-antibody has strong tissue penetrating ability and can enter dense tissues. Excess unbound Nano-antibodies can be quickly cleared, which is conducive to the diagnosis and treatment of diseases. (4) Low cost and large-scale production. Nano-antibodies are small in mass and simple in structure, can be encoded by a single gene, and can be expressed in large amounts in microorganisms such as yeast and E. coli by genetic engineering.

Due to its special structural properties, Nanobodies combine the advantages of traditional antibodies and small molecule drugs, and almost completely overcome the shortcomings of traditional antibody development cycle, low stability and harsh storage conditions. This single-domain antibody with a molecular weight of only 1/10 of the conventional antibody has gradually become an emerging force in the diagnosis and treatment of a new generation of antibodies. Therefore, the application of nano-antibody technology to develop CD47 therapeutic antibody drugs has broad prospects.

Summary of the invention

In view of the above prior art, the present invention provides a single domain antibody directed against the extracellular domain of CD47, and provides a coding sequence of the single domain antibody, a vector containing the coding sequence, and a host cell.

The invention is achieved by the following technical solutions:

A single domain antibody directed against CD47, which is a single domain antibody directed against the extracellular domain of CD47, the amino acid sequence of which is represented by SEQ ID NO: 9, consisting of four framework regions and three complementarity determining regions, wherein four frameworks The amino acid sequences of the regions are as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, and the amino acid sequences of the three complementarity determining regions are respectively SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. The complementarity determining region is mainly responsible for the recognition of antigens, and the structure of the framework region is relatively stable, mainly supporting the maintenance of protein structure.

A nucleic acid encoding the above single domain antibody, the nucleotide sequence of which is one of the following sequences:

(1) a nucleotide sequence as shown in SEQ ID NO. 8;

(2) a sequence in which a nucleotide sequence shown by SEQ ID NO. 8 is added, substituted, deleted or inserted into one or several nucleotides;

(3) a nucleotide sequence that hybridizes to the nucleotide sequence of (1) or (2) under stringent conditions;

(4) The nucleotide sequence of the nucleotide sequence of (1), (2), and (3) is distinguished by the degeneracy of the genetic code.

The nucleotide sequence or at least a portion of the sequences of the invention can be expressed by a suitable expression system to yield the corresponding protein or polypeptide. These expression systems include bacteria, yeasts, filamentous fungi, animal cells, insect cells, plant cells, or cell-free expression systems.

An expression vector comprising the above nucleic acid encoding a single domain antibody against CD47.

A host cell comprising the above expression vector.

The recipient bacteria of the host cell are preferably Escherichia coli.

The use of the single domain antibody against CD47 for detecting CD47, in specific application, using the ability of the single domain antibody against CD47 of the present invention to specifically bind to CD47, using an enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay) ELISA), fluorescence immunoassay (Fluoroimmunoassay, FIA), immunochip method and affinity chromatography were performed.

The use of the single domain antibody against CD47 for the preparation of a reagent or kit for detecting CD47; the reagent or kit for detecting CD47 contains a single domain antibody against CD47.

The use of the single domain antibody against CD47 for the preparation of a medicament for the treatment of CD47.

The present invention first synthesizes a CD47 polypeptide (extracellular segment) and makes it immunogenic, and then couples the CD47 polypeptide molecule to the ELISA plate to display the correct spatial structure of the protein, and the antigen-utilizing phage display technology in this form The immune nanobody gene library (the camelid heavy chain antibody phage display gene library) was screened to obtain the CD47-specific Nanobody gene, which was transferred to E. coli to establish a Nanobody capable of high expression in E. coli. Strain. Needle of the present invention The single domain antibody to CD47 has broad application prospects in the development of CD47 therapeutic antibody drugs.

DRAWINGS

Figure 1: The insertion rate of the CD47 camel single-domain antibody library. The bands from left to right are: the first is the DNA molecular marker, and the other is the PCR product of the VHH insert (single domain antibody gene fragment). It is about 500 bp in size.

Figure 2: Purification map of CD47 single domain antibody, electrophoresis pattern of SDS-PAGE after purification by nickel column resin gel affinity chromatography, the results show that CD47 single domain antibody can achieve purity of more than 90% after the purification process.

detailed description

The present invention will be further described below in conjunction with the embodiments.

The instruments, reagents, materials and the like involved in the following examples are conventional instruments, reagents, materials and the like which are available in the prior art unless otherwise specified, and can be obtained by a formal commercial route. The experimental methods, detection methods, and the like involved in the following examples are conventional experimental methods, detection methods, and the like which have been known in the prior art unless otherwise specified.

Example 1 Construction of a Nanobody Library Against CD47

(1) Firstly synthesize CD47 polypeptide, mix 1mg CD47 with Freund's adjuvant in equal volume, immunize a Xinjiang dromedary, once a week, a total of 7 times, stimulate B cells to express antigen-specific Nanobodies;

(2) After 7 immunizations, 100 mL of camel peripheral blood was taken, lymphocytes were isolated and total RNA was extracted;

(3) synthesizing cDNA and amplifying VHH by nested PCR;

(4) using restriction endonucleases PstI and NotI to digest 16 μg of pCMECS3 phage display vector and 8 μg of VHH and ligating the two fragments;

(5) The ligation product was transformed into electroporation competent cell TG1, and the CD47 Nanobody library was constructed and the storage capacity was determined. The storage capacity was 5.85×10 ⁸ CFU. In addition, the insertion rate of the constructed library was detected, and 24 monoclonals were randomly selected. PCR analysis of the insert was performed to determine the insertion rate of the VHH fragment, and the result showed that the insertion rate of the constructed library reached 95.8% (Fig. 1).

Example 2 Nanobody Screening Process for CD47

(1) 20 μg of CD47 dissolved in 100 mM NaHCO ₃ , pH 8.2 was coupled to a NUNC plate, and left at 4 ° C overnight;

(2) Add 100 μL of 0.1% BSA the next day, and block at room temperature for 2 h;

(3) After 2 h, 100 μL of phage (2 × 10 ¹¹ immune camel single-domain antibody phage) was added, and allowed to stand at room temperature for 1 h;

(4) Washing 5 times with PBS + 0.05% Tween-20 to wash away unbound phage;

(5) The phage specifically binding to CD47 was eluted with 100 mM TEA (triethylamine), and the E. coli TG1 cells growing in log phase were infected and cultured at 37 ° C for 1 h to produce and purify the phage for the next round of screening. , The same screening process was repeated for 3 to 4 rounds, and the enrichment was gradually obtained.

Example 3 Screening for specific single positive clones by ELISA using phage enzyme-linked immunosorbent assay (ELISA)

(1) From the cell culture dishes containing phage after the above 3 to 4 rounds of screening, 96 single colonies were selected and inoculated into 1 mL of TB medium containing 100 ug/mL of ampicillin (2.3 L of KH _{2 in} 1 L of TB medium). PO ₄ , 12.52 g K ₂ HPO ₄ , 12 g peptone, 24 g yeast extract, 4 mL glycerol), after growth to log phase, IPTG was added to a final concentration of 1 mM, and cultured overnight at 28 °C.

(2) A crude antibody was obtained by an infiltration method, and the antibody was transferred to an antigen-coated ELISA plate and allowed to stand at room temperature for 1 hour.

(3) Unbound antibody was washed away with PBST, and mouse anti-HA tag antibody (anti-mouse anti-HA antibody, purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) was added and allowed to stand at room temperature for 1 h.

(4) Unbound antibody was washed away with PBST, and anti-mouse alkaline phosphatase conjugate (goat anti-mouse alkaline phosphatase-labeled antibody, purchased from Amytech Co., Ltd.) was added and allowed to stand at room temperature for 1 hour.

(5) The unbound antibody was washed away with PBST, an alkaline phosphatase coloring solution was added, and placed on a microplate reader, and the absorption value was read at a wavelength of 405 nm.

(6) When the OD value of the sample well is more than 3 times the OD value of the control well, it is judged as a positive clone.

(7) The positive cloned bacteria were shaken in a LA liquid containing 100 ug/mL to extract a plasmid and perform sequencing.

The gene sequence of each clone was analyzed according to the sequence alignment software Vector NTI, and the strains with the same CDR3 sequence were regarded as the same clone, and the strains with different sequences were regarded as different clones, and the amino acid sequence of the VHH chain of the antibody was SEQ ID. NO: 9 is composed of 4 framework regions and 3 complementarity determining regions, wherein the amino acid sequences of the four framework regions are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID, respectively. As shown by NO: 4, the amino acid sequences of the three complementarity determining regions are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

Example 4 Nanobody expression and purification in host strain Escherichia coli

(1) The plasmids of the different clones obtained by the above sequencing analysis were electrotransformed into Escherichia coli WK6, and coated on LA+2% Glμcose (ie containing ampicillin and glucose) culture plates, and cultured at 37 ° C overnight;

(2) selecting a single colony inoculated in 5 mL of LB medium containing ampicillin and incubating at 37 ° C overnight;

(3) Inoculate 1 mL of the overnight strain into 330 mL TB culture medium, incubate at 37 ° C, culture until the OD value reaches 0.6-0.9, add IPTG, and shake overnight at 28 ° C (about 12-16 h);

(4) centrifugation, collecting bacteria;

(5) Obtaining the crude crude extract by using the infiltration method: treating the cells with 4 mL of high sugar solution TES for 2 h, then 8 mL of 1/4 TES Treated for 2 h; after centrifugation, the supernatant was taken for subsequent purification;

(6) Purification of single domain antibody by nickel column ion affinity chromatography: protein solution was mixed with nickel column for 1 hour; washed 10 times with 10×PBS, washed twice with PBS containing 20 mM imidazole, and once with 50 mM PBS. 1 mL of PBS containing 100 mM imidazole was washed 3 times with PBS containing 500 mM imidazole; the eluted protein solution was collected, the purity was observed by gel (Figure 2), and it was ultrafiltered into 1×PBS. Store at -80 °C.

Claims

A single domain antibody against CD47, characterized in that the amino acid sequence thereof is represented by SEQ ID NO: 9, consisting of four framework regions and three complementarity determining regions, wherein the amino acid sequences of the four framework regions are respectively SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, the amino acid sequences of the three complementarity determining regions are SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO, respectively. :7 is shown.
Use of the single domain antibody against CD47 of claim 1 for detecting CD47.
Use of a single domain antibody against CD47 according to claim 1 for the preparation of a reagent or kit for detecting CD47.
Use of a single domain antibody against CD47 according to claim 1 for the preparation of a medicament for the treatment of a CD47 therapeutic antibody.
A nucleic acid encoding the single domain antibody of claim 1, wherein the nucleotide sequence is one of the following sequences:

(1) a nucleotide sequence as shown in SEQ ID NO. 8;

(2) a sequence in which a nucleotide sequence shown by SEQ ID NO. 8 is added, substituted, deleted or inserted into one or several nucleotides;

(3) a nucleotide sequence that hybridizes to the nucleotide sequence of (1) or (2) under stringent conditions;

(4) The nucleotide sequence of the nucleotide sequence of (1), (2), and (3) is distinguished by the degeneracy of the genetic code.
An expression vector comprising the nucleic acid of claim 4.
A host cell comprising the expression vector of claim 6.
The host cell according to claim 7, wherein the recipient cell of the host cell is Escherichia coli.