CN109021104B - Antibody for resisting human platelet-derived growth factor beta receptor and application thereof - Google Patents

Abstract

The invention discloses an antibody for resisting a platelet-derived growth factor beta receptor and application thereof. Comprising a heavy chain variable region comprising CDR1, CDR2 and/or CDR 3; the amino acid sequence of CDR1 is shown in SEQ ID NO.2, the amino acid sequence of CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 4. The antibody can be combined with an extracellular region of human PDGFR beta, and can effectively inhibit or block the combination of PDGFb and PDGFR beta, so as to down-regulate or cut off a corresponding signal path; the application of the PDGFR beta-related protein inhibitor comprises, but not limited to, inhibiting PDGFR beta-mediated signal pathways and preparing medicines for diseases related to the PDGFR beta-mediated signal pathways, and the like.

Description

Antibody for resisting human platelet-derived growth factor beta receptor and application thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to an antibody for resisting a platelet-derived growth factor beta receptor and application thereof.

Background

Platelet-derived growth factor (PDGF), a ubiquitous growth factor encoded by the sis proto-oncogene, was initially isolated from Platelet α, synthesized primarily by megakaryocytes, and plays a very important role in tissue damage repair, angiogenesis and cell differentiation. PDGF molecules are glycoprotein dimers and can be classified into-AA, -BB and-AB. PDGF and its receptor (PDGFR) are highly expressed in many human tumors, such as pancreatic cancer, small cell lung cancer, gastric cancer and breast cancer. PDGFR is a cell surface located Receptor Tyrosine Kinase (RTK) that can be divided into two subtypes: PDGFR α and PDGFR β (Matsui et al 1989). PDGFR β can be activated by specific binding to PDGF-BB or-AB (Herdanan et al 1991). After being activated, two PDGFRs can form a dimer and activate a downstream signal to transfer to a pathway through autophosphorylation so as to realize the function of regulating the differentiation and growth of cells. PDGF signaling has been implicated in a variety of human diseases, including diseases associated with pathological neovascularization, vascular and fibrotic diseases, tumor growth and ocular diseases. Therefore, inhibitors of PDGF signaling have been proposed for use in a variety of therapeutic settings. For example, inhibitors of PDGFR β have been proposed for the treatment of various diseases and disorders. (Andrae et al (2008) Genes Dev 22(10): 1276-. PDGFR β inhibitors include non-specific small molecule tyrosine kinase inhibitors such as imatinib mesylate, sunitinib malate, and CP-673451, as well as anti-PDGFR β antibodies (see, e.g., U.S. patent nos. 7,060,271; 5,882,644; 7,740,850; and U.S. patent application publication No. 2011/0177074). Anti-ligand aptamers (e.g., anti-PDGF-B) have also been proposed for therapeutic applications. However, there is a need in the art for new, highly specific and potent PDGF signaling inhibitors.

The present monoclonal antibody is mostly derived from mice, so that the antibody has antigenicity in human body as a heterologous protein, which greatly limits its use in clinical treatment. To overcome this drawback, many solutions have been proposed, including human/human or human/mouse hybridoma technology, chimeric antibody technology, phage display technology. The human/human or human/mouse hybridoma technology has exited the market due to the technical difficulty, and the humanized antibody is mainly prepared by an in vitro antibody engineering method at present, but the antibody obtained by the method is often poor in affinity and easy to aggregate, and the humanized or fully human antibody prepared from the in vivo has the advantages of high affinity, good water solubility and difficulty in aggregation, so that the preparation of the human fully human antibody from the fully human antibody transgenic mouse has the most research and development advantages and is the mainstream of antibody development in the future. The heavy chain antibody is an antibody without two light chains, and compared with the traditional antibody, the heavy chain antibody is easier to penetrate through a blood vessel wall and enter the interior of a solid tumor, has more advantages in the aspect of developing anti-tumor monoclonal antibody medicines, and in addition, the single domain antibody derived from the heavy chain antibody is the minimum antibody molecule discovered so far, and can be widely applied in the aspects of tumor imaging, treatment and diagnosis. Based on the modular nature of the heavy chain antibody, a further application is the production of bispecific antibodies directed against two different target antigens simultaneously.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of low specificity and low affinity of the existing anti-human platelet derived growth factor beta receptor (PDGFR beta) antibody in the prior art and to provide an anti-PDGFR beta antibody, in particular a monoclonal heavy chain antibody and application thereof by utilizing the characteristics of high cost of preparing an antibody, in particular a heavy chain antibody, by phage display screening. The antibody can be combined with an extracellular region of human PDGFR beta, and can efficiently inhibit or block the combination of PDGFb and PDGFR beta, thereby down-regulating or cutting off a corresponding signal path; the application of the PDGFR beta-related protein inhibitor comprises, but not limited to, inhibiting PDGFR beta-mediated signal pathways and preparing medicines for diseases related to the PDGFR beta-mediated signal pathways, and the like.

One of the technical solutions for solving the above technical problems of the present invention is: an antibody against human platelet-derived growth factor beta receptor comprising a heavy chain variable region comprising CDR1, CDR2 and/or CDR 3; the amino acid sequence of the CDR1 is shown as SEQ ID NO.2, the nucleotide sequence coding the CDR1 is preferably shown as 76 th to 99 th positions of SEQ ID NO.5, the amino acid sequence of the CDR2 is shown as SEQ ID NO.3, the nucleotide sequence coding the CDR2 is preferably shown as 151 th to 174 th positions of SEQ ID NO.5, the amino acid sequence of the CDR3 is shown as SEQ ID NO.4, and the nucleotide sequence coding the CDR3 is preferably shown as 289 th to 312 th positions of SEQ ID NO. 5. Preferably, the heavy chain variable region comprises CDR1, CDR2 and CDR 3; more preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 1; more preferably, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 5.

The antibodies of the invention may also comprise a heavy chain constant region, which is conventional in the art, preferably of human or mouse origin, more preferably a human antibody heavy chain constant region; the heavy chain variable region is conventional in the art, and is preferably of human origin.

According to the present invention, the antibody refers to an antibody conventional in the art, preferably a monoclonal antibody, an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (scFv), a single-domain antibody (sdAb), or a single-domain antibody (sign-domain antibody); the monoclonal antibody can be developed by various means and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the monoclonal antibody is prepared from wild-type or transgenic mice by the hybridoma technology in the mainstream.

The antibody full-length protein is conventional in the art and includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. Preferably, the heavy chain variable region and the light chain variable region of the antibody and the mouse-derived heavy chain constant region and the mouse-derived light chain constant region constitute a full-length protein of the antibody. Alternatively, more preferably, the heavy chain variable region and the light chain variable region of the antibody and the human heavy chain constant region and the human light chain constant region constitute a fully human antibody full-length protein. Most preferably, the full length protein of the antibody is IgG1, IgG2, IgG3, or IgG 4.

The single-chain antibody is a conventional single-chain antibody in the field and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.

The antigen-antibody binding domain protein fragment is an antigen-antibody binding domain protein fragment conventional in the art, which includes a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragments are Fab and F (ab') ₂ 。

Single domain antibodies are conventional in the art and include a heavy chain variable region and a heavy chain constant region.

The single domain antibody is a single domain antibody that is conventional in the art and includes only the heavy chain variable domain.

The existing antibody is in a common antibody form with two heavy chains and two light chains, while the heavy chain antibody is in a novel antibody form without the light chains, the existing method for preparing the heavy chain antibody is to obtain the antibody by an immune camel through phage display screening, the cost is higher, the fully human heavy chain antibody can be obtained only by humanization in the later period, and the fully human platelet-derived growth factor receptor heavy chain antibody does not exist in China at present. Another difficulty in the development of PDGFR β heavy chain antibodies is that because transgenic mice are different from normal mice, conventional monoclonal antibody development techniques cannot achieve the desired effect when applied to transgenic mice intact. Therefore, it is necessary to specially optimize transgenic mice, i.e. to obtain high serum titer by multiple immunizations, and establish a matched development technology of high-efficiency immunization and fusion monoclonal antibodies and a sensitive and rapid screening and analysis technology and platform.

In a preferred embodiment of the invention, the antibody is a heavy chain antibody comprising a human heavy chain variable region and a mouse heavy chain constant region.

Among them, the antibody is prepared by a conventional method in the art. The preparation method is preferably as follows: obtained by separating from an expression transformant for recombinant expression of the vascular endothelial growth factor antibody or obtained by artificially synthesizing a protein sequence. The following method is preferably obtained by isolating an expression transformant which recombinantly expresses the antibody: cloning the nucleic acid molecule which codes the protein and has point mutation into a recombinant vector, transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant, and culturing the obtained recombinant expression transformant to obtain the antibody through separation and purification.

The second technical scheme for solving the technical problems is as follows: a nucleic acid encoding said antibody against human platelet-derived growth factor beta receptor; preferably, the amino acid sequence of the antibody encoded by the nucleic acid comprises the sequence shown as SEQ ID NO. 1; more preferably, the nucleotide sequence encoding the antibody comprises the sequence shown in SEQ ID NO. 5.

Those skilled in the art know that the base sequence encoding the amino acid sequence of the above antibody may be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homolog. The polynucleotide homologue of the present invention may be prepared by substituting, deleting or adding one or more bases of a gene encoding the antibody sequence within a range in which the activity of the antibody is maintained.

The third technical scheme for solving the technical problems is as follows: a recombinant expression vector comprising said nucleic acid.

Wherein the recombinant expression vector is obtainable by methods conventional in the art, i.e.: the nucleic acid molecule of the present invention is constructed by ligating it to various expression vectors. The expression vector is a variety of vectors which are conventional in the art, so long as they are capable of carrying the aforementioned nucleic acid molecule. The carrier preferably comprises: various plasmids, cosmids, bacteriophages or viral vectors, etc.

The fourth technical scheme for solving the technical problems is as follows: a recombinant expression transformant comprising the recombinant expression vector.

Among them, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, and preferably: transforming the recombinant expression vector into a host cell. The host cell is a variety of host cells conventional in the art, provided that the recombinant expression vector is stably self-replicating and the nucleic acid is efficiently expressed. Preferably, the host cell is an e.coli tg1 or BL21 cell (expressing a single chain antibody or Fab antibody), or a CHO-K1 cell (expressing a full length IgG antibody). The recombinant expression plasmid is transformed into a host cell to obtain a recombinant expression transformant preferred in the present invention. Wherein the above-mentioned transformation method is a transformation method conventional in the art, and is preferably a chemical transformation method, a thermal shock method or an electric transformation method.

The fifth technical scheme for solving the technical problems is as follows: a preparation method of the antibody for resisting the human platelet-derived growth factor beta receptor comprises the following steps: culturing the recombinant expression transformant, and obtaining an antibody against a human platelet-derived growth factor beta receptor from the culture.

The sixth technical scheme for solving the technical problems of the invention is as follows: the application of the antibody for resisting the human platelet-derived growth factor beta receptor in preparing the medicines for treating diseases related to the platelet-derived growth factor and/or the platelet-derived growth factor beta receptor; preferably, the drug is an anti-tumor drug.

The seventh technical scheme for solving the technical problems of the invention is as follows: a pharmaceutical composition comprises the antibody as an active ingredient. Preferably, the pharmaceutical composition is a pharmaceutical composition for preventing or treating tumors.

The administration route of the above-mentioned pharmaceutical composition of the present invention is preferably injection administration or oral administration. The above injection preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a granule, an injection or an infusion, etc.

Preferably, the aforementioned pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical excipients are conventional in the art, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the antibody and 0.01-99.99% of a pharmaceutically acceptable carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

Preferably, the aforementioned pharmaceutical composition is administered in an effective amount, which is an amount that alleviates or delays the progression of the disease, degenerative or damaging condition. Such effective amounts may be determined on an individual basis and will be based in part on consideration of the condition to be treated and the results sought. One skilled in the art can determine an effective amount by using such factors as an individual basis and using no more than routine experimentation.

The eighth technical scheme for solving the technical problems of the invention is as follows: the antibody is applied to the preparation of medicines for preventing or treating diseases related to platelet-derived growth factors or receptors thereof. The disease is preferably a tumor, preferably pancreatic cancer, small cell lung cancer, gastric cancer and breast cancer, a vascular disease and a fibrotic disease.

The ninth technical scheme for solving the technical problems is as follows: the application of the pharmaceutical composition in preparing a medicament for preventing or treating diseases related to platelet-derived growth factors or receptors thereof.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the invention utilizes a heavy chain antibody transgenic mouse to comprehensively apply a plurality of immunization methods to develop an antibody of an anti-human platelet-derived growth factor beta receptor with high affinity and high biological activity, in particular a monoclonal heavy chain antibody. The antibody shows excellent properties, can be combined with an extracellular region of human PDGFR beta, and can effectively inhibit or block the combination of PDGFb and PDGFR beta, thereby down-regulating or cutting off a corresponding signal path. The antibody has the application of inhibiting PDGFR beta mediated signal path and preparing medicines for diseases caused by PDGFR beta signal path.

Drawings

Figure 1 is the serum titers of 5 HCAb mice.

FIG. 2A shows the results of ELISA for heavy chain antibody binding to immunogen; FIG. 2B is an ELISA binding curve of heavy chain antibody to other unrelated proteins with hFc.

FIG. 3 is a graph of binding of a heavy chain antibody to an overexpressed target using flow cytometry with gradient dilution.

FIG. 4 shows that the Western blotting method detects that the heavy chain antibody blocks the phosphorylation ERK level up-regulated by ligand PDGFb on human brain perivascular cells.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 preparation of PDGFR beta antibody

Preparation of immunogen (human PDGFR beta ECD-hFc protein)

1. Immunogen structure design

PDGFR β is a receptor tyrosine kinase whose extracellular domain includes 5 immunoglobulin-like loops. The hFc is a protein macromolecule with high immunogenicity, and is used as a carrier protein for preparing immunogen, and is crosslinked to other antigens to enhance the immunogenicity of compounds. Here, the PDGFR β extracellular domain (ECD) and human constant domain (hFc) products were designed with a His tag at the C-terminus. Numbered PDGFR beta ECD-hFc-His. The specific information is shown in table 1.

TABLE 1 structural information of immunogens

2. Expression and purification of immunogens

A pCpC vector (purchased from Invitrogen, V044-50) containing a nucleotide sequence encoding the amino acid sequence of table 1 above, wherein the nucleotide sequence encoding the human PDGFR β protein was numbered NC 000005.10 in Genebank, was cloned into the pCpC vector with a human IgG Fc fragment (hFc) and 6 xhis (both hFc and His tag were cloned into the pCpC vector by PCR technology), and plasmids were prepared according to established standard molecular biology methods. See Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). This plasmid was used to infect HEK293 cells (purchased from ATCC, USA) by transient transfection method, and after 2 weeks of amplification, the supernatant was applied to a protein A affinity chromatography column (10ml MabSelect Sure, purchased from GE healthcare, cat. No. 17-5438), while changes in the ultraviolet absorbance (A280nm) were monitored by an Ultraviolet (UV) detector. After loading, the protein a affinity column was washed with PBS phosphate buffer (pH 7.4) until the uv absorbance returned to baseline, and then eluted with 50mM sodium citrate buffer (pH 3.0) to collect the hFc-bearing PDGFR β ECD eluted from the protein a affinity column. Dialyzed overnight at 4 ℃ in a refrigerator with PBS phosphate buffer (pH 7.4). The dialyzed protein is sterilized and filtered by 0.22 micron, and then subpackaged at-80 ℃ for storage, thus obtaining the purified immunogen hPDGFR beta ECD-hFc-his.

(II) preparation of hybridoma cells

1. Immunogen immunized mice

Heavy chain antibody transgenic mice (HCAb mice, purchased from Harbour, Netherlands) were immunized with the immunogen prepared as described above, and the mice were bred under SPF (Specific pathogen Free) conditions. For the initial immunization, the immunogen was emulsified in Freund's complete adjuvant and injected intraperitoneally with 25. mu.l, i.e., 50. mu.g of immunogen per mouse. In boosting, the immunogen is emulsified with Freund's incomplete adjuvant and injected intraperitoneally with 25. mu.l, i.e., 50. mu.g of immunogen per mouse. The interval between the primary and the first booster was 2 weeks, followed by 3 weeks between each booster. The immunization can be performed 1 to 7 times, preferably 4 to 6 times.

2. Indirect ELISA method for detecting immune serum titer

ELISA is a short name of enzyme-linked immunosorbent assay, is an immunoenzyme technology developed after immunofluorescence and radioimmunoassay, develops rapidly since the beginning of the 70 s, and is widely used in many fields of biology and medical science at present. In the invention, after 1 week of boosting immunization, blood is collected from the cheek, and the antibody titer and specificity of immunogen in serum are detected by indirect ELISA.

The specific operation is as follows:

the hPDGFR beta ECD-hFc-His prepared in 1 mu g/mL (preparation of I) immunogen preparation is dissolved in phosphate buffer solution, coated on an ELISA plate, 100 mu L/hole and kept at 4 ℃ overnight. The next day, the plate was washed three times with PBS buffer (containing 0.1% (V/V) Tween-20), 200. mu.L/well with 0.5% gelatin blocking solution, 1 hour at 37 ℃, three times with PBS buffer, 7 days after the second immunization, the mouse cheek blood was collected, the mouse immune serum was diluted with 10mM PBS buffer containing 2% newborn bovine serum, the microplate was added, 100. mu.L/well at 37 ℃ for 1 hour, and 1:10 after three times with plate washing ⁴ Diluting horseradish peroxidase labeled goat anti-mouse IgG by times, carrying out reaction at 37 ℃ for 1 hour at 100 mu L/hole after washing the plate, adding TMB for color development at 100 mu L/hole, keeping the plate away from light for 20min at room temperature, adding 2M HCl at 50 mu L/hole to terminate the reaction, measuring the absorption value at 450nm, taking the serum of the mouse before immunization as a negative control, and judging the titer of the immune serum by taking the ratio of the measured value to the control value as positive or more than 2.1.

The results are shown in FIG. 1 and Table 2. FIG. 1 shows that the serum titers obtained are high; table 2 shows that the sera of mice immunized with the extracellular domain of PDGFR β all bound the immunogen to different extents after immunization, showing antigen-antibody responses with the highest dilution around one hundred thousand. The blank was 1% (w/w) BSA, where the batch refers to mouse sera at day seven after the 1 st booster immunization, and the data in the table are OD450nm values. In 9396 example, the serum was diluted 1:100 and strongly bound to the antigen coated ELISA plates, showing an OD of up to 3.3, which gradually decreased after 10-fold dilution.

TABLE 2 ELISA detection of serum antibody titers of mice immunized with PDGFR beta extracellular region

3. Cell fusion

Each of the selected mice was subjected to a final intraperitoneal injection of 100. mu.g of the PDGFR beta extracellular region protein prepared in the first part (i) above (i) (mice immunized against the immunogen), and the mice were sacrificed 5 days later, and splenocytes were collected. Addition of NH ₄ OH to a final concentration of 1% (w/w), erythrocytes adulterated in the spleen cells were lysed, and spleen cell suspension was obtained. Cells were washed 3 times by centrifugation at 1000 rpm in DMEM basal medium, then mixed with mouse myeloma cell SP20 (purchased from ATCC) at a ratio of 4:1 in the number of viable cells, and fused by the PEG method.

4. Identification of hybridoma cells

The fused cells were diluted in DMEM medium containing 20% fetal bovine serum, 1 × HAT, in mass%. Then press 1X 10 ⁵ Per 200. mu.l per well into a 96 well cell culture plate, 5% CO ₂ And in a 37 ℃ incubator, the percentage is volume percentage. After 14 days, screening cell fusion plate supernatants by using ELISA (microplate protein detection) plates coated with antigen PDGFR beta extracellular region protein-hFc-his and hFc respectively, and amplifying positive clones with positive negative ratio of more than 10 in ELISA to 24-hole clonesPlates containing 10% (w/w) HT fetal bovine serum, DMEM (invitrogen) at 37 deg.C, 5% (v/v) CO ₂ And (5) carrying out amplification culture under the condition. After 3 days of culture, the culture medium in the 24-well plate was subjected to centrifugation, the supernatant was collected, and antibody subtype analysis was performed on the supernatant to determine the binding activity to PDGFR β -positive cells by FACS (see example 3 (one) and example 3 (two), respectively, for methods of detecting the binding activity). Selecting MFI value in FACS experiment according to 24-pore plate screening result>50 of hybridoma cells were eligible positive clones, and the eligible hybridoma cells were selected for subcloning in 96-well plates by limiting dilution in DMEM medium containing 10% (w/w) FBS (ex invitrogen) at 37 deg.C and 5% (v/v) CO ₂ Culturing under the condition. After 10 days of subcloning, primary screening was performed by ELISA, and single positive monoclonal was selected and amplified to 24-well plates for further culture. Antigen binding positivity was determined after 3 days by FACS and bioactivity was assessed by PDGFR β receptor ligand binding assay (assessment criteria is MFI value in FACS assay>50)。

5. Hybridoma cell expansion

Based on the results of the 24-well plate sample assay, the best clones were selected and grown in DMEM medium containing 10% (w/w) FBS (ex invitrogen) at 37 deg.C and 5% (v/v) CO ₂ And (3) performing amplification culture on the optimal clone under the condition, and performing freezing storage by liquid nitrogen to obtain the hybridoma cell, wherein the hybridoma cell can be used for subsequent antibody production and purification.

EXAMPLE 2 production and purification of antibodies

The antibody produced by the hybridoma cells is at a low concentration, approximately only 1-10. mu.g/mL, with large variations in concentration. And various proteins produced by cell culture in a culture medium and fetal calf serum components contained in the culture medium interfere with various biological activity analysis methods to different degrees, so that small-scale (1-5mg) antibody production and purification are required.

The Hybridoma cells obtained in example 1 were inoculated into a T-75 cell culture flask and acclimatized for passage 3 with a production medium (Hybridoma serum free medium, purchased from Invitrogen). Inoculating a cell culture rotary bottle when the growth state is good. 500mL of production medium was added to each 2L of the culture spinner flask, and the seeded cells were grown at a density of 1.0X 10 ⁵ /mL。And (4) tightly covering the bottle cap, and placing the rotary bottle on a rotary bottle machine in an incubator at 37 ℃ at the rotating speed of 3 revolutions per minute. After continuous rotation culture for 14 days, the cell culture broth was collected, filtered to remove cells, and filtered with a 0.45 μm filter until the culture supernatant was clarified. The clarified culture supernatant can be immediately purified or frozen at-30 ℃.

Monoclonal antibodies in the clarified hybridoma culture supernatant (300mL) were purified using a 2mL protein a column (purchased from GE Healthcare). The protein A column was equilibrated with an equilibration buffer (PBS phosphate buffer, pH7.2), and then the clarified culture supernatant was applied to the protein A column at a flow rate of 3 mL/min. And (4) washing the protein A column by using an equilibrium buffer solution after the loading is finished, wherein the volume of the equilibrium buffer solution is 4 times of that of a column bed of the protein A column. The PDGFR β antibody bound to the protein G column was eluted with an eluent (0.1M glycine hydrochloride buffer, pH2.5) and the elution was monitored with an ultraviolet detector (A280 ultraviolet absorption peak). The eluted antibody was collected, neutralized pH by adding 10% 1.0M Tris-HCl buffer, as a percentage by volume, and immediately dialyzed overnight with PBS phosphate buffer, followed by 1 exchange of the next day and dialysis continued for 3 hours. Collecting the PDGFR beta antibody after dialysis, performing sterile filtration by using a 0.22 mu m filter, and performing sterile storage to obtain the purified heavy chain PDGFR beta antibody.

The purified PDGFR β antibody was analyzed for protein concentration (a280/1.4), purity, and the like, and the results are shown in table 3.

TABLE 3 purified PDGFR beta antibody detection assay

Clone number	Purity of antibody	Protein concentration (mg/mL)
			39E10E12B3	>90％	1.2

EXAMPLE 3 assay of antibodies

Enzyme-linked immunosorbent assay (ELISA) detection of antigen-antibody binding sites

The purified PDGFR β antibody obtained in example 2 was conjugated to hPDGFR β -hFc-his protein obtained in example 1.

The purified immunogen obtained in example 1 was diluted with PBS to a final concentration of 5.0. mu.g/mL, and then added to a 96-well ELISA plate at 100. mu.l per well. After incubation overnight at 4 ℃ with plastic membrane, the plate was washed 2 times the next day with plate wash [ PBS + 0.01% (v/v) Tween20], and blocked for 2 hours at room temperature by adding blocking solution [ PBS + 0.01% (v/v) Tween20+ 1% (w/w) BSA ]. The blocking solution was decanted off and 100. mu.l of the purified PDGFR β antibody obtained in example 2 was added to each well. After incubation for 2 hours at 37 ℃, the plate was washed 3 times with wash solution [ PBS + 0.01% (v/v) Tween20 ]. HRP (horseradish peroxidase) -labeled secondary antibody (purchased from Sigma) was added, and after incubation at 37 ℃ for 2 hours, the plate was washed 3 times with a washing solution [ PBS + 0.01% (v/v) Tween20 ]. 100. mu.l of TMB substrate per well was added, and after incubation at room temperature for 30 minutes, 100. mu.l of stop buffer (1.0N HCl) per well was added. A450nm values were read using an ELISA reader (SpectraMax 384plus from Molecular devices) as shown in FIG. 2 and Table 4, where the IgG control was mouse IgG and the data in the table was OD450nm values. The results of table 4 and fig. 2 illustrate: ELISA plate antigen, 100nM heavy chain antibody of the invention as the initial concentration for 1:10 gradient dilution, table 4 is the reaction after the OD value, figure 2A; due to the presence of the hFc-tagged protein in the immunogen, the ELISA plate of FIG. 2B was coated with other hFc-bearing proteins that did not react with the antibody, indicating that the heavy chain antibody specifically binds to the antigen rather than to the tagged protein.

Table 4 ELISA detection of PDGFR β antibody binding to PDGFR β extracellular domain

(II) flow cytometry assay (FACS) detection of antibody binding to PDGFR beta expressing cells

A nucleotide sequence (see Genebank ID: NC-000005.10 for details) containing the sequence encoding the full-length amino acid of human PDGFR beta (see Uniprot P09619 for details) was introduced into HEK293 cell line to obtain a HEK293 stable cell line containing human PDGFR beta (herein referred to as HEK293-hPDGFR beta stable cell line), which was then expanded to 90% confluence in a T-75 cell culture flask, the medium was aspirated, washed 2 times with PBS buffer (Invitrogen), and then treated with an enzyme-free cell dissociation solution (Versene solution: from Life technology) and collected. Cells were washed 2 times with PBS buffer, and after cell counting, cells were diluted to 2X 10 with PBS buffer ⁶ cells/mL, 2% calf serum blocking solution was added, the percentages are mass percentages, incubated for 15 minutes at room temperature, and then washed 2 times by centrifugation with PBS buffer. The collected cells were suspended to 3X 10 with FACS buffer (PBS + 2% FBS, the percentages are by mass) ⁶ cells/mL were added to a 96-well FACS reaction plate at 100. mu.l per well, and 100. mu.l per well of the purified PDGFR β antibody test sample obtained in example 2 was added and incubated at 4 ℃ for 1 hour. The cells were washed 2 times by centrifugation in FACS buffer, 100. mu.l of a fluorescently (Alexa 488) -labeled secondary antibody (from Invitrogen) was added to each well, and incubated for 1 hour at 4 ℃. Washed 3 times by centrifugation in FACS buffer and 100. mu.l of fixative [ 4% (v/v) paraformaldehyde ] per well was added]Cells were suspended and washed 2 times after 10 minutes by centrifugation in FACS buffer. Cells were suspended in 100. mu.l FACS buffer, and the results were detected and analyzed by FACS (FACS Calibur, from BD). The results are shown in fig. 3 and table 5, and table 5 illustrates that the test antibody binds to PDGFR β on the cell surface. Where the IgG control was murine IgG (mIgG) and the data in the table are the mean fluorescence intensity values (MFI) for the cell populations tested. Figure 3 illustrates that incubation of cells with graded dilutions of antibodies and re-incubation with an alxa-488 labeled secondary antibody (purchased from Life technologies) can measure good binding of 39E10E12B3 to PDGFR β overexpressing cell lines, whereas the negative control mIgG gave no fluorescent signal.

TABLE 5 FACS detection of binding reactions of PDGFR β antibodies to HEK293-hPDGFR β

Example 4 western blot experiments to detect blockade of PDGFR β antibody on PDGFR β -mediated downstream signaling pathway by PDGFb

For analysis by Western blotting, HBVP (human cerebrovascular perivascular cells) was treated with lysis buffer (1% (w/v) SDS,1mM Tris (pH 7.4),2mM sodium vanadate, 2mM EGTA,2mM EDTA, I mM phenylmethylsulfonyl fluoride and mM sodium fluoride) to obtain a lysate, and the lysate was boiled and centrifuged at 10000g for 5 minutes at 4 ℃ to remove insoluble precipitate. The supernatant was mixed with SDS sample buffer and boiled for 10 minutes. SDS-PAGE and Western blotting were carried out according to methods widely used in the art, using the following samples: 12% SDS-polyacrylamide gel, PVDF membrane (Millipore # IPVH00010, usa), anti-phosphorylation ERK (Cell Signaling technology, usa) and anti-ERK antibody (Cell Signaling technology, usa) used as the primary antibody for phosphorylation ERK; and HRP conjugated goat anti-mouse IgG antibody (Santa Cruze B1technology, usa) as a secondary antibody. See fig. 4. FIG. 4 illustrates: heavy chain antibody 39E10E12B3 blocks ERK phosphorylation in HBVP by PDGFb, whereas pERK phosphorylation is an indicator of various cellular functions, such as proliferation.

EXAMPLE 5 determination of amino acid sequence of heavy chain variable region

Total RNA isolation: after the supernatants obtained by subclone culture in the amplification of hybridoma cells according to the second step 5 of example 1 were examined for antigen binding (i.e., after the assays and activity assays of examples 3-5), 5X 10 cells were collected by centrifugation ⁷ Adding 1mL of Trizol into each hybridoma cell, uniformly mixing, transferring into a 1.5mL centrifuge tube, and standing for 5 minutes at room temperature; adding 0.2mL of chloroform, oscillating for 15 seconds, standing for 2 minutes, centrifuging at 12000g for 5 minutes at 4 ℃, taking supernatant, and transferring the supernatant into a new 1.5mL centrifuge tube; adding 0.5mL of isopropanol, gently mixing the liquid in the tube, standing at room temperature for 10 minutes, centrifuging at 12000g for 15 minutes at 4 ℃, and removing the supernatant; 1mL of 75% ethanol (the percentages are by volume) was added, and the precipitate was gently washed at 4 ℃ and 12000g centrifuging for 5 min, discarding the supernatant, air drying the precipitate, adding DEPC treated H ₂ Dissolving in O (water bath at 55 ℃ for promoting the solvent for 10 minutes) to obtain the total RNA.

Reverse transcription and PCR: mu.g of total RNA was taken and prepared into a 20. mu.l system, and after reverse transcriptase was added, the reaction was carried out at 42 ℃ for 60 minutes and at 7 ℃ for 10 minutes to terminate the reaction. Preparing 50 mu l of PCR system, including 1 mu lcDNA, 25pmol of each primer, 1 mu l of DNA polymerase and matched buffer system, 250 mu moldNTPs; setting PCR program, pre-denaturing at 95 deg.c for 3 min, denaturing at 95 deg.c for 30 sec, annealing at 55 deg.c for 30 sec, extending at 72 deg.c for 35 sec, and extending at 72 deg.c for 5 min after 35 cycles to obtain PCR product. Wherein the kit used for reverse transcription is PrimeScript RT Master Mix purchased from Takara under the cat # RR 036; the kit used for PCR included Q5 super fidelity enzyme, purchased from NEB under cat number M0492.

Cloning and sequencing: taking 5 mul of PCR product to carry out agarose gel electrophoresis detection, and purifying a positive sample to be detected by using a column recovery kit, wherein the recovery kit is

Gel&PCRclean-up, available from MACHEREY-NAGEL under cat # 740609. Carrying out a ligation reaction: 50ng of sample, 50ng of T vector, 0.5 mu l of ligase, 1 mu l of buffer solution and 10 mu l of reaction system, and reacting for half an hour at 16 ℃ to obtain a ligation product, wherein the ligation kit is T4DNA ligase purchased from NEB under the product number of M0402; mu.l of the ligation product was added to 100. mu.l of competent cells (Ecos 101 component cells, from Yeaster, cat # FYE607), ice-washed for 5 minutes, heat-shocked in a water bath at 42 ℃ for 1 minute, returned to ice for 1 minute, added to 650. mu.l of the antibiotic-free SOC medium, revived at 37 ℃ for 30 minutes on a shaker at 200RPM, removed 200. mu.l of the antibiotic-containing LB solid medium and plated on an incubator at 37 ℃ overnight; the next day, a 30. mu.l PCR system was prepared using primers M13F (nucleotide sequence: GTAAAACGACGGCCAGT) and M13R (nucleotide sequence: CAGGAAACAGCTATGAC) on T-vector (purchased from Takara, cat # 6011), colony PCR was performed, the colony was dipped with the tip of a pipette and aspirated into the PCR reaction system, and 0.5. mu.l of the colony was spotted on another LB solid culture dish containing 100nM ampicillin to preserve the strain(ii) a After the PCR reaction was completed, 5. mu.l of the sample was subjected to agarose gel electrophoresis, and the positive sample was subjected to sequencing and analysis [ see Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)]. The sequencing results are shown in tables 6-7.

TABLE 6 PDGFR beta antibody protein amino acid sequence numbering

TABLE 7 PDGFR beta antibody gene (DNA) sequence numbering

Clone number	Heavy chain protein variable region
		39E10E12B3	5

Wherein, the numbers in tables 6 and 7 are sequence numbers in the sequence table, for example, the amino acid sequence of the heavy chain protein variable region of 39E10E12B3 is SEQ ID No.1, the amino acid sequence of CDR1 in the heavy chain protein variable region of 39E10E12B3 is SEQ ID No.2, the amino acid sequence of CDR2 is SEQ ID No.3, and the amino acid sequence of CDR3 is SEQ ID No. 4; the nucleotide sequence of the variable region of the heavy chain protein of 39E10E12B3 is SEQ ID No. 5.

Wherein, the nucleotide sequence of CDR1 in the variable region of the heavy chain protein of the code 39E10E12B3 is from the 76 th position to the 99 th position in the sequence table SEQ ID No. 5;

the nucleotide sequence of CDR2 in the variable region of the heavy chain protein of the code 39E10E12B3 is from the 151 th position to the 174 th position in the sequence table SEQ ID No. 5;

the nucleotide sequence of CDR3 in the variable region of the heavy chain protein of the code 39E10E12B3 is 289 th site to 312 th site in the sequence table SEQ ID No. 5.

It should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> Shanghai Ruizi chemical research, Inc.; kai Hui Rui Zhi Biotechnology (Shanghai) Co., Ltd

<120> antibody for resisting human platelet-derived growth factor beta receptor and application thereof

<130> P1710588C

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 115

<212> PRT

<213> Homo sapiens

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

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

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Thr Gly Asp Val Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 2

<211> 8

<212> PRT

<213> Homo sapiens

<400> 2

Gly Phe Thr Phe Ser Ser Tyr Gly

1 5

<210> 3

<211> 8

<212> PRT

<213> Homo sapiens

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Ile Trp Tyr Asp Gly Ser Asn Lys

1 5

<210> 4

<211> 8

<212> PRT

<213> Homo sapiens

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Ala Val Thr Gly Asp Val Asp Tyr

1 5

<210> 5

<211> 345

<212> DNA

<213> Homo sapiens

<400> 5

gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc ggtaactggg 300

gatgttgact actggggcca gggaaccctg gtcaccgtct cctca 345

Claims (16)

1. A heavy chain antibody against human platelet-derived growth factor β receptor comprising a heavy chain variable region comprising CDR1, CDR2 and CDR 3; the amino acid sequence of the CDR1 is shown as SEQ ID NO.2, the amino acid sequence of the CDR2 is shown as SEQ ID NO.3, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 4.

2. The heavy chain antibody of claim 1, wherein the nucleotide sequence encoding the CDR1 is shown in positions 76-99 of SEQ ID NO.5, the nucleotide sequence encoding the CDR2 is shown in positions 151-174 of SEQ ID NO.5, and the nucleotide sequence encoding the CDR3 is shown in positions 289-312 of SEQ ID NO. 5.

3. The heavy chain antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 1.

4. The heavy chain antibody of claim 3, wherein the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID NO. 5.

5. The heavy chain antibody of claim 1, further comprising a heavy chain constant region.

6. The heavy chain antibody of claim 5, wherein the heavy chain constant region is of human or mouse origin; and/or, the heavy chain variable region is human.

7. A nucleic acid encoding the heavy chain antibody against human platelet-derived growth factor beta receptor according to any one of claims 1 to 6.

8. The nucleic acid of claim 7, wherein the nucleic acid encodes a heavy chain antibody having an amino acid sequence comprising the sequence set forth in SEQ ID NO. 1.

9. The nucleic acid of claim 8, wherein the nucleotide sequence encoding the heavy chain antibody comprises the sequence set forth in SEQ ID NO. 5.

10. A recombinant expression vector comprising the nucleic acid of any one of claims 7 to 9.

11. A recombinant expression transformant comprising the recombinant expression vector of claim 10.

12. A method for preparing a heavy chain antibody against human platelet-derived growth factor beta receptor according to any one of claims 1 to 6, comprising the steps of: culturing the recombinant expression transformant according to claim 11, and obtaining a heavy chain antibody against a human platelet-derived growth factor β receptor from the culture.

13. A pharmaceutical composition comprising as an active ingredient a heavy chain antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition comprises 0.01 to 99.99% of the heavy chain antibody of any one of claims 1 to 6 and 0.01 to 99.99% of a pharmaceutically acceptable carrier, wherein the percentages are by weight of the pharmaceutical composition.

15. Use of a heavy chain antibody according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 13 or 14 for the manufacture of a medicament for the prevention or treatment of a disease associated with human platelet-derived growth factor or a receptor thereof;

the disease is tumor, vascular disease or fibrosis disease.

16. The use of claim 15, wherein the tumor is pancreatic cancer, small cell lung cancer, gastric cancer or breast cancer.

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