Fully humanized Anti-human CD36 antibody and use thereof

Abstract

Provided is a fully humanized anti-human CD36 antibody, comprising a heavy chain and a light chain. The amino acid sequence of the variable region of the heavy chain comprises a sequence as set forth in SEQ ID NO. 2, and the amino acid sequence of the variable region of the light chain comprises a sequence as set forth in SEQ ID NO. 4. Both the light chain and the heavy chain of the fully humanized anti-human CD36 antibody are derived from humans, resulting in a lower immunogenicity and better safety.

Description

Fully human anti-human CD36 antibody and its application Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a fully human anti-human CD36 antibody and its application. Background Art

[0002] CD36, also known as platelet membrane glycoprotein IV, is a highly glycosylated transmembrane protein. It belongs to the B-type scavenger receptor family and is expressed on the surfaces of many human cells, such as platelets, monocytes / macrophages, microglia, dendritic cells, microvascular endothelial cells, and retinal pigment epithelial cells.

[0003] Loss of CD36 expression is associated with disease. As a ligand recognized by pattern recognition receptors, CD36 is closely linked to the development of diseases such as atherosclerosis and Alzheimer's disease. Individuals with CD36 loss may develop anti-CD36 alloantibodies during pregnancy, transplantation, or blood transfusion. Individuals with these antibodies may develop fetal / neonatal alloimmune thrombocytopenia (FNAIT), platelet transfusion ineffectiveness (PTR), post-transfusion purpura (PTP), and transfusion-related acute lung injury (TRALI) during pregnancy or re-transfusion.

[0004] In disease diagnosis and treatment, mouse and rabbit monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies are commonly used. However, in actual clinical applications, heterologous monoclonal antibodies can cause HAMA reactions due to their high immunogenicity, resulting in a short half-life of the monoclonal antibody drug, poor therapeutic efficacy, and also prone to safety issues. To overcome this difficulty, the development of therapeutic antibodies has gone through multiple stages, including mouse antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. Fully human antibodies have 100% human amino acid sequences, thus offering the advantages of lower immunogenicity and better safety.

[0005] Prior art CD36 antibodies are all mouse or humanized mouse antibodies, with no fully humanized CD36 antibodies. For example, patent application number CN201810600518.7 discloses a mouse CD36 antibody obtained through a hybridoma method; patent application number CN202180034204.5 discloses a humanized CD36 antibody and its use in cancer treatment.

[0006] Fully human antibodies, derived entirely from humans, minimize rejection and offer the best safety. Fully humanized antibodies, whose light and heavy chains are both human, are a growing trend in therapeutic antibodies. However, fully human anti-human CD36 antibodies do not currently exist.

[0007] The disclosure of the above background technology content is only used to assist in understanding the inventive concept and technical solution of the present invention. It does not necessarily belong to the prior art of this application. In the absence of clear evidence showing that the above content has been disclosed before the filing date of this application, the above background technology should not be used to evaluate the novelty and creativity of this application. Summary of the Invention

[0008] In view of this, in order to overcome the deficiencies of the prior art, the present invention aims to provide a fully human anti-human CD36 antibody and corresponding sequences and applications.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention provides a fully human anti-human CD36 antibody, comprising a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain is the sequence shown in SEQ ID NO.2, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID NO.4.

[0011] The present invention provides a nucleic acid encoding the fully human anti-human CD36 antibody described above.

[0012] According to some preferred embodiments of the present invention, the nucleotide sequence encoding the antibody heavy chain variable region is the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the antibody light chain variable region is the sequence shown in SEQ ID NO.3.

[0013] The present invention provides a fully human anti-human CD36 antibody, comprising a heavy chain and a light chain, wherein the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the nucleotide sequence of the light chain variable region is the sequence shown in SEQ ID NO.3.

[0014] The present invention provides a nucleic acid encoding the fully human anti-human CD36 antibody described above.

[0015] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0016] According to some preferred embodiments of the present invention, the expression vector comprising the heavy chain antibody has an amino acid sequence corresponding to the nucleotide sequence of its expression cassette as shown in SEQ ID NO.7.

[0017] Preferably, the amino acid sequence corresponding to the nucleotide sequence of the heavy chain antibody expression vector is obtained by constructing the fully human anti-human CD36 antibody heavy chain variable region amino acid sequence shown in SEQ ID NO.2 and the human IgG1 amino acid sequence shown in SEQ ID NO.5 into the pcDNA3.4 vector.

[0018] According to some preferred embodiments of the present invention, the expression vector comprising the light chain antibody has an amino acid sequence corresponding to the nucleotide sequence of its expression cassette as shown in SEQ ID NO.8.

[0019] Preferably, the amino acid sequence corresponding to the nucleotide sequence of the expression vector is obtained by constructing the fully human anti-human CD36 antibody light chain variable region amino acid sequence shown in SEQ ID NO.4 and the human Kappa amino acid sequence shown in SEQ ID NO.6 into the pcDNA3.4 vector.

[0020] The present invention also provides a cell transformed with the above recombinant expression vector.

[0021] The present invention also provides a use of the above-mentioned antibody, nucleic acid, recombinant expression vector, and cell in the preparation of drugs or related kits for central nervous system diseases such as stroke, Alzheimer's disease, Parkinson's disease, and spinal cord injury.

[0022] The present invention also provides a pharmaceutical composition comprising the above-mentioned antibody, and / or the above-mentioned nucleic acid, and / or the above-mentioned expression vector, in combination with one or more pharmaceutically acceptable excipients.

[0023] The present invention provides an amino acid sequence of a fully human anti-human CD36 antibody that specifically binds to human CD36, comprising an amino acid sequence of a heavy chain variable region and an amino acid sequence of a light chain variable region; the amino acid sequence of the heavy chain variable region comprises the sequence shown in SEQ ID NO.2; and the amino acid sequence of the light chain variable region comprises the sequence shown in SEQ ID NO.4.

[0024] The present invention provides a nucleotide sequence of a fully human anti-human CD36 antibody that specifically binds to human CD36, comprising a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region; the nucleotide sequence encoding the heavy chain variable region comprises the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the light chain variable region comprises the sequence shown in SEQ ID NO.3.

[0025] Due to the adoption of the above technical solution, compared with the existing technology, the present invention is beneficial in that the light chain and heavy chain of the fully human anti-human CD36 antibody of the present invention are both derived from humans, and therefore have lower immunogenicity and better safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] FIG1 is an SDS-PAGE non-reducing gel electrophoresis diagram of the fully human CD36 antibody in Example 2 of the present invention;

[0028] FIG2 is an affinity curve of the binding of the fully human anti-human CD36 antibody to human CD36 in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0030] Mouse monoclonal antibodies are commonly used in disease diagnosis and treatment. However, in actual clinical applications, heterologous monoclonal antibodies can induce human anti-mouse antibody (HAMA) reactions due to their high immunogenicity, resulting in a short half-life of mouse monoclonal antibodies, poor therapeutic efficacy, and safety issues. To overcome this difficulty, this application uses yeast display technology to screen human natural antibody libraries to prepare and obtain fully human antibodies. The amino acid sequence of the antibodies is 100% human, thus having the advantages of lower immunogenicity and better safety.

[0031] Example 1 Screening of fully human anti-human CD36 antibodies

[0032] 1.1 First round of magnetic bead enrichment of the library

[0033] (1) Take 100 OD of yeast library strain (human antibody scFv yeast display library) from the -80℃ refrigerator, add it to 100 ml of SD-Trp-deficient culture medium, and culture at 30℃ and 225 rpm for 3-4 h.

[0034] (2) After culturing for 3-4 hours, measure the absorbance at OD600, remove the defective culture medium by centrifugation, and collect the yeast cells. Add the yeast cells to 250 ml of SG-CAA medium and incubate at 20°C and 225 rpm for 24 hours. Collect yeast cells at 500 OD by centrifugation.

[0035] (3) Resuspend the yeast cells in 40 ml PBS + 1% BSA.

[0036] (4) Centrifuge and discard the supernatant, resuspend the yeast in 5 ml PBS + 1% BSA, add 100 nM CD36-biotin, and incubate with shaking at room temperature for 1 h.

[0037] (5) After incubation for 1 hour, centrifuge at 3000 rpm for 3 minutes and discard the supernatant.

[0038] (6) Add 40 ml of PBS + 1% BSA and centrifuge at 3000 rpm for 3 minutes.

[0039] (7) Repeat step (6) twice.

[0040] (8) Add 5 ml PBS + 1% BSA to resuspend the cells, then add 40 μL SA-beads and mix thoroughly.

[0041] (9) Incubate for 20 minutes.

[0042] (10) Place the magnetic column on the magnetic rack, pass the incubated cells through the column, and then wash the column three times with 5 ml PBS + 1% BSA.

[0043] (11) Remove the magnetic column from the magnetic rack and incubate the cells with 5 ml of defective culture medium.

[0044] (12) The eluted cells were cultured overnight at 30°C and 225 rpm.

[0045] (13) After overnight culture, the cells were replaced with SG-CAA medium and cultured at 20°C and 225 rpm for 24 h before flow cytometry analysis.

[0046] 1.2 Second round of FACS enrichment of the library

[0047] (1) Take 8 OD of bacterial solution and place it in a 15 ml centrifuge tube. Centrifuge and discard the supernatant. Wash the tube three times with 10 ml PBS + 1% BSA (3000 rpm, 3 min). Then, reselect the bacteria with 1000 μL PBS + 1% BSA.

[0048] (2) Add 1 μL anti-FLAG and a final concentration of 50 nM CD36-Biotin to 1000 μL bacterial solution.

[0049] (3) Incubate at room temperature for 60 minutes.

[0050] (4) Centrifuge at 3000 rpm for 3 min and remove the supernatant.

[0051] (5) Add 5 mL of PBS + 1% BSA to the cells to reselect the cells, centrifuge at 3000 rpm for 3 min, and remove the supernatant.

[0052] (6) Repeat step (5) twice.

[0053] (7) Reselect the cells with 1000 μL of PBS + 1% BSA, add 1 μL of SA-PE and 1 μL of Goat antimouse-647, and incubate at room temperature for 60 min.

[0054] (8) Centrifuge at 3000 rpm for 3 min and remove the supernatant.

[0055] (9) Repeat step (8) twice.

[0056] (10) Reselect the cells with 1000 μL of PBS + 1% BSA, and use flow cytometry to separate the PE and 647 double-positive cell populations.

[0057] 1.3 Clone FACS identification

[0058] (1) The enriched yeast cells were spread on SD-Trp plates and cultured in a 30°C incubator until a single colony grew.

[0059] (2) Select clones and inoculate them into SD-Trp medium, and culture them at 30°C with shaking overnight.

[0060] (3) Transfer the overnight cultured cells to SG-CAA medium and culture at 25°C for 48 h.

[0061] (4) Perform FACS detection according to step example 1.2.

[0062] 1.4 Yeast clone sequencing

[0063] (1) Yeast cloning: Extract plasmids using a yeast plasmid extraction kit.

[0064] (2) The plasmid was transformed into Top10 Escherichia coli competent cells.

[0065] (3) The clones were selected for sequencing to obtain the fully human anti-human CD36 antibody sequence. The obtained fully human anti-human CD36 antibody heavy chain variable region nucleotide sequence is shown in SEQ ID NO.1; the fully human anti-human CD36 antibody heavy chain variable region amino acid sequence is shown in SEQ ID NO.2; the fully human anti-human CD36 antibody light chain variable region nucleotide sequence is shown in SEQ ID NO.3; the fully human anti-human CD36 antibody light chain variable region amino acid sequence is shown in SEQ ID NO.4.

[0066] Example 2 Expression and Verification of Fully Human Anti-human CD36 Antibody

[0067] 2.1 Expression and purification of fully human anti-human CD36 antibodies

[0068] The amino acid sequence of the fully human anti-human CD36 antibody heavy chain variable region shown in SEQ ID NO.2 and the human IgG1 amino acid sequence shown in SEQ ID NO.5 were constructed into the pcDNA3.4 vector to obtain the amino acid sequence shown in SEQ ID NO.7. This amino acid sequence was transformed to obtain the nucleotide sequence of the expression cassette corresponding to the expression vector of the heavy chain antibody.

[0069] The fully human anti-human CD36 antibody light chain variable region amino acid sequence shown in SEQ ID NO.4 and the human Kappa amino acid sequence shown in SEQ ID NO.6 were constructed into the pcDNA3.4 vector to obtain the amino acid sequence shown in SEQ ID NO.8. This amino acid sequence was transformed to obtain the nucleotide sequence of the expression cassette corresponding to the expression vector of the light chain antibody.

[0070] The light chain plasmid and heavy chain plasmid were transformed into 293 cells at a ratio of 1:1 and then the antibody was purified.

[0071] 2.2 SDS-PAGE of fully human anti-human CD36 antibody

[0072] (1) Add 4 μL of loading buffer to 16 μL of sample and denature at 100°C for 5-10 min.

[0073] (2) Load 10 μL of sample and run SDS-PAGE electrophoresis at 150 V for 45 min.

[0074] (3) After electrophoresis, turn off the power, remove the gel, and place it in a large culture dish for staining. Use rapid staining solution at room temperature for 10 minutes, discard the staining solution, and decolorize with distilled water overnight.

[0075] (4) Purified fully human anti-human CD36 antibodies were obtained, as shown in Figure 1. Band M is a marker; band 1 is a purified fully human anti-human CD36 antibody. In Figure 1, the markers from top to bottom are: 270 kDa, 175 kDa, 130 kDa, 95 kDa, 65 kDa, 50 kDa, 35 kDa, 30 kDa, 15 kDa, and 5 kDa.

[0076] 2.3 Binding analysis of fully human anti-human CD36 antibodies

[0077] (1) Add 100 μL of 2 μg / ml human CD36 antigen to each well of the Elsia plate and coat at 4°C overnight.

[0078] (2) The next day, wash three times with PBST.

[0079] (3) Add 300 μL of 2% BSA and incubate at room temperature for 60 min.

[0080] (4) Wash three times with PBST.

[0081] (5) Add gradient dilutions of CD36 antibody and incubate at room temperature for 60 min.

[0082] (6) Wash 3 times with PBST.

[0083] (7) Add 5000-fold diluted goat anti-human HRP antibody and incubate at room temperature for 60 min.

[0084] (8) Wash 3 times with PBST.

[0085] (9) Add 100 μL of color development solution.

[0086] (10) Add 100 μL of stop solution.

[0087] (11) Read the plate using an enzyme-linked microplate reader.

[0088] (12) Data Analysis (CD36 Antibody Affinity Curve) As shown in FIG2 , the fully human anti-human CD36 antibody binds to human CD36.

[0089] Fully humanized antibodies have both light and heavy chains derived from humans, so they have the lowest rejection reaction and the best safety, which is the development trend of therapeutic antibodies. This application uses yeast display to obtain fully human antibodies with lower immunogenicity and better safety. This antibody is obtained by screening a yeast display library with human CD36 antigen, and its research and development cycle is shorter than that of hybridomas. And because it is a human antibody library, the obtained antibody sequence is fully human, which has the advantages of lower immunogenicity and better safety.

[0090] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

[0091] The corresponding table of the sequence numbers and specific sequences of the present invention is as follows:

Claims

1. A fully human anti-human CD36 antibody, characterized in that: It comprises a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain comprises the sequence shown in SEQ ID NO.2, and the amino acid sequence of the variable region of the light chain comprises the sequence shown in SEQ ID NO.

4.

2. A nucleic acid encoding the fully human anti-human CD36 antibody according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence encoding the antibody heavy chain variable region includes the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the antibody light chain variable region includes the sequence shown in SEQ ID NO.

3.

4. A fully human anti-human CD36 antibody, characterized in that: It comprises a heavy chain and a light chain, wherein the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the nucleotide sequence of the light chain variable region is the sequence shown in SEQ ID NO.

3.

5. A nucleic acid encoding the fully human anti-human CD36 antibody according to claim 4.

6. A recombinant expression vector comprising the nucleic acid according to claim 2, claim 3 or claim 5.

7. The recombinant expression vector according to claim 6, characterized in that The expression vector comprising the heavy chain antibody has an amino acid sequence corresponding to the nucleotide sequence of its expression cassette including the sequence shown in SEQ ID NO.

7.

8. The recombinant expression vector according to claim 7, characterized in that The amino acid sequence corresponding to the nucleotide sequence of the heavy chain antibody expression vector is obtained by constructing the fully human anti-human CD36 antibody heavy chain variable region amino acid sequence shown in SEQ ID NO.2 and the human IgG1 amino acid sequence shown in SEQ ID NO.5 into the pcDNA3.4 vector.

9. The recombinant expression vector according to claim 8, characterized in that The expression vector comprising the light chain antibody has an amino acid sequence corresponding to the nucleotide sequence of its expression cassette including the sequence shown in SEQ ID NO.

8.

10. The recombinant expression vector according to claim 9, characterized in that The amino acid sequence corresponding to the nucleotide sequence of the light chain antibody expression vector is obtained by constructing the fully human anti-human CD36 antibody light chain variable region amino acid sequence shown in SEQ ID NO.4 and the human Kappa amino acid sequence shown in SEQ ID NO.6 into the pcDNA3.4 vector.

11. A cell transformed with the recombinant expression vector according to any one of claims 6 to 10.

12. Use of the antibody according to claim 1 or 4, the nucleic acid according to claim 2, 3 or 5, the recombinant expression vector according to any one of claims 6 to 10, or the cell according to claim 11 in the preparation of a drug or kit for related diseases.

13. A pharmaceutical composition comprising the antibody of claim 1 or 4, and / or the nucleic acid of claim 2, 3 or 5, and / or the recombinant expression vector of any one of claims 6 to 10, in combination with one or more pharmaceutically acceptable excipients.

14. An amino acid sequence of a fully human anti-human CD36 antibody that specifically binds to human CD36, characterized in that: It includes the amino acid sequence of the variable region of the heavy chain and the amino acid sequence of the variable region of the light chain; the amino acid sequence of the variable region of the heavy chain includes the sequence shown in SEQ ID NO.2; the amino acid sequence of the variable region of the light chain includes the sequence shown in SEQ ID NO.

4.

15. A nucleotide sequence of a fully human anti-human CD36 antibody that specifically binds to human CD36, characterized in that: It includes a nucleotide sequence encoding the variable region of the heavy chain and a nucleotide sequence encoding the variable region of the light chain; the nucleotide sequence encoding the variable region of the heavy chain includes the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the variable region of the light chain includes the sequence shown in SEQ ID NO.3.

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