High-affinity anti-human and monkey PCSK9 antibody and application thereof

Abstract

The invention discloses a high-affinity anti-human and monkey PCSK9 antibody and application thereof, and belongs to the field of biological medicine. The anti-human PCSK9 antibody comprises a heavy chain variable region of which the sequence is SEQ ID NO.1 and a light chain variable region of which the sequence is SEQ ID NO.2, or a heavy chain variable region of which the sequence is SEQ ID NO.3 and a light chain variable region of which the sequence is SEQ ID NO.4. The six anti-human PCSK9 antibodies are obtained by immunizing mice with human PCSK9 protein and combining with a hybridoma cell line and screening, and can be combined with human PCSK9 with high affinity and high selectivity, so that the combination of the human PCSK9 and LDLR (Low Density Lipoprotein Receptor) on a cytoplasmic membrane is blocked, and the degradation of the LDLR is reduced. PCSK9 is highly expressed in tumors, tumor immune recognition and CD8 + T cell anti-tumor activity are inhibited, and the PCSK9 participates in tumor immune escape. The anti-human PCSK9 antibody provided by the invention can provide candidate antibody molecules for tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a high-affinity anti-human and monkey PCSK9 antibody and its application. Background Art

[0002] Immunotherapy is currently an important means for treating malignant tumors following traditional treatment methods such as surgery, radiotherapy, and chemotherapy. Different from traditional therapies, immunotherapy focuses on the body's own immune system and clears tumor cells by enhancing or reconstructing immune capabilities. However, tumor metabolic reprogramming generates an inhibitory microenvironment, including nutrient deficiency, metabolite accumulation, acidity, and hypoxia, which mediates metabolic stress in microenvironmental immune cells and inhibits their anti-tumor functions. This is the key factor and bottleneck problem leading to tumor immune escape and the limitation of existing immunotherapies in the treatment of solid tumors and low clinical responsiveness.

[0003] As a basic component of cell membranes, the remodeling of cholesterol metabolism in the tumor microenvironment leading to immunosuppression is a key factor in tumor immune escape. Tumor cells and tumor-associated macrophages (TAMs) consume cholesterol in the microenvironment, resulting in cholesterol deficiency in tumor-infiltrating CD8 + T cells, ultimately leading to dysfunction and exhaustion. Therefore, targeting cholesterol metabolism in the tumor microenvironment for cancer treatment has attracted much attention.

[0004] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is mainly expressed in the liver and is an important regulatory molecule for cholesterol metabolism homeostasis. In the absence of PCSK9, the low-density lipoprotein receptor (LDLR) on the surface of hepatocytes binds free low-density lipoprotein cholesterol (LDL-C) in the blood, mediates its endocytosis into cells for degradation and metabolism, and the LDLR is then transported back to the cell membrane for recycling. However, PCSK9 can bind to LDLR through its catalytic domain, mediate the degradation of LDLR in lysosomes, thereby reducing the ability of the liver to clear LDL-C, leading to an increase in the level of LDL-C in the blood and inducing cardiovascular diseases. Therefore, PCSK9 has become an important target for cardiovascular diseases. In addition, studies in recent years have shown that targeting PCSK9 has broad application potential and development prospects in enhancing tumor immunotherapy. Previous studies by the research group found that PCSK9 is highly expressed ectopically in tumor tissues, inhibiting the anti-tumor activity of CD8 T cells by blocking the recycling of the T cell receptor TCR and TCR signaling. Another research report showed that PCSK9 directly binds to MHC I on the surface of tumor cells and mediates its degradation, inhibiting antigen recognition and CD8 T cell infiltration, and promoting the occurrence and development of tumors. In addition, PCSK9 can also regulate PTEN protein degradation, activate caspase-3, regulate cholesterol levels, etc., and participate in the tumor process. Given the regulatory effects of PCSK9 on tumor immune recognition, the anti-tumor function of CD8 T cells, and tumor cell proliferation and apoptosis, the tumor treatment strategy targeting PCSK9 has important clinical translational value and potential.

[0005] Therapeutic monoclonal antibodies have shown broad application prospects in modern medicine due to their strong targeting and significant efficacy. At present, the State Drug Administration of China has approved the marketing of 3 monoclonal antibodies targeting PCSK9 for the treatment of cardiovascular diseases: Evolocumab, Alirocumab, and Tafolecimab. However, there is still a need for alternative PCSK9 antibodies to provide potential new clinical treatment drugs during the process of expanding indications. In the present invention, mice were immunized with human PCSK9 protein, and through the screening of hybridoma cell lines and humanization, a monoclonal antibody against human PCSK9 with an epitope different from that of existing antibodies was obtained, which can provide a potential new clinical treatment plan for the preparation of a preparation with monoclonal antibody against PCSK9 as the core component for the treatment of solid tumors or hematological tumors with high expression of PCSK9. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a high-affinity anti-human and anti-monkey PCSK9 antibody and its application.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] In a first aspect of the present invention, there is provided an anti-human PCSK9 antibody, comprising:

[0009] Sequence combination A: comprising:

[0010] A heavy chain variable region with complementary determining regions HCDR1, HCDR2, and HCDR3 having sequences GYTFSSYW, ILRGSGST, and ASRGYGYDY, respectively;

[0011] And a light chain variable region with complementary determining regions LCDR1, LCDR2, and LCDR3 having sequences QNVNTD, SAS, and HQYNNYPYT, respectively;

[0012] Sequence combination B: comprising:

[0013] A heavy chain variable region with complementary determining regions HCDR1, HCDR2, and HCDR3 having sequences GYAFTDYY, IWPGRVNT, and ARRSYYGSFDV (SEQ NO ID.21), respectively;

[0014] And a light chain variable region with complementary determining regions LCDR1, LCDR2, and LCDR3 having sequences QDINTY, RAN, and LQYDDFPWT, respectively;

[0015] Sequence combination C, comprising:

[0016] A heavy chain variable region with complementary determining regions HCDR1, HCDR2, and HCDR3 having more than 90% identity with the HCDR1, HCDR2, and HCDR3 of the said sequence A;

[0017] And a light chain variable region with complementary determining regions LCDR1, LCDR2, and LCDR3 having more than 90% identity with the LCDR1, LCDR2, and LCDR3 of the said sequence A;

[0018] Or, sequence combination D, comprising:

[0019] A heavy chain variable region with complementary determining regions HCDR1, HCDR2, and HCDR3 having more than 90% identity with the HCDR1, HCDR2, and HCDR3 of the said sequence B;

[0020] And a light chain variable region with complementary determining regions LCDR1, LCDR2, and LCDR3 having more than 90% identity with the LCDR1, LCDR2, and LCDR3 of the said sequence B.

[0021] Optionally, the sequences of the heavy chain variable region and the light chain variable region of the sequence combination A are SEQ ID NO.1 and SEQ ID NO.2 respectively; the sequences of the heavy chain variable region and the light chain variable region of the sequence combination B are SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0022] The second aspect of the present invention relates to a nucleic acid encoding the anti-human PCSK9 antibody described above.

[0023] The third aspect of the present invention relates to a recombinant vector carrying the nucleic acid described above.

[0024] The fourth aspect of the present invention relates to a cell capable of expressing the recombinant vector described above.

[0025] The fifth aspect of the present invention relates to a method for the anti-human PCSK9 antibody described above, comprising the following steps:

[0026] Introducing the recombinant vector described above into a cell;

[0027] Culturing the cell to obtain the method for the anti-human PCSK9 antibody.

[0028] The sixth aspect of the present invention relates to a drug for treating PCSK9-related diseases, comprising the anti-human PCSK9 antibody described above, the nucleic acid described above or the vector described above.

[0029] The seventh aspect of the present invention relates to a kit for detecting PCSK9, comprising the anti-human PCSK9 antibody described above.

[0030] The eighth aspect of the present invention relates to the application of the anti-human PCSK9 antibody described above, the nucleic acid described above or the vector described above in the preparation of a drug for treating PCSK9-related diseases.

[0031] The ninth aspect of the present invention relates to the application of the anti-human PCSK9 antibody described above in the preparation of a kit for detecting PCSK9.

[0032] Advantages of the present invention:

[0033] The antibody of the present application can bind to human PCSK9 with high affinity and high selectivity, block its binding to LDLR on the cell membrane, and reduce the degradation of LDLR. Moreover, it binds to a different epitope of the PCSK9 protein from that of Alirocumab, and can restore the level of cell surface LDLR and the ability of cell LDL uptake. Therefore, the anti-human PCSK9 antibody of the present application can provide a new treatment plan for PCSK9-related diseases and has potential application value. Brief Description of the Drawings

[0034] The present invention will be further described below with reference to the accompanying drawings.

[0035] Figure 1 For the binding of mouse serum to human PCSK9;

[0036] Figure 2 For the ability of the parental clone supernatant to restore LDLR;

[0037] Figure 3 For the affinity of the subcloned antibody in the examples of this application to human PCSK9;

[0038] Figure 4 For the binding of the PCSK9 antibody detected by BLI in the examples of this application to human / monkey / mouse PCSK9 protein;

[0039] Figure 5 For the binding of the PCSK9 antibody detected by ELISA in the examples of this application to human / monkey / mouse PCSK9 protein;

[0040] Figure 6 For the epitopes of the 6 PCSK9 antibodies binding to human PCSK9 protein in the examples of this application being different from those of Alirocumab;

[0041] Figure 7 For the effect of the PCSK9 antibody on the cell surface LDLR level and LDL uptake in the examples of this application. Detailed implementation manners

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] (1) Animal immunization: Select 5 female Balb / c and SJL mice at 8 weeks old each, and perform immunization injections according to Table 1. The antigen enters the peripheral immune organs through the blood circulation or lymphatic circulation, stimulates the corresponding B lymphocyte clones, activates, proliferates them, and differentiates them into sensitized B lymphocytes.

[0044] Table 1 Immunization Schedule

[0045]

[0046] (2) Serum detection: Collect blood at the time points in Table 1, separate the serum, coat the human PCSK9 protein (0.5 μg / ml, 100 μl / well) in a 96-well plate, detect the immune status of the mice, and select 1 mouse each from Balb / c and SJL mice for hybridoma cell fusion (Figure 1 )

[0047] (3) Cell fusion and screening of master clones: After enriching CD138 in mouse plasma cells, two rounds of fusion will be performed by electrofusion. The hybridoma cells obtained after each fusion will be plated on approximately 30 96-well plates. All clones will be screened for the human target PCSK9 protein by ELISA. Positive master clone supernatants will be selected according to the screening results for LDLR rescue experiments ( Figure 2 )

[0048] (4) Subclone screening and antibody variable region sequencing: Up to 50 master clones will be subcloned by limiting dilution to ensure that each subclone is derived from a single master clone. All subclones will be screened for the human target PCSK9 protein and LDLR rescue experiments by ELISA. Ten subclones will be selected for variable region sequencing, and the above antibodies will be recombinantly expressed (Table 2).

[0049] Table 2 Amino acid sequence of antibody variable region

[0050]

[0051]

[0052]

[0053] (5) Affinity test: Fix the above 10 antibodies with protein A biosensor, and detect their binding ability to human PCSK9 protein by biomembrane interference technology (BLI). It is found that 6 of the antibodies can bind human PCSK9 with high affinity, and the KD value reaches 10 -11 M( Figure 3 , Table 3).

[0054] Table 3 Affinity of subcloned antibodies to human PCSK9

[0055]

[0056] (6) Binding ability of PCSK9 antibodies to PCSK9 proteins of different species: Use BLI to detect the binding ability of the above 6 PCSK9 antibodies to human / monkey / mouse PCSK9 proteins. It is found that 4 of the antibodies can bind monkey PCSK9 but not mouse PCSK9 ( Figure 4 , Table 4). The same conclusion was obtained by ELISA coating human / monkey / mouse PCSK9 proteins ( Figure 5 ).

[0057] Table 4 Affinity of PCSK9 antibodies to human / monkey / mouse PCSK9 proteins

[0058]

[0059] (7) Comparison of the binding epitopes of PCSK9 antibodies with human PCSK9 protein: The biotinylated human PCSK9 protein was immobilized using a streptavidin (SA) biosensor, incubated with Alirocumab until no further binding occurred, and then incubated with the above 6 PCSK9 antibodies (Alirocumab as a control). It was found that the epitopes of these 6 antibodies binding to human PCSK9 were inconsistent with those of Alirocumab ( Figure 6 A in). The same conclusion was also obtained by ELISA coating with biotinylated human PCSK9 protein ( Figure 6 B in).

[0060] (8) Functional verification of PCSK9 antibodies: Using HepG2 cells as model cells, 5 antibodies with strong binding ability were selected from the above antibodies for LDLR rescue experiments. It was found that as the antibody concentration increased, these five antibodies could all restore the LDLR level on the cell surface ( Figure 7 A in), and could promote the uptake of LDL by cells ( Figure 7 B in). We also detected whether the above antibodies could regulate the level of LDLR on the surface of T cells in Jurkat T cells, and the results were basically the same as those in HepG2 ( Figure 7 C in).

[0061] In summary, the present invention aims to develop new anti-human PCSK9 antibodies in order to provide potential new clinical treatment drugs during the process of expanding indications. For this purpose, on the one hand, the present invention prepared PCSK9 antibodies with high affinity, and disclosed several anti-human PCSK9 antibodies and their antigen-binding fragments, including the heavy chain variable region (VH) and the light chain variable region (VL). Among them, the VH includes the antigen-determining regions VH CDR1, VH CDR2, and VH CDR3, and the VL includes the antigen-determining regions VL CDR1, VL CDR2, and VL CDR3; on the other hand, the binding abilities of the above PCSK9 antibodies with human, monkey, and mouse PCSK9 proteins were explored, the differences between the binding epitopes of the above PCSK9 antibodies with human PCSK9 protein and those of Alirocumab were compared, and the abilities of the above PCSK9 antibodies to restore the LDLR level on the cell surface and the LDL uptake by cells were detected. The present invention can provide a potential new clinical treatment plan for the preparation of preparations with PCSK9 monoclonal antibody as the core component to treat solid tumors or hematological tumors with high expression of PCSK9.

[0062] Therefore, based on the conclusions of the above embodiments, the present application proposes the use of the anti-PCSK9 antibody of the above embodiments in reducing blood lipids and tumor immunotherapy. More generally, it includes PCSK9-related diseases in the art, that is, various diseases that can be treated, improved or prevented by blocking the binding of PCSK9 to LDLR. On the other hand, since the PCSK9 antibody of the present application can bind to human PCSK9 with high affinity and high selectivity, when the antibody of this embodiment is combined with a detection reagent (such as a fluorescent reagent), selective labeling and detection of PCSK9 can be achieved, and thus it can be applied in a detection kit for PCSK9.

[0063] In the description of this specification, the description with reference to terms such as "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0064] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. An anti-human PCSK9 antibody, characterized in that, Comprising: Sequence combination A: Comprising: Heavy chain variable region, with complementary determining regions HCDR1, HCDR2, HCDR3, and the sequences are respectively: GYTFSSYW, ILRGSGST, ASRGYGYDY; And, light chain variable region, with complementary determining regions LCDR1, LCDR2, LCDR3, and the sequences are respectively: QNVNTD, SAS, HQYNNYPYT; Sequence combination B: Comprising: Heavy chain variable region, with complementary determining regions HCDR1, HCDR2, HCDR3, and the sequences are respectively: GYAFTDYY, IWPGRVNT, SEQ NO ID.21; And, light chain variable region, with complementary determining regions LCDR1, LCDR2, LCDR3, and the sequences are respectively: QDINTY, RAN, LQYDDFPWT; Sequence combination C, comprising: Heavy chain variable region, whose complementary determining regions HCDR1, HCDR2, HCDR3 have more than 90% identity with the HCDR1, HCDR2, HCDR3 of the said sequence A; And, light chain variable region, whose complementary determining regions LCDR1, LCDR2, LCDR3 have more than 90% identity with the LCDR1, LCDR2, LCDR3 of the said sequence A; Or, sequence combination D, comprising: Heavy chain variable region, whose complementary determining regions HCDR1, HCDR2, HCDR3 have more than 90% identity with the HCDR1, HCDR2, HCDR3 of the said sequence B; And, light chain variable region, whose complementary determining regions LCDR1, LCDR2, LCDR3 have more than 90% identity with the LCDR1, LCDR2, LCDR3 of the said sequence B.

2. The anti-human PCSK9 antibody sequence according to claim 1, characterized in that, The sequences of the heavy chain variable region and the light chain variable region of the said sequence combination A are SEQ ID NO.1 and SEQ ID NO.2 respectively; The sequences of the heavy chain variable region and the light chain variable region of the said sequence combination B are SEQ ID NO.3 and SEQ IDNO.4 respectively.

3. A nucleic acid, characterized in that, Encoding the anti-human PCSK9 antibody as claimed in claim 1 or 2.

4. A recombinant vector, characterized in that, Carrying the nucleic acid as claimed in claim 3.

5. A cell, characterized in that, Capable of expressing the recombinant vector as claimed in claim 4.

6. A method for preparing the anti-human PCSK9 antibody according to claim 1 or 2, characterized in that, Comprising the following steps: Introducing the recombinant vector as claimed in claim 4 into a cell; Culturing the said cell to obtain the method for the anti-human PCSK9 antibody.

7. A drug for treating PCSK9-related diseases, characterized in that, Comprising the anti-human PCSK9 antibody as claimed in claim 1 or 2, the nucleic acid as claimed in claim 3 or the vector as claimed in claim 4.

8. A kit for detecting PCSK9, characterized in that, Comprising the anti-human PCSK9 antibody as claimed in claim 1 or 2.

9. Use of the anti-human PCSK9 antibody as claimed in claim 1 or 2, the nucleic acid as claimed in claim 3 or the vector as claimed in claim 4 in the preparation of a medicament for treating PCSK9-related diseases.

10. Use of the anti-human PCSK9 antibody as claimed in claim 1 or 2 in the preparation of a kit for detecting PCSK9.

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