High-affinity anti-human and monkey PCSK9 antibody

Abstract

The invention discloses a high-affinity anti-human and monkey PCSK9 antibody, 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.13 and a light chain variable region of which the sequence is SEQ ID NO.14, or a heavy chain variable region of which the sequence is SEQ ID NO.15 and a light chain variable region of which the sequence is SEQ ID NO.16. 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 in particular to a high-affinity anti-human and monkey PCSK9 antibody. Background Art

[0002] Immunotherapy is currently an important means of treating malignant tumors after traditional treatments such as surgery, radiotherapy, and chemotherapy. Unlike traditional therapies, immunotherapy focuses on the body's own immune system to eliminate tumor cells by enhancing or rebuilding immune capacity. However, tumor metabolic reprogramming produces an inhibitory microenvironment, including nutrient deficiency, metabolite accumulation, acidity, and hypoxia, which mediate metabolic stress in microenvironmental immune cells and inhibit their anti-tumor function. This is a key factor and bottleneck problem that leads to tumor immune escape and the limited clinical responsiveness of existing immunotherapies in the treatment of solid tumors.

[0003] As a basic component of cell membranes, cholesterol metabolism remodeling in the tumor microenvironment leads to immunosuppression, which is a key factor in tumor immune escape. Tumor cells and tumor-associated macrophages (TAMs) consume cholesterol in the microenvironment, leading to tumor-infiltrating CD8 + T cells are cholesterol-deficient and eventually become dysfunctional and exhausted. Therefore, targeting cholesterol metabolism in the tumor microenvironment for cancer therapy has attracted much attention.

[0004] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is mainly expressed by the liver and is an important cholesterol metabolism homeostasis regulator. In the absence of PCSK9, the low-density lipoprotein receptor (LDLR) on the surface of hepatocytes binds to free low-density lipoprotein cholesterol (LDL-C) in the blood, mediates its endocytosis into cells for degradation and metabolism, and LDLR is transported back to the cell membrane for recycling. PCSK9 can bind to LDLR through the catalytic domain, mediate LDLR to enter lysosomes for degradation, thereby reducing the liver's ability to clear LDL-C, leading to increased LDL-C levels in the blood and inducing cardiovascular disease. Therefore, PCSK9 has become an important target for cardiovascular disease. In addition, studies in recent years have shown that targeting PCSK9 has shown broad application potential and development prospects in enhancing tumor immunotherapy. The research team's previous studies found that PCSK9 is highly expressed ectopically in tumor tissues and inhibits the anti-tumor activity of CD8T cells by blocking T cell receptor TCR recycling and TCR signals. Other studies have reported 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 tumor progression. Given the regulatory role of PCSK9 in tumor immune recognition, CD8 T cell anti-tumor function, and tumor cell proliferation and apoptosis, tumor treatment strategies targeting PCSK9 have important clinical translation 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, my country's Food and Drug Administration has approved the marketing of three monoclonal antibodies targeting PCSK9 for the treatment of cardiovascular diseases: Evolocumab, Alirocumab and Tafolecimab. However, there is still a demand for alternative PCSK9 antibodies in order to provide potential new clinical treatment drugs in the process of expanding indications. The present invention uses human PCSK9 protein to immunize mice, and obtains anti-human PCSK9 monoclonal antibodies with different binding epitopes from existing antibodies through hybridoma cell line screening and humanization, which can provide a potential new clinical treatment plan for the preparation of preparations with PCSK9 monoclonal antibodies as the core component to treat solid tumors or blood tumors with high PCSK9 expression. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a high-affinity anti-human and monkey PCSK9 antibody.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] The first aspect of the present invention relates to an anti-human PCSK9 antibody, comprising:

[0009] Sequence combination A: includes:

[0010] The heavy chain variable region, its complementarity determining regions HCDR1, HCDR2, and HCDR3, have the following sequences: GYTFSRYW, ILPGSISY, and ARRDYGRDY, respectively;

[0011] and, the light chain variable region, whose complementarity determining regions LCDR1, LCDR2, and LCDR3, have the sequences: QNVGTD, SAS, and HQYNSYPYT, respectively;

[0012] Sequence combination B: includes:

[0013] The heavy chain variable region, its complementarity determining regions HCDR1, HCDR2, and HCDR3, have the sequences: GFNIKDTY, IDPANGHI, and SEQ ID NO. 21 (ARSYYGSWFAY), respectively;

[0014] and, the light chain variable region, whose complementarity determining regions LCDR1, LCDR2, and LCDR3, have the following sequences: SSVTY, RTS, QQYHSYPPT, respectively;

[0015] Sequence combination C, including:

[0016] A heavy chain variable region, wherein the complementarity determining regions HCDR1, HCDR2, and HCDR3 thereof have more than 90% identity with the HCDR1, HCDR2, and HCDR3 of the sequence A;

[0017] and, a light chain variable region, wherein the complementarity determining regions LCDR1, LCDR2, and LCDR3 thereof have more than 90% identity with the LCDR1, LCDR2, and LCDR3 of the sequence A;

[0018] Alternatively, a sequence combination D comprising:

[0019] A heavy chain variable region, wherein the complementarity determining regions HCDR1, HCDR2, and HCDR3 thereof have more than 90% identity with the HCDR1, HCDR2, and HCDR3 of the sequence B;

[0020] And, the light chain variable region, its complementarity determining regions LCDR1, LCDR2, LCDR3 have more than 90% identity with the LCDR1, LCDR2, LCDR3 of the 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.13 and SEQ ID NO.14, respectively;

[0022] The sequences of the heavy chain variable region and the light chain variable region of the sequence combination B sequence are SEQ ID NO.15 and SEQ ID NO.16 respectively.

[0023] The second aspect of the present invention relates to a nucleic acid encoding the above-mentioned anti-human PCSK9 antibody.

[0024] The third aspect of the present invention relates to a recombinant vector carrying the above-mentioned nucleic acid.

[0025] The fourth aspect of the present invention relates to a cell capable of expressing the above-mentioned recombinant vector.

[0026] A fifth aspect of the present invention relates to a method of producing the above-mentioned anti-human PCSK9 antibody, comprising the following steps:

[0027] Introducing the above-mentioned recombinant vector into cells;

[0028] The cells are cultured to obtain the anti-human PCSK9 antibody.

[0029] The sixth aspect of the present invention relates to a drug for treating PCSK9-related diseases, comprising the above-mentioned anti-human PCSK9 antibody, the above-mentioned nucleic acid or the above-mentioned vector.

[0030] The seventh aspect of the present invention relates to a kit for detecting PCSK9, comprising the above-mentioned anti-human PCSK9 antibody.

[0031] The eighth aspect of the present invention relates to the use of the above-mentioned anti-human PCSK9 antibody, the above-mentioned nucleic acid or the above-mentioned vector in the preparation of a drug for treating PCSK9-related diseases.

[0032] The ninth aspect of the present invention relates to the use of the above-mentioned anti-human PCSK9 antibody in the preparation of a kit for detecting PCSK9.

[0033] Beneficial effects of the present invention:

[0034] The antibody of the present application can bind to human PCSK9 with high affinity and high selectivity, blocking its binding to LDLR on the cell plasma membrane and reducing the degradation of LDLR. It also binds to a different epitope of PCSK9 protein than Alirocumab, and can restore the cell surface LDLR level and the ability of cellular LDL uptake. Therefore, the anti-human PCSK9 antibody of the present application can provide a new treatment for PCSK9-related diseases and has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present invention will be further described below in conjunction with the accompanying drawings.

[0036] Figure 1 is the binding of mouse serum to human PCSK9;

[0037] Figure 2 The ability of the parent clone supernatant to restore LDLR;

[0038] Figure 3 The affinity of the subclone antibody in the examples of this application to human PCSK9;

[0039] Figure 4 The BLI method in the present application detects the binding of PCSK9 antibody to human / monkey / mouse PCSK9 protein;

[0040] Figure 5 The ELISA in the examples of this application detects the binding of PCSK9 antibodies to human / monkey / mouse PCSK9 proteins;

[0041] Figure 6 The epitopes of the human PCSK9 protein bound by the 6 PCSK9 antibodies in the examples of this application are different from those of Alirocumab;

[0042] Figure 7 The PCSK9 antibody in the examples of this application affects the cell surface LDLR level and LDL uptake. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions 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 creative work are within the scope of protection of the present invention.

[0044] (1) Animal immunization: Five female Balb / c and SJL mice aged 8 weeks were selected and immunized according to Table 1. Antigens enter the peripheral immune organs through the blood circulation or lymphatic circulation, stimulate the corresponding B lymphocyte clones, activate them, proliferate, and differentiate into sensitized B lymphocytes.

[0045] Table 1 Immunization process table

[0046]

[0047] (2) Serum detection: Blood was collected at the time points in Table 1, and serum was separated. Human PCSK9 protein (0.5 μg / ml, 100 μl / well) was coated in a 96-well plate to detect the immune status of mice. One Balb / c mouse and one SJL mouse were selected for hybridoma cell fusion ( Figure 1 ).

[0048] (3) Cell fusion and mother clone screening: Mouse plasma cells will be enriched for CD138 and then fused for two rounds by electrofusion. Each hybridoma cell obtained after fusion will be plated on about 30 96-well plates. All clones will be screened for human PCSK9 protein by ELISA. Positive mother clone supernatants will be selected based on the screening results for LDLR rescue experiments ( Figure 2 ).

[0049] (4) Subclone screening and antibody variable region sequencing: Up to 50 parent clones were subcloned by limiting dilution to ensure that each subclone was derived from a single parent clone. All subclones were screened for human PCSK9 protein and LDLR rescue experiments by ELISA. Ten subclones were selected for variable region sequencing and recombinant expression of the above antibodies (Table 2).

[0050] Table 2 Antibody variable region amino acid sequence

[0051]

[0052]

[0053]

[0054] (5) Affinity test: The above 10 antibodies were fixed using protein A biosensor, and their binding ability to human PCSK9 protein was detected by biomembrane interferometry (BLI). It was found that 6 of them could bind to human PCSK9 with high affinity, with a KD value of 10 -11 M( Figure 3 , Table 3).

[0055] Table 3 Affinity of subcloned antibodies to human PCSK9

[0056]

[0057] (6) Binding ability of PCSK9 antibodies to PCSK9 proteins of different species: BLI was used to detect the binding ability of the above 6 PCSK9 antibodies to human / monkey / mouse PCSK9 proteins. It was found that 4 of them could bind to monkey PCSK9 but not to mouse PCSK9 ( Figure 4, Table 4). The same conclusion was obtained by ELISA with human / monkey / mouse PCSK9 protein ( Figure 5 ).

[0058] Table 4 Affinity of PCSK9 antibodies to human / monkey / mouse PCSK9 proteins

[0059]

[0060] (7) Comparison of binding epitopes of PCSK9 antibodies and human PCSK9 protein: Biotinylated human PCSK9 protein was immobilized using a streptavidin (SA) biosensor and incubated with Alirocumab until it no longer bound, and then incubated with the above six PCSK9 antibodies (Alirocumab was used as a control). It was found that the epitopes of human PCSK9 bound by these six antibodies were inconsistent with that of Alirocumab ( Figure 6 The same conclusion was obtained by ELISA with biotinylated human PCSK9 protein ( Figure 6 B).

[0061] (8) PCSK9 antibody function verification: Using HepG2 cells as model cells, five 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 restore the cell surface LDLR level ( Figure 7 A), and can promote the uptake of LDL by cells ( Figure 7 We also tested whether the above antibodies can regulate the level of LDLR on the surface of T cells in Jurkat T cells, and the results were basically consistent with those in HepG2 ( Figure 7 C).

[0062] In summary, the present invention aims to develop new anti-human PCSK9 antibodies in order to provide potential new clinical therapeutic drugs in the process of expanding indications. To this end, the present invention, on the one hand, prepares high-affinity PCSK9 antibodies, discloses several anti-human PCSK9 antibodies and antigen-binding fragments thereof, including heavy chain variable regions (VH) and light chain variable regions (VL), wherein the VH includes antigenic determining regions VH CDR1, VH CDR2 and VH CDR3, and the VL includes antigenic determining regions VL CDR1, VL CDR2 and VL CDR3; on the other hand, the binding ability of the above-mentioned PCSK9 antibodies to human, monkey and mouse PCSK9 proteins is explored, the difference between the above-mentioned PCSK9 antibodies and human PCSK9 protein binding epitopes and Alirocumab is compared, and the ability of the above-mentioned PCSK9 antibodies to restore cell surface LDLR levels and cell LDL uptake is detected. The present invention can provide a potential new clinical treatment scheme for the preparation of preparations with PCSK9 monoclonal antibodies as the core component for the treatment of solid tumors or blood tumors with high PCSK9 expression.

[0063] Therefore, based on the conclusions of the above embodiments, the present application proposes the use of the anti-PCSK9 antibodies of the above embodiments in lipid-lowering and tumor immunotherapy. More broadly speaking, 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), PCSK9 selective labeling and detection can be achieved, and then it can be used in a detection kit for PCSK9.

[0064] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction 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 any one or more embodiments or examples in a suitable manner.

[0065] 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 to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. An anti-human PCSK9 antibody, characterized in that include: Sequence combination A: includes: The heavy chain variable region, its complementarity determining regions HCDR1, HCDR2, and HCDR3, have the following sequences: GYTFSRYW, ILPGSISY, and ARRDYGRDY, respectively; and, the light chain variable region, whose complementarity determining regions LCDR1, LCDR2, and LCDR3, have the sequences: QNVGTD, SAS, and HQYNSYPYT, respectively; Sequence combination B: includes: The heavy chain variable region, its complementarity determining regions HCDR1, HCDR2, and HCDR3, have the sequences: GFNIKDTY, IDPANGHI, SEQ ID NO.21, respectively; and, the light chain variable region, whose complementarity determining regions LCDR1, LCDR2, and LCDR3, have the following sequences: SSVTY, RTS, QQYHSYPPT, respectively; Sequence combination C, including: A heavy chain variable region, wherein the complementarity determining regions HCDR1, HCDR2, and HCDR3 thereof have more than 90% identity with the HCDR1, HCDR2, and HCDR3 of the sequence A; and, a light chain variable region, wherein the complementarity determining regions LCDR1, LCDR2, and LCDR3 thereof have more than 90% identity with the LCDR1, LCDR2, and LCDR3 of the sequence A; Alternatively, a sequence combination D comprising: A heavy chain variable region, wherein the complementarity determining regions HCDR1, HCDR2, and HCDR3 thereof have more than 90% identity with the HCDR1, HCDR2, and HCDR3 of the sequence B; And, the light chain variable region, its complementarity determining regions LCDR1, LCDR2, LCDR3 have more than 90% identity with the LCDR1, LCDR2, LCDR3 of the 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 sequence combination A are SEQ ID NO.13 and SEQ ID NO.14 respectively; The sequences of the heavy chain variable region and the light chain variable region of the sequence combination B sequence are SEQ ID NO.15 and SEQ ID NO.16 respectively.

3. A nucleic acid, characterized in that Encodes the anti-human PCSK9 antibody according to claim 1 or 2.

4. A recombinant vector, characterized in that: Carrying the nucleic acid of claim 3.

5. A cell, characterized in that A recombinant vector capable of expressing the recombinant vector of claim 4.

6. A method for preparing the anti-human PCSK9 antibody according to claim 1 or 2, characterized in that: The following steps are involved: Introducing the recombinant vector according to claim 4 into a cell; The cells are cultured to obtain the anti-human PCSK9 antibody.

7. A drug for treating PCSK9-related diseases, characterized in that: It comprises the anti-human PCSK9 antibody according to claim 1 or 2, the nucleic acid according to claim 3 or the vector according to claim 4.

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

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

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

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