CN118005787A

CN118005787A - Anti-human PD-1 monoclonal antibody and preparation method and application thereof

Info

Publication number: CN118005787A
Application number: CN202211497428.2A
Authority: CN
Inventors: 赵同标; 程晨曦; 曹佳妮; 李晓燕; 张琳
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2022-11-08
Filing date: 2022-11-08
Publication date: 2024-05-10

Abstract

The invention belongs to the technical field of antibody preparation, and discloses an anti-human PD-1 monoclonal antibody which has strong specificity and higher affinity, can effectively block the combination of PD-1 and PDL-1, effectively activate CD4T cells and CD8T cells, obviously stimulate the secretion of IL-2 and IFN-gamma by the CD8T, and enhance the killing effect of the T cells on tumor cells.

Description

Anti-human PD-1 monoclonal antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-human PD-1 monoclonal antibody, and a preparation method and application thereof.

Background

The transmembrane receptor PD-1 (programmed CELL DEATH, programmed factor 1) is one of the CD28 gene family members, PD-1 being expressed predominantly in macrophages, NK cells, B cells, activated T cells and dendritic cells, but its expression is up-regulated in depleted T cells. The ligands PD-L1 and PD-L2 of PD-1 belong to the B7 superfamily, PD-L1 is highly expressed in inflammatory tissues and cancer cells, and PD-L2 is expressed only in B lymphocytes and antigen presenting cells such as dendritic cells and macrophages, which play an important role in T cell clearance of viruses and bacteria. PD-1 and PD-L1 binding is primarily through PI3K/AKT and Ras/MEK/ERK signaling pathways, which impair T cell proliferation, differentiation and activation during effector phase. In addition to tumor cells, PD-L1 can be expressed on tumor-infiltrating lymphocytes (TILs), including tumor-associated macrophages (TAMs), dendritic Cells (DCs), regulatory T cells (tregs), cancer-associated fibroblasts (CAFs), mast cells, and tumor stromal cells (astrocytes, vascular endothelial cells, keratinocytes). Thus, overexpression of PD-L1 on tumor cells, TIL or tumor stromal cells inevitably triggers T cell failure, creating an immunosuppressive microenvironment and promoting tumor growth. PD-1 is only expressed on activated T cells, and a PD-1 immune checkpoint can play a better treatment effect in solid tumors, and clinical results show that targeting PD-1/PD-L1 can effectively enhance anti-tumor immune response, and at present, development of a novel anti-PD-1 antibody with a better treatment effect is still needed to effectively block the combination of PD-1 and PD-L1 for clinical treatment.

Disclosure of Invention

The invention prepares a novel and specific PD-1 monoclonal antibody.

In the invention, a PD-1 over-expressed cell line is used as an immunogen to immunize a mouse, hybridoma cells are obtained by fusion of spleen cells and myeloma cells of the mouse, a large number of hybridoma cell lines are obtained by screening, the hybridoma can produce a monoclonal antibody specifically combined with the PD-1, can effectively block the combination of the PD-1 and the PD-L1, and can be applied to anti-tumor, anti-infection and autoimmune diseases.

The invention provides a monoclonal antibody for encoding anti-PD-1, which can be a full-length sequence of the antibody or a fragment of the anti-PD-1 antibody, wherein the protein and the antibody comprise recombinant protein, recombinant antibody, scFv antibody, humanized antibody, chimeric antibody, bispecific antibody single domain antibody, ADCC conjugated antibody and protein, and the antibody can further provide a derivative of the anti-hPD-1 antibody, wherein the derivative is a fragment of hPD-1 antibody, an antibody/antibody fragment-factor fusion protein and an antibody/antibody fragment-chemical conjugate.

The invention newly discovers that 1 monoclonal antibody 8A8 with remarkably high affinity to hPD-1 can not only specifically bind to hPD-1 antigen, but also block the binding capacity of hPD-1 to its ligand when EC ₅₀ is similar to that of a reference. The invention obtains the gene sequence of the target antibody from the monoclonal cell strain, can be used for constructing a eukaryotic expression vector, and can reconstruct the activity of the antibody after expression to obtain the anti-hPD-1 monoclonal antibody.

The anti-hPD-1 monoclonal antibody can be used for preparing anti-tumor (including lung cancer, liver cancer, breast cancer, squamous cell carcinoma, ovarian cancer, colorectal cancer, gastric cancer, gastrointestinal stromal tumor, bladder cancer, thyroid cancer, melanoma, neck cancer and prostate cancer which are highly expressed hPD-1) medicines, anti-infective (infectious diseases including diseases caused by HIV, HBV, HCV and other virus infections) medicines and medicines for preparing medicines for treating autoimmune diseases (including systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, psoriasis, multiple sclerosis and ulcerative colitis).

The advantages of the present invention are mainly, but not limited to, the following:

The antibody has high affinity to hPD-1. The high-efficiency expression in animal cells can be used for industrial production. Experiments prove that the anti-hPD-1 monoclonal antibody can block hPD-1 receptor from combining with the ligand PD-L1 thereof, so that the antibody has wide application prospect in the treatment of tumors, infectious diseases and autoimmune diseases.

Drawings

Fig. 1: binding assays to the 8A8 antibody and 3T3-hPD-1 cell lines were performed in a flow assay.

Fig. 2: detection of 8A8 antibody EC ₅₀.

Fig. 3: detection of 8A8 antibody affinity.

Fig. 4: detection of 8A8 antibody IC ₅₀.

Fig. 5: post-electrophoresis images were obtained by PCR of the 8A8 antibody heavy chain light chain variable region.

Fig. 6: effects of the combination of 8A8 antibody and CAR-T therapy on IL-2 secretion and IFN-gamma secretion by CAR-T cells.

Fig. 7: effect of 8A8 antibody and CAR-T therapy in combination on CAR-T cell killing tumors.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be more clearly and completely described in the following in conjunction with specific embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present application without making any inventive effort, shall fall within the scope of the present application.

Example 1

3T3 cell membrane surface over-expression hPD-1 protein

In order to obtain a cell strain which over-expresses hPD-1 protein on the surface of a 3T3 cell membrane as an immunogen, cDNA clone (Genbank accession No. NM-005018.2) of the full-length open reading frame of the human PD-1 is directionally inserted into a PCDH vector, a core plasmid and a packaging plasmid are transfected into 293T cells after the sequencing is correct, viruses are harvested after 48 hours of transfection, the obtained lentivirus is infected with the 3T3 cells, the obtained lentivirus is subjected to liquid exchange after 12 hours of infection, and the 3T3 cells which stably express hPD-1 are obtained by flow sorting after 48 hours and are named as 3T3-hPD-1.

Example 2

Immunization of animals

15 Female BALB/c mice, 6-8 weeks old, were housed in 3 cages, divided into 3 groups, and 3T3 cells and 3T3-hPD-1 cell lines were immunized against the mice, and 5 other mice were injected subcutaneously with PBS by the same method as negative controls. The method comprises the following specific steps:

(1) First, 3T3 and 3T3-hPD-1 cell lines 1X 10 ⁷/mouse were immunized by subcutaneous injection at 200. Mu.L.

(2) Two weeks later, each of the 3T3 and 3T3-hPD-1 cell lines 1X 10 ⁷ was subjected to the 2 nd immunization by the same method, and the third immunization was performed in the same manner after two weeks.

(3) The immunized mice were subjected to orbital blood collection one week after the 3 rd immunization, placed at 37℃for 2 hours, left at 4℃overnight, centrifuged at 4℃for 20min to separate serum, and the serum titers were determined by an indirect ELISA method.

(4) Mice with highest serum titers were selected for 2X 10 ⁷/mouse intraperitoneal injection 3 days before fusion, boosted, and after 3 days of final immunization, 2 mice with high titers were selected for cell fusion. The spleens of the mice and the sp2/0 cells prepared in advance were taken out aseptically, and after cell counting, the mixture was mixed in a ratio of 10:1, and 50% PEG Solution was added for cell fusion.

Example 3

Cell fusion

1. Preparation of feeder cells

(1) When the cells are fused, a BALB/c mouse which is not immune and has good health condition is used for preparing feeder cells, firstly, the orbit is exsanguinated, the serum of the mouse is collected as negative serum, then, the mouse is subjected to neck removal and sacrifice, and the mouse is soaked in 75% alcohol solution for 10min.

(2) After fixing the limbs of the mice, the abdominal skin was gripped with forceps. The scissors are opened gently, the peritoneum is exposed, a small opening is cut on the peritoneum, 2mL of RPM 1640 basic solution is added into the abdominal cavity of the mouse by using a pipette, the operation is repeated for several times, the solution is placed into a 50mL centrifuge tube, and the solution in the 50mL centrifuge tube is the abdominal cavity macrophage.

(3) The supernatant was removed by centrifugation at 1000r/min for 10min, and the cells were suspended in HAT medium and added to a cell culture flask, and cultured in a 5% C0 ₂ incubator at 37℃for cell fusion.

2. Preparation of spleen cells and cell fusion

Fusion process:

(1) The BALB/c mice with the enhanced immunity are killed by orbital exsanguination, the orbital blood is collected and serum is prepared, and the mice are soaked in 75% alcohol for disinfection.

(2) The sterilized mice were fixed to the dissecting plate, and the spleen cells were removed and added to a sterilized homogenizer prepared in advance.

(3) Adding 5mL of RPMI1640 base solution into a homogenizer, separating spleen cells by using a homogenizing rod, dripping 10mL of the RPMI1640 base solution, standing for 2min, transferring the supernatant into another sterile 50mL centrifuge tube, then adding 10mL of the RPMI1640 base solution, repeating the same steps for 2 times, taking prepared SP2/0 cells, discarding the culture medium and flushing 2 times by using a proper amount of the RPMI1640 base solution, adding 5mL of the RPMI1640 base solution, blowing down the SP2/0 and counting, transferring 1-2X 10 ⁷ SP2/0 cells into a 50mL centrifuge tube containing the spleen cells, fully mixing, 1000r/min, and centrifuging for 8min.

(4) The supernatant was discarded and placed in a 37℃water bath. 50% PEG, 0.8mL, preheated in advance, was then slowly added, after 1min, with constant stirring.

(5) The mixture was stirred continuously for 30s, allowed to stand for 30s, and then the fusion reaction was terminated by slowly adding a preheated RMPI1640 stop solution at 37 ℃.

(6) Centrifuging at 1000r/min for 5min, removing supernatant, mixing HAT medium containing abdominal cavity macrophage with fused cells, adding appropriate amount of HAT medium, spreading into 96-well cell culture plate, and culturing in cell culture box at about 250 μl/well.

3. Indirect ELISA screening of positive hybridoma cells

The following day after fusion was observed for contamination and HT medium was changed after 4 days. The colony of the cells to be fused can be selected by taking 100 mu L of cell culture supernatant from each well under aseptic condition, and initially screening positive hybridoma cells by using purified ex-hPD-1 protein as coating antigen by using an indirect ELISA method, and simultaneously setting positive control, negative control and blank control. And selecting fusion cells with higher OD450nm values according to ELISA results to perform cell subcloning, and simultaneously, timely amplifying and freezing when hybridoma cells have higher positive values. After 3 times of subcloning, positive cells with high OD450nm value are selected for gradual expansion culture, and the collected positive hybridoma cell supernatant is subjected to indirect ELISA detection by taking ex-hPD-1 protein as a coating antigen.

4. Subcloning of hybridoma cells

The cells in the wells were gently blown up by adding an appropriate amount of culture medium to the wells with higher OD450nm measurement, and the cells were diluted ten-fold in a 1.5mL EP tube and counted by a cell counting plate. 100 cells are sucked by a pipette for the 1 st subcloning, diluted by 10mL Clone easy culture solution, added to a 96-well cell culture plate for re-culture after being uniformly mixed, 100mL is added to each well, and the rest cells are frozen in time. After the first subcloning, when the cell culture solution turns yellow, the culture solution in the hole is sucked for indirect ELISA detection and the 2 nd subcloning, the 3rd subcloning and the 1 st subcloning are subcloned by the same method, and after the 3rd subcloning, if all the cell hole detection results are positive, the positive hybridoma cell strain capable of stably secreting the anti-human PD-1 protein is obtained, and the hybridoma cells are timely amplified and frozen for preservation.

5. Preparation of ascites from hybridoma cells

The mice with 8-10 weeks age are sensitized by sterilized paraffin, after one week, hybridoma cells in good growth state are blown up by PBS, centrifuged for 5min, repeated three times, cell sediment is suspended and diluted by PBS, 1x10 ⁶/hybridoma cells are injected by abdominal cavity, ascites of the mice are extracted after one week, supernatant is centrifugally taken, and the titer is measured and stored at-80 ℃.

6. Purification of antibodies

The specific experimental steps are as follows:

The collected ascites is filtered by a 0.45 mu M filter, equal amount of PBS is added, the purification is carried out by a AKATA instrument, the heteroprotein is eluted by PBS after the sample is loaded, the antibody is eluted by Elusion Buffer of 0.1M Gly PH=2.7 after 10 column volumes of PBS are used, 150 mu L of neutralization Buffer (0.1M Tris PH=9.0) is added into a sample collecting tube, the collected sample is concentrated by an ultrafiltration concentration tube and replaced by PBS, and 30% -50% of glycerol is added into the purified antibody for long-term storage at-20 ℃ or-80 ℃.

Example 4

Identification of antibody function

1.8A8 antibody to 3T3-hPD-1 cell line flow assay

The 3T3-hPD-1 cell line was digested with pancreatin, diluted in 1X 10 ⁶ tube, resuspended in 100. Mu.L of PBS, the 8A8 antibody was added at 1. Mu.g/mL while positive and negative antibodies were added, reacted at 4℃for 40min, washed twice with PBS containing 2% FBS, FITC-labeled goat anti-mouse secondary antibody was added, reacted at 4℃for 20min, washed twice with PBS containing 2% FBS, and split into flow tubes for flow analysis using a BD FACS Aria instrument. As shown in FIG. 1, the binding of 8A8 antibody to the 3T3-hPD-1 cell line was detected in a flow assay, which showed that 8A8 antibody was effective in recognizing the 3T3-hPD-1 cell line.

Binding of 2.8A8 antibodies to PD-1 recombinant proteins

Ex-hPD-1 was coated onto 96-well ELISA plates: purified antibodies were 10-fold diluted at 10 ³ μg/mL to 10 ^-4 each with three replicates, positive, negative and blank controls were set simultaneously, igG-FC-HRP was secondary antibody, after addition of chromogenic and stop solutions, OD450nm was read with BioTek microplate reader and EC50 concentrations were calculated using Graphpad software. As shown in FIG. 2, the 8A8 antibody EC ₅₀ was tested with EC ₅₀ of 0.0001391 μg/mL.

3. Affinity detection

The method comprises the steps of detecting the monoclonal antibody 8A8 antibody affinity Chip of the invention by adopting the Biacore technology, selecting CM5 Chip, carrying out the Chip surface pretreatment, carrying out antibody fixation by adopting an amino coupling mode, carrying out sample regeneration, determining the concentration gradient and dissociation time of antigen sample injection before sample injection, determining post-dilution antigen, carrying out measurement, and carrying out software analysis to generate dissociation constant KD after all procedures are completed. As shown in FIG. 3, the dissociation constant KD of the detection of the affinity of the 8A8 antibody is 1.05X10 ^-9.

4.8A8 antibody and PD-L1 Competition ELISA

ELISA plates were coated with PD-1-FC at a concentration of 1. Mu.g/mL, the purified antibodies were diluted 2-fold at 50. Mu.g/mL, each group was repeated three times, positive control, negative control and blank control were simultaneously set, the reaction was performed at 4℃for 1h, botin-PD-L1 was diluted at a concentration of 10. Mu.g/mL, 10. Mu.L/well, mixed well, reacted at 37℃for 1h, PBST washed 3 times, HRP-labeled streptomycin 1:400 was diluted, reacted at 37℃for 1h, PBST washed 3 times, after addition of chromogenic solution and stop solution, OD450nm was read with BioTek microplate reader, and IC ₅₀ concentration was calculated with Graphpad software. As shown in FIG. 4, the detection of the 8A8 antibody IC ₅₀, IC ₅₀, was 3.101 μg/mL.

Example 5

The heavy chain and light chain variable region genes of the antibodies with blocking effect were amplified and sequenced with reference to the instructions of the mouse antibody ScFv kit (PPL, P001Z); as shown in FIG. 5, the 8A8 antibody heavy chain light chain variable region PCR was obtained and then electrophoresed (nucleic acid molecular weight standard: DL5000 Marker). The heavy chain variable region, the light chain variable region and the CDR sequences of the 8A8 antibody are shown in the sequence table.

Example 6

1. Secretion of cytokines

CAR-T centrifugation 1500r/min was performed for 5min, after centrifugation, the HCC827, H23, a549 cells were digested with pancreatin after 24H, HCC827, H23, a549, K562 cells were collected, resuspended at 1×10 ⁵/mL, diluted cells were seeded at 50 μl in U96-well plates, CAR-T cells were centrifuged at 1500r/min for 5min, after centrifugation, resuspended at 4×10 ⁵/mL with X-VIVO medium and medium containing 8A8 antibody, diluted cells were seeded at 50 μl in U96-well plates containing tumor cells, three replicates were set per set, after seeding, after centrifugation at 1500r/min for 3min, cultured at 37 ℃ 5% co ₂, 48, 72H and cell culture supernatant was collected to detect IL-2, ifn- γ. Before ELISA detection, all reagents and samples are balanced to room temperature, 300 μl of 1 Xwashing liquid is added for standing and soaking for 30s, PBST is removed, 100 μl of standard substance diluted by 2 times is added into standard substance holes, 100 μl of standard substance culture medium is added into blank holes, and 100 μl of cell culture supernatant is added into sample holes. 50 μl of diluted detection antibody (1:100) was added to each well and the plates were sealed using a seal plate membrane. Incubating for 2 hours at room temperature of 100-300 r/min. The liquid was discarded, 300. Mu.l of PBST was added to each well, and washed 6 times. 100. Mu.L of diluted horseradish peroxidase-labeled streptavidin (1:100) was added to each well, shaken at 100-300r/min and incubated for 45min at room temperature. The liquid was discarded, 300. Mu.l of PBST was added to each well, and washed 6 times. mu.L of chromogenic substrate TMB was added to each well and incubated at room temperature for 5-30min in the absence of light. 100 mu L of stop solution is added into each hole, and the mixture is fully and uniformly mixed. Within 30min, a dual wavelength detection was performed using an enzyme-labeled instrument, and the maximum absorption wavelengths of 450nm and 570nm were determined. As shown in fig. 6, 8A8 antibody in combination with CAR-T cells was effective in increasing secretion of CAR-T cells IL-2 and IFN- γ.

2. Antibody combined T cell in vitro killing tumor

CAR-T cells and Control-T cells were centrifuged at 1500r/min for 5min, after centrifugation, resuspended in 5% fbs X-VIVO medium without IL-2 and the beads were removed for 24H, after 24H HCC827, H23, a549 were digested with pancreatin, HCC827, H23, a549 and K562 cells were collected, resuspended at 1×10 ⁵/mL, diluted cells were inoculated to a white opaque 96-well plate at 50 μl, CAR-T cells and Control-T cells were centrifuged at 1500r/min for 5min, after centrifugation, resuspended in X-VIVO medium and medium with 8A8 antibody at 2×10 ⁵/mL,4×10⁵/mL,8×10⁵/mL,16×10⁵/mL, diluted cells were inoculated to a 96-well plate with tumor cells at 50 μl, three replicates were set up per experiment, after inoculation, centrifuged at 3min at 37 ℃ for 5% co ₂ for 16-18H, after incubation, D-substrate was added to a light-sensitive substrate at 150 μl per well for 25 μl of substrate, and a substrate was detected at a light-shielding value of Bioluminescence μl per well. As shown in fig. 7, the results demonstrate that the 8A8 antibody can effectively increase the killing of tumor cells by CAR-T cells.

Calculating the killing percentage, wherein the Bioluminescence value of the co-hatched hole of the tumor cells and the Control T cells is used as a background value, and the Bioluminescence value of the decrease of the co-hatched hole of the CAR-T cells and the tumor cells is used as the killing efficiency of the T cells. The calculation formula is as follows: killing efficiency (%) = (background value-experiment Kong Shuzhi)/background value) ×100

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds human PD-1, wherein the antibody or antigen-binding fragment thereof comprises 3 CDRs of a light chain variable region and 3 CDRs of a heavy chain variable region, wherein the 3 CDRs of the light chain variable region are: SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, the 3 CDRs of the heavy chain variable region are: SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the sequence of the light chain variable region is set forth in SEQ ID No. 7.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region has the sequence set forth in SEQ ID No. 8.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region has a coding sequence as set forth in SEQ ID No. 9.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region has a coding sequence as set forth in SEQ ID No. 10.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antigen-binding fragment comprises, but is not limited to, fab ', F (ab') 2, fv, or scFV.

7. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-6, an expression cassette or host comprising the nucleic acid.

8. Use of the antibody or antigen binding fragment thereof of any one of claims 1-6 in the manufacture of a medicament for:

a) Improving the activation of immune cells,

B) Can be used for treating cancer, such as cancer,

C) Treating infection or infectious diseases.

9. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6 in the preparation of a diagnostic or detection reagent.

10. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-6.

11. The application according to claim 9, characterized in that it comprises: binding or blocking activity is determined by ELISA, FACS, surface plasmon resonance, GST pull down, epitope tag, immunoprecipitation, fluorescence resonance energy transfer, time resolved fluorescence immunoassay (TR-FIA), radioimmunoassay (RIA), enzyme immunoassay, latex agglutination, immunohistochemistry, or a combination of any two or more of the foregoing.