CN116396387A

CN116396387A - PD-L1 monoclonal antibody, heavy chain, light chain variable region, monoclonal cell strain, application and kit

Info

Publication number: CN116396387A
Application number: CN202211559839.XA
Authority: CN
Inventors: 周嘉颖; 徐震宇; 林晓东
Original assignee: Guangzhou Zhaorui Medical Biotechnology Co ltd
Current assignee: Guangzhou Zhaorui Medical Biotechnology Co ltd
Priority date: 2021-12-07
Filing date: 2022-12-06
Publication date: 2023-07-07
Also published as: CN114057879A

Abstract

The present application relates to the field of monoclonal antibodies, and more particularly, to PPD-L1 monoclonal antibodies, heavy chain, light chain variable regions, monoclonal cell lines, and uses and kits, the heavy chain variable regions comprising the following complementarity determining regions: CDRH1: SYWIE; CDRH2: EILPGSGNTNYNEKLKG; CDRH3: ERRGDY; the light chain variable region comprises the following complementarity determining regions: CDRL1: SASQGIYNYLN; CDRL2: YTSSLHS; CDRL3: QQYSKLPYT. The antibody in the application has good specificity and binding property to PD-L1 antigen, and has wide application prospect.

Description

PD-L1 monoclonal antibody, heavy chain, light chain variable region, monoclonal cell strain, application and kit

The present application claims priority to chinese patent application No. 202111485905.9 (invention name "PD-L1 monoclonal antibody, heavy chain, light chain variable region, monoclonal cell line and uses and kits") filed at 12 and 7 of 2021. Which is incorporated by reference herein in its entirety.

Technical Field

The present application relates to the field of monoclonal antibodies, and more particularly, to PD-L1 monoclonal antibodies, heavy and light chain variable regions, monoclonal cell lines, and uses and kits.

Background

Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule with two natural ligands, PD-L1 and PD-L2, respectively, and PD-L1 can detect expression in a variety of tumor cells, such as non-small cell lung cancer, malignant melanoma, gastric cancer, breast cancer, esophageal cancer, glioma, and the like. Therefore, the method has important clinical significance for PD-L1 serving as a target point for tumor treatment and diagnosis.

Currently, the method commonly used in the market for detecting PD-L1 is an immunohistochemical method, wherein PD-L1 antigen and retrograde immunorecognition are mainly performed by a PD-L1 antibody, and the antigen-antibody is developed by other developers, so that the PD-L1 is characterized. Meanwhile, through specific targeting of PD-L1 targets, the method has good potential of targeted drug administration, so that the development of the PD-L1 antibodies with high affinity and high specificity has important significance in the aspects of accurate diagnosis and reasonable drug administration.

Disclosure of Invention

In order to provide the PD-L1 monoclonal antibody, the application has the advantages of high affinity and high specificity, and simultaneously, the application provides a heavy chain variable region, a light chain variable region, a monoclonal cell strain, application and a kit of the monoclonal antibody.

First, the present application provides a heavy chain and a light chain variable region of a PD-L1 monoclonal antibody, wherein the heavy chain variable region comprises the following complementarity determining regions:

CDRH1：SYWIE；

CDRH2：EILPGSGNTNYNEKLKG；

CDRH3：ERRGDY；

the light chain variable region comprises the following complementarity determining regions:

CDRL1：SASQGIYNYLN；

CDRL2：YTSSLHS；

CDRL3：QQYSKLPYT。

alternatively, the heavy chain sequence is shown as SEQ ID No.2, and the light chain sequence is shown as SEQ ID No. 3.

The heavy chain and light chain variable regions with the complementarity determining regions have better binding and selectivity for the antigen, and have significant advantages in the field of practical detection and diagnostic treatment.

Meanwhile, the application also provides PD-L1 monoclonal antibodies, comprising the heavy chain and light chain variable regions.

The antibody with the heavy chain and light chain variable regions has the advantages of high selectivity, better affinity and specificity, high titer, high sensitivity, good linearity and the like.

The antibody having the complementarity determining region may be a murine antibody, a chimeric human-murine antibody may be used with a variable region having the complementarity determining region, or a humanized modified antibody may be used with the complementarity determining region.

Meanwhile, the above antibody may be subjected to surface remodeling, or modified or substituted for the amino acid sequence of a non-complementarity determining region portion thereof.

The antibody can be IgM, igD, igG, igA and IgE type

Alternatively, the heavy chain constant region is a murine IgG1 constant region.

The sequence of the heavy chain is shown as SEQ ID No.4, and the sequence of the light chain is shown as SEQ ID No. 5.

The antibody has high affinity and high specificity, and has better linearity and sensitivity through experimental formalism.

Optionally, the antibody is humanized.

After humanized transformation, the human immune system can be activated, the HAMA reaction is avoided being induced, and the method is suitable for immunotherapy.

Humanized alterations include replacement of the constant region by a human source, or direct implantation of a complementarity determining region into a human antibody in place of the complementarity determining region of a human antibody, or other modifications that help reduce HAMA reactions without affecting the specific binding properties of the antibody.

In addition, the application also provides a monoclonal cell strain for expressing the monoclonal antibody.

The monoclonal cell line can be a fusion cell of B cells and myeloma cells after antigen activation of rodents, or can be other cell lines with the capacity of expressing the antibodies.

In addition, the application also provides application of the PD-L1 monoclonal antibody, which is applied to the field of expression detection of PD-L1 antigen or immune killing of tumor cells.

The antigen expression detection can be an enzyme-linked immunosorbent assay, an immunohistochemical assay or other assay methods using antigen and antibody quality detection combined with each other, and also comprises an assay method for forming sandwich pairing between the monoclonal antibody and other monoclonal antibodies and detecting the antigen.

The immune killing field can be used for different areas in vivo and in vitro, and the action targets can be human, animals except human, cells, stops, organs or other bioactive components, and aims at inhibiting, killing, inducing apoptosis, limiting diffusion, relieving symptoms and the like of tumor bodies, tumor cells and cancer cells.

In addition, the application also provides a kit comprising the monoclonal antibody or any monoclonal antibody containing the heavy chain and light chain variable regions.

The kit can be an ELISA kit, an immunofluorescence kit or other kits for detecting products after the monoclonal antibodies or the monoclonal antibodies containing the heavy chain and the light chain variable regions react with antigens in a specific immunoreaction way, and has the advantages of high specificity and high sensitivity, and has a good application prospect.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of PD-L1 antigen before purification and after purification;

FIG. 2 is an SDS-PAGE electrophoresis of antibody 1 and antibody 2, wherein antibody 2 is purified and antibody 1 is purified in this order from left to right;

FIG. 3 is a linear analysis of antigen titers detected by sandwich immunoreactions of antibody 1 and antibody 2.

Detailed Description

The present application is described in further detail below with reference to the drawings and examples.

Example 1, PD-L1 monoclonal antibody, heavy chain, light chain variable region, monoclonal cell line and use, wherein the sequence of PD-L1 monoclonal antibody is specifically shown in Table 1.

Table 1, sequence listing of antibody PD-L1

The PD-L1 monoclonal antibody is prepared by the following steps.

1. Construction of PD-L1 expression vectors

The N-terminal (1-238 aa) amino acid fragment was selected from the gene sequence (NP-054862) of human PD-L1 in the National Center for Biotechnology Information (NCBI), cloned into pcDNA3.1 (+) expression vector by gene synthesis, and finally constructed as pcDNA3.1 (+) -PD-L1 vector by synthesis. Meanwhile, during gene synthesis, 6 histidine (His) tags are added at the C end of the PD-L1 amino acid, the molecular weight of the recombinant PD-L1 expressed by the vector is about 32KD, and the amino acid sequence is SEQ ID NO. 1.

2. Expression of immunogenic cells

1. Transformation and extraction of plasmids

The pcDNA3.1 (+) -PD-L1 vector is transformed into competent cells of escherichia coli DH5 alpha (Tiangen Co.) and uniformly spread on an LB agarose plate containing 100ug/ml ampicillin, and cultured overnight in an incubator at 37 ℃; selecting a monoclonal colony on a flat plate, adding the colony into 5ml of LB medium containing 100ug/ml ampicillin, and continuously culturing by a constant-temperature shake culture shaker (250 rpm,37 ℃) for about 6 hours; 1ml of the cultured bacterial liquid is taken to be cultivated in an enlarged way into an LB medium containing 200ml, and the bacterial liquid is continuously cultivated for 18 hours by a constant-temperature shake cultivation shaking table (200 rpm,37 ℃); the cultured bacterial liquid was centrifuged (8000 rpm,5 min), and the supernatant was discarded, and the plasmid was extracted according to the direction of endotoxin plasmid extraction kit (Tiangen Co.) and stored at-20℃for use.

2. Cell culture and transfection

By FreeStyle ^TM 293 expression medium (Thermo Fisher) cultures 293F cells, the temperature of the cell incubator was set to 37℃and the rotational speed was 90rpm; ensuring that 293F cells were at a density of 0.6X10 at 24 hours prior to transfection ⁶ -0.7×10 ⁶ Cell density was controlled at 1.0X10 cells/mL on day of transfection ⁶ –1.5×10 ⁶ Cell viability was greater than 90% per mL, 1ul of FreeStyle was used per 1ug of PD-L1 plasmid at transfection ^TM MAX transfection reagent, about 1ug of the above plasmid is added per 1ml of cell fluid, and the maximum expression level can be achieved by culturing the cells about 5-7 days after transfection. 7 days after transfection, the cell fluid was centrifuged (8000 rpm,10 min) and the supernatant was collected and sent to the next purification step.

3. And (5) purifying the PD-L1 antigen.

The supernatant was purified by Ni Sepharose 6Fast Flow (Cytiva), and the antigen was purified.

Pretreating the affinity chromatographic column before loading, regulating the flow rate to 3ml/min, flowing out 20% ethanol in the chromatographic packing, and balancing the nickel affinity chromatographic column with a balancing solution (specific formula shown in Table 4) until the conductivity baseline is stable; sampling and purifying the collected PD-L1 cell supernatant, and regulating the speed of a purifier to be 1ml/min; washing with washing solution (specific formula shown in Table 4) for removing non-specifically bound impurity protein by more than 5 times of column volume after sample loading, and adjusting the speed of the purifier to 3ml/min; after the washing, PD-L1 protein is eluted by using an eluent (the specific formula is shown in Table 4), the eluting speed of a purifier is regulated to be 1ml/min, and the eluent is collected according to the eluting peak value of UV (A280).

Dialyzing the collected eluent (50 mM Tris-HCl,200mM NaCl,pH 8.0) for 3 times, wherein the dialysis time is more than or equal to 4 hours each time; the dialyzed samples were subjected to SDS-PAGE to determine the protein purification. As shown in FIG. 1, the molecular weight of the protein after dialysis is about 32kDa, which is consistent with the size of the PD-L1 recombinant protein, and the purity of the protein is more than 95 percent without foreign proteins.

4. Experiments on immunized mice by the antigen

The experimental animals Balb/c mice were prepared, and the individual body weight was required to be 20g or more. PD-L1 antigen and Freund's complete adjuvant are mixed into emulsion according to the proportion of 1:1, and subcutaneous multipoint injection is carried out, wherein the injection quantity is 500 ul/patient; the immunization is carried out once every 14 days later, the dose of PD-L1 antigen which is added into each mouse for the primary immune reaction is 50ug, the subsequent dose of each injection is 25ug, and the antigen and Freund's incomplete adjuvant are mixed into emulsion according to the proportion of 1:1, and the total immunization is carried out for 4 times; the mice with higher titers were screened by detecting immune antibody titers in serum by enzyme-linked immunosorbent assay (ELISA) by tail vein blood sampling of the mice on day 10 after the 4 th immunization, and the results are shown in table 2.

TABLE 2 detection of serum titers of PD-L1 antigen immunized mice

As can be seen from the results of the serum titer detection in Table 2, the serum titer can reach 1:1562500 when the 2ug/ml PD-L1 antigen is coated, and the cell fusion experiment can be carried out.

5. Hybridoma fusion

1. Preparation of myeloma cells

3 bottles of T175 cell culture flasks SP2/0 cells were cultured in advance, and prior to fusion, the growth state of the cells was observed with a microscope to ensure that the cells were free of bacterial and fungal contamination.

2. Spleen cell preparation

The mice in the fourth step are euthanized by CO2 or cervical dislocation method, and soaked in 75% alcohol for sterilization for 5min. The spleens of mice obtained in aseptic operation are washed by sterilized PBS, redundant adipose tissues on the spleens are trimmed, the trimmed spleens tissues are placed on a 40-mesh stainless steel net membrane, and the spleens cell suspension is prepared by gentle grinding.

3. Cell fusion and subclone screening

SP2/0 myeloma cells and spleen cells were counted, mixed in an amount of SP2/0 to spleen cells 1:5, and the system SP2/0 and spleen cells were used with the electrofusion apparatus BTX ECM 2001. The fused cells are spread into a 96-well plate for culture, and the cell culture plate is placed into CO ₂ The incubator was continuously cultured, and the growth state of the fused cells was observed.

Changing the cell fusion into HAT selective medium about 10 days after cell fusion, culturing for 10 days again, taking cell supernatant, and detecting and identifying PD-L1 antibody secretion by an enzyme-linked immunosorbent assay; under the condition of antibody secretion, 5 rounds of subcloning screening are carried out by utilizing a limiting dilution method, and finally monoclonal cell strains are screened out.

4. Preparation of ascites in mice

Balb/c mice with the age of 8-10 weeks are selected and sensitized with paraffin oil one week in advance; culturing the selected monoclonal cell strains until the cells grow in logarithmic phase, injecting each clone strain into abdominal cavity of 3 mice, and injecting hybridoma cell number of 1×10 into abdominal cavity of each mouse ⁶ . After hybridoma cells are injected for about 7 days, ascites and survival conditions of the mice are observed, the abdomen of the mice is touched to have obvious swelling, and the ascites can be collected by a syringe and collected again every other day. The collected ascites was stored at-20℃and used for purification of monoclonal antibodies.

5. Monoclonal antibody purification of mouse ascites

Centrifuging (12000 rpm,4 deg.C, 20 min) the collected ascites of mice, removing impurities and cell debris, filtering with qualitative filter paper after centrifuging, measuring the volume of ascites with a measuring cylinder, adding equal volume of 1×PBS (10 mM, pH 7.4), and mixing. Pretreating a Protein A affinity chromatographic column with 1 XPBS (phosphate buffer solution) of 5 times of column volume before loading, adding treated ascites into the chromatographic column, incubating for 30min at room temperature, and periodically re-suspending Protein A filler to fully combine monoclonal antibody with a purification medium; after the incubation, the impurities are washed by 10 times of column volume 5 XPBS, and the impurities which are not specifically adsorbed are removed.

Adding 100ul of 1M Tris-HCl (pH=9.0) into a centrifuge tube, eluting a column by using 100mM glycine water solution (pH 3.0), collecting 1ml of each tube, dialyzing the collected monoclonal antibody eluent by using 1 XPBS, and dialyzing for more than or equal to 4 hours each time for three times; the purity of the antibody was measured by SDS-PAGE after dialysis, and the results are shown in FIG. 2.

Example 2 the use of the PD-L1 monoclonal antibody as shown in example 1 for antigen detection adopts a double-antibody sandwich detection mode, the PD-L1 monoclonal antibody in example 1 is used as a coating antibody, the heavy chain sequence of the labeled antibody is shown as SEQ ID No.6, and the light chain sequence is shown as SEQ ID No. 7.

For convenience of description, the PD-L1 monoclonal antibody in example 1 was recorded as antibody 1, and the antibody used for preparing the coated antibody was recorded as antibody 2.

The specific detection steps are as follows:

1. antibody coating: antibody 1 was diluted to a concentration of 4ug/ml with 1 XPBS, pipetted into 96 empty ELISA plates with 100ul per well and chilled overnight at 4 ℃.

2. And (3) sealing the ELISA plate: removing the coating liquid, washing with PBST, adding 250ul of the coating liquid into each hole, washing for 4 times, and drying; 250ul of blocking solution (1 XPBS, 2% BSA) was added to each well of the ELISA plate and incubated at 37℃for 2 hours.

3. After closing, washing with PBST, adding 250ul of the mixture into each hole, washing for 4 times, and drying; PD-L1 antigen was diluted from 50ng/ml for a total of 7 gradients: 50ng/ml, 10ng/ml, 2ng/ml, 400pg/ml, 80pg/ml, 16pg/ml, 3.2pg/ml, 100ul of diluted antibody 1 was added to each microplate well and incubated in an incubator at 37℃for 1 hour.

4. After incubation, washing with PBST, adding 250ul of the PBST into each hole, washing for 4 times, and drying; antibody 2-labeled HRP, diluted 5000-fold, 100ul of each ELISA plate well was added and incubated in an incubator at 37℃for 1 hour.

5. After incubation, washing with PBST, adding 250ul of the PBST into each hole, washing for 4 times, and drying; adding 100ul of display liquid (TMB) into each hole, and developing for 10-15min in dark; after the completion of the color development, 50ul of 2M H was added ₂ SO ₄ Terminate and determine the final sampleOD450, experimental results are shown in table 3 and fig. 3.

By computer fitting, the detection limit of the antibody 1 serving as a coated antibody and the antibody 2 serving as a labeled antibody is lower than 16pg/mL, and by computer program fitting, the antigen linear relationship R2= 0.99988 has better linearity.

In conclusion, the antibody can be used for monitoring the prognosis PD-L1 expression of a tumor patient by taking plasma as a sample, can be used for modifying a monoclonal antibody, and can be used for enhancing the immune killing of tumor cells, thereby having good popularization prospect and application value.

Table 4 shows the proportions of the experimental materials in the examples.

Table 4, a list of partial experimental Material formulas

Experimental materials	Composition of the components
		Balancing liquid	50mM Tris-HCl,200mM NaCl,5mM imidazole, pH 8.0
Washing liquid	50mM Tris-HCl,200mM NaCl,50mM imidazole, pH 8.0
		Eluent (eluent)	50mM Tris-HCl,200mM NaCl,300mM imidazole, pH 8.0

The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.

Claims

A pd-L1 monoclonal antibody heavy chain and light chain variable region, characterized in that the heavy chain variable region comprises the following complementarity determining regions:

CDRH1：SYWIE；

CDRH2：EILPGSGNTNYNEKLKG；

CDRH3：ERRGDY；

the light chain variable region comprises the following complementarity determining regions:

CDRL1：SASQGIYNYLN；

CDRL2：YTSSLHS；

CDRL3：QQYSKLPYT。
2. the heavy and light chain variable region of the PD-L1 monoclonal antibody according to claim 1, wherein the heavy chain is represented by SEQ ID No.2 and the light chain is represented by SEQ ID No. 3.
A pd-L1 monoclonal antibody according to any one of claims 1-2, characterized in that it comprises a heavy chain, light chain variable region.
4. The PD-L1 monoclonal antibody according to claim 3, wherein the heavy chain constant region is a murine IgG1 constant region.
5. The PD-L1 monoclonal antibody according to claim 3 or 4, wherein the heavy chain has the sequence shown in SEQ ID No.4 and the light chain has the sequence shown in SEQ ID No. 5.
6. The PD-L1 monoclonal antibody according to claim 3, wherein the antibody is humanized.
7. A monoclonal cell line expressing the PD-L1 monoclonal antibody of any one of claims 3-6 or a monoclonal antibody having the heavy and light chain variable regions of the PD-L1 monoclonal antibody of any one of claims 1-2.
8. The use of the PD-L1 monoclonal antibody according to any one of claims 3-6, which is used in the field of expression detection of PD-L1 antigen or immune killing of tumor cells.
9. Kit comprising the PD-L1 monoclonal antibody according to any one of claims 3-6 or a monoclonal antibody having the heavy chain and light chain variable region of the PD-L1 monoclonal antibody according to any one of claims 1-2.