CN109232740B - An anti-PD-L1 antibody and its application in anti-tumor therapy - Google Patents

Abstract

The present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, which can specifically bind to PD-L1 molecules, block the binding of PD-L1 and PD-1 after binding, and can activate T cells and anti-tumor and other biological effects.

Description

An anti-PD-L1 antibody and its application in anti-tumor therapy

technical field

The invention belongs to the field of medicine, in particular to an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof specifically recognizes programmed cell death 1 ligand 1 (programmed cell death 1 ligand 1, PD-L1), and can be used as a The immune activator stimulates the immune response of the body, thereby producing the effect of anti-tumor and other diseases.

Background technique

In 2011, cancer surpassed heart disease as the leading cause of death worldwide. WHO announced in December 2013 that the number of new cancer patients worldwide has exceeded 14 million every year, which is a significant increase compared with the 2008 statistics of 12.7 million. Deaths from cancer patients also increased over the same period, from 7.6 million in the past to 8.2 million. In 2013, immune anti-cancer therapy was rated as the top 10 scientific and technological breakthroughs of the year by Science magazine. Since then, tumor immunotherapy research has continuously made breakthroughs, and its clinical application has also achieved great success. The promising treatment method is expected to become a new conventional treatment method after surgery, radiotherapy and chemotherapy.

In the early 1980s, Allison and others determined the genetic structure of the alpha beta T cell receptor (TCR) on the surface of T cells responsible for recognizing antigens. In the late 1980s, Boone, Rosenberg, Old and others found that there are some tumor-specific antigens in different tumor patients, which can be recognized by T cells and specifically kill tumor cells, rekindling the hope of tumor immunotherapy. A great deal of research is devoted to the research and development of therapeutic vaccines for tumors. However, Schwartz et al. found that the TCR signal alone is not enough to activate antigen-specific T cells, and the activation of T cells also requires the participation of other molecules, that is, the synergy of the so-called second signal "costimulatory molecules". At the same time, it was found that only specific antigen-presenting cells (APCs) can express costimulatory molecules, while most cells, including tumor cells, cannot provide costimulatory molecule signals. In the early 1990s, Allison et al. discovered the CD28 molecule, which provides the second signal required for T cell activation. Later, Linsley et al. found that the B7 molecule expressed on the surface of APCs cells is the ligand of CD28 molecule, while Allison et al. studied in a mouse model, and the tumor cells that can express B7 molecule after modification can be quickly cleared by the mouse immune system. Therefore, the lack of expression of B7 molecule in tumor cells may be an important factor that the body cannot effectively stimulate T cell immunity.

Studies in the 1990s showed that cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4) played a completely opposite function to CD28 in vivo. If the role is the "accelerator" of a car, then CTLA-4 such molecules play the "brake" function. When the body's T cells are activated, these molecules will "check" the degree of activation of immune cells, and their expression will be up-regulated in the activated cells and play an immunosuppressive function, so that the body's T cells will not be excessively proliferated and activated to damage normal cells. Therefore, Such molecules are also known as "immune checkpoint" molecules. Cancer cells use the immunosuppressive mechanisms of these molecules to evade the body's immune system. Studies have shown that the use of CTLA-4-specific monoclonal antibodies to block CTLA-4 signals can significantly improve T cell activity, and in mouse model studies of various tumors, it has been found that monoclonal antibodies block CTLA-4 after blocking CTLA-4. It can greatly improve the tumor suppressive ability of mice. In addition to CTLA-4, immune checkpoint molecules also include programmed cell death receptor 1 (PD-1) and its ligands PD-L1 (PD-1 ligand 1), TIM-3 , LAG-3, TIGIT and other B7 superfamily and CD28 superfamily molecules. Blocking these "inhibitory" signals by specific monoclonal antibodies can re-release the activity of T cells, thereby enabling these T cells to play an anti-tumor effect. The contribution of tumor immune checkpoint therapy to anti-tumor strategies is: On the one hand, immune checkpoint therapy does not directly target tumor cells, but acts on the patient's immune system to release T cell activity by releasing signals that limit the function of T cells. On the other hand, this activation of T cells is not antigen-specific, but the reactivation of the entire immune system, so it can be applied to the treatment of a variety of different tumors, and can be used as a general therapy for tumors. Not only that, the success of CTLA-4 antibody blocking therapy has pioneered the development and application of immunosuppressive-related molecular blockade in tumor treatment, based on the development and application of immunosuppressive molecules represented by PD-1 and PD-L1. Antibodies have also made major breakthroughs.

As the ligand of PD-1 molecule, PD-L1 plays an important role in the process of T cells exerting their effects. Studies have shown that its high expression in tumor cells can induce the up-regulation of PD-1 molecule expression on tumor-specific T cells, thereby inhibiting the function of T cells to a certain extent, resulting in immune escape of tumors. Therefore, targeting PD-L1 and blocking the interaction between PD-1/PD-L1 can antagonize the inhibitory signal of T cells mediated by PD-1 molecules, thereby restoring the tumor-killing activity of T cells and achieving tumor treatment. It is an important target of tumor immunotherapy.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide an anti-PD-L1 antibody or an antigen-binding fragment thereof capable of specifically binding to a PD-L1 molecule, the anti-PD-L1 antibody comprising SEQ ID NO: 3, SEQ ID NO: 4 and The heavy chain CDRs shown in SEQ ID NO:5; and the light chain CDRs shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. wherein the fragment is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab') ₂ , diabodies and polypeptides comprising CDRs, and the anti-PD-L1 antibody or antigen-binding fragment thereof is capable of blocking Binding of PD-L1 to PD-1.

In a specific embodiment, the anti-PD-L1 antibody comprises the heavy chain sequence shown in SEQ ID NO:9 and the light chain sequence shown in SEQ ID NO:10, or the anti-PD-L1 antibody comprises SEQ ID NO : heavy chain sequence shown in 1 and light chain sequence shown in SEQ ID NO:2.

In a specific embodiment, the anti-PD-L1 antibody comprises the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12, or, the anti-PD The -L1 antibody comprises the heavy chain variable region shown in SEQ ID NO:13 and the light chain variable region shown in SEQ ID NO:14.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is a murine or humanized anti-PD-L1 monoclonal antibody, preferably the humanized PD-L1 monoclonal antibody comprises a human Fc region, More preferred is the Fc region of IgG1.

In a specific embodiment, the invention relates to a polypeptide whose sequence is set forth in SEQ ID NO: 11, wherein the polypeptide is part of an antibody that specifically binds a PD-L1 molecule, and wherein the antibody further comprises SEQ ID NO : the polypeptide shown in 12.

In a specific embodiment, the invention relates to a polypeptide whose sequence is set forth in SEQ ID NO: 13, wherein the polypeptide is part of an antibody that specifically binds a PD-L1 molecule, and wherein the antibody further comprises SEQ ID NO : the polypeptide shown in 14.

In a specific embodiment, the invention relates to a polypeptide whose sequence is set forth in SEQ ID NO: 12, wherein the polypeptide is part of an antibody that specifically binds a PD-L1 molecule, and wherein the antibody further comprises SEQ ID NO : the polypeptide shown in 11.

In a specific embodiment, the present invention relates to a polypeptide, the sequence of which is set forth in SEQ ID NO: 14, wherein the polypeptide is part of an antibody that specifically binds a PD-L1 molecule, and wherein the antibody further comprises SEQ ID NO : the polypeptide shown in 13.

A second aspect of the present invention is to provide an isolated polynucleotide encoding the anti-PD-L1 antibody or antigen-binding fragment thereof, wherein the anti-PD-L1 antibody comprises the heavy chain sequence shown in SEQ ID NO: 9 and SEQ ID NO: 9 The light chain sequence shown in ID NO: 10, or the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain sequence shown in SEQ ID NO: 1 and the light chain sequence shown in SEQ ID NO: 2.

In specific embodiments, the present invention relates to polynucleotides encoding said anti-PD-L1 antibodies or antigen-binding fragments thereof.

In a specific embodiment, the invention relates to a polynucleotide encoding the polypeptide set forth in SEQ ID NO: 11, wherein the polypeptide is part of an antibody that specifically binds a PD-L1 molecule, and wherein the antibody further comprises The polypeptide shown in SEQ ID NO: 12, preferably, the polynucleotide sequence is shown in SEQ ID NO: 15.

In a specific embodiment, the invention relates to a polynucleotide encoding the polypeptide set forth in SEQ ID NO: 12, wherein the polypeptide is part of an antibody that specifically binds a PD-L1 molecule, and wherein the antibody further comprises The polypeptide shown in SEQ ID NO: 11, preferably, the polynucleotide sequence is shown in SEQ ID NO: 16.

In a specific embodiment, the present invention relates to a polynucleotide encoding the polypeptide set forth in SEQ ID NO: 13, wherein the polypeptide is part of an antibody that specifically binds a PD-L1 molecule, and wherein the antibody further comprises The polypeptide shown in SEQ ID NO: 14, preferably, the polynucleotide sequence is shown in SEQ ID NO: 17.

In a specific embodiment, the invention relates to a polynucleotide encoding the polypeptide set forth in SEQ ID NO: 14, wherein the polypeptide is part of an antibody that specifically binds a PD-L1 molecule, and wherein the antibody further comprises The polypeptide shown in SEQ ID NO: 13, preferably, the polynucleotide sequence is shown in SEQ ID NO: 18.

A third aspect of the present invention is to provide an expression vector comprising the polynucleotide.

A fourth aspect of the present invention is to provide a host cell comprising the above-mentioned expression vector.

A fifth aspect of the present invention is to provide a method for preparing the anti-PD-L1 antibody or antigen-binding fragment thereof, the method comprising: 1) culturing the host cell; 2) recovering the anti-PD-L1 antibody from the host cell or culture medium The anti-PD-L1 antibody or antigen-binding fragment thereof.

The sixth aspect of the present invention is to provide a pharmaceutical composition containing the anti-PD-L1 antibody or its antigen-binding fragment, and a pharmaceutical carrier.

A seventh aspect of the present invention is to provide use of the anti-PD-L1 antibody or antigen-binding fragment thereof in the preparation of a medicament for increasing the level of IFN-γ secreted by T cells.

The eighth aspect of the present invention is to provide the use of the anti-PD-L1 antibody or its antigen-binding fragment in the preparation of an anti-tumor drug for treating cancer, especially non-small cell lung cancer and the like.

The anti-PD-L1 antibody or its fragment provided by the present invention can specifically bind to PD-L1 molecule, and after binding, can block the binding of PD-L1 and PD-1, and can produce a series of biological effects. These biological effects include, for example, the ability to increase the level of IFN-γ secreted by tumor-specific T cells in tumor cases, and especially to inhibit tumor growth in mice.

In the present invention, the expression "PD-L1 antibody" or "murine PD-L1 antibody" refers to a murine monoclonal antibody against PD-L1, in a specific embodiment, a PD-L1.A antibody. The expression "humanized PD-L1 antibody" is prepared by humanization on the basis of murine PD-1 antibody, and in a specific embodiment, it is prepared by humanizing PD-L1.A antibody.

PD-1 molecule is an important member of CD28 family, which also includes CD28, CTLA-4, ICOS and so on. PD-1 molecule is expressed in activated B cells, T cells and bone marrow cells. It is a type I membrane protein with a molecular weight of about 55kDa. It is currently found that PD-1 has two ligands, namely PD-L1 and PD-L2. PD-L1 -1 inhibits T cell activity through interaction with (PD-L1 and/or PD-L2). PD-L1 can be highly expressed on the surface of various tumor cells. Through the interaction between PD-1 and PD-L1, it can inhibit the functional activity of tumor-infiltrating lymphocytes, including TCR-mediated T cell proliferation and cytokine secretion levels. etc., and thus are utilized by a variety of tumors as an important mechanism for tumor cells to escape the surveillance of the immune system. The use of antibodies to specifically block the interaction between PD-1 and (PD-L1 and/or PD-L2) can activate T cells in an inhibited state, release the function of T cells, and restore the function of T cells, thereby To achieve the effect of using the body's immune system to kill tumor cells and then carry out tumor treatment.

The present invention is made based on the above principles. The anti-PD-L1 antibody or its antigen-binding fragment in the present invention specifically binds to the PD-L1 molecule to block the combination of PD-L1 and PD-1, thereby enabling T cells Activation increases the level of IFN-γ secreted by T cells.

The present application includes antibodies or derivatives that specifically bind to PD-L1, and also includes antigen-binding fragments that exhibit substantially the same antigen-binding specificity as the original antibody. "Fragments of antibodies" or "antigen-binding fragments" refer to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding or variable regions of the parent antibody, eg, one or more CDRs. Fragments of antibodies retain at least some of the binding specificity of the parent antibody. Antigen-binding fragments include those selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab') ₂ , diabodies, peptides comprising CDRs, and the like.

A "Fab fragment" consists of the CH1 and variable regions of one light chain and one heavy chain.

The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

A "Fab' fragment" contains a light chain and a portion of a heavy chain comprising the VH and CH1 domains and the region between the CH1 and CH2 domains, the two heavy chains of two Fab' fragments forming an interchain dyad Sulfur bonds to form F(ab')2 molecules.

An "F(ab') ₂ fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, an F(ab') ₂ fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

"Fv regions" comprise variable regions from both heavy and light chains, but lack constant regions.

"Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, these domains being present in a single polypeptide chain. In general, scFv polypeptides contain a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

"Diabodies" are small antigen-binding fragments with two antigen-binding sites. The fragment comprises a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using a linker that is too short to pair between the two domains of the same chain, the domains are allowed to pair with the complementary domains of the other chain and form two antigen binding sites.

"Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. The majority of humanized antibodies are human immunoglobulins in which the hypervariable region residues of the recipient antibody are replaced by residues in the hypervariable region of a non-human species with the desired specificity, affinity and capacity, such as small Mouse, rat, rabbit or non-human primate. In certain instances, Fv framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not present in either the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance.

"Specific" binding when referring to a ligand/receptor, antibody/antigen, or other binding pair refers to determining the presence or absence of binding of a protein such as PD-L1 in a heterogeneous population of proteins and/or other biological agents reaction. Thus, under the specified conditions, a specific ligand/antigen binds to a specific receptor/antibody, and does not bind to other proteins present in the sample in significant amounts.

The present invention also provides a pharmaceutical composition containing the PD-L1 antibody or antigen-binding fragment thereof of the present invention. To prepare pharmaceutical compositions, various desired dosage forms can be prepared by admixing the antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier or excipient. Examples of the dosage form of the pharmaceutical composition of the present invention include tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, Sublingual tablets, ointments, etc., as non-oral preparations, injections, suppositories, transdermal preparations, ointments, plasters, external liquid preparations, etc., can be listed, and those skilled in the art can select appropriate drugs according to the route of administration and the object of administration, etc. dosage form.

The dosage of the active ingredient of the pharmaceutical composition of the present invention varies depending on the administration object, target organ, symptoms, administration method, etc., and can be considered in consideration of the type of dosage form, administration method, age and weight of the patient, The patient's symptoms, etc., are determined by the doctor's judgment.

The pharmaceutical compositions of the present invention may also contain other agents, including but not limited to cytotoxic agents, cytostatic agents, anti-angiogenic or anti-metabolite drugs, targeted tumor drugs, immunostimulatory or immunomodulatory agents or in combination with cytotoxic agents, cellular Antibodies bound to growth inhibitors or other toxic drugs.

Description of drawings

Figure 1 is a graph showing the identification of PD-L1 protein produced by the 293T cell expression system by molecular sieve chromatography and SDS-PAGE.

Fig. 2 is a graph showing the results of expression and purification of PD-L1.A antibody protein and detection of purity by SDS-PAGE.

3 is a graph showing that the PD-L1.A antibody can block the binding of PD-1 to PD-L1.

Fig. 4 is a graph showing that PD-L1.A antibody can activate T cells.

Fig. 5 is a graph showing the results of expression and purification of humanized PD-L1.A antibody protein and detection of purity by SDS-PAGE.

Fig. 6 is a graph showing the binding affinity of PD-L1.A antibody and humanized PD-L1.A antibody to PD-L1 protein detected by SPR

FIG. 7 is a graph showing the results of the inhibition experiment of the humanized PD-L1.A antibody NCG mouse HCC-827 tumor model.

Detailed ways

Hereinafter, the present invention will be described in more detail by way of examples. However, those skilled in the art can understand that the following examples are only for the purpose of illustrating the present invention, rather than for limiting the present invention.

Example 1. Preparation of PD-L1.A antibody

1. Construction of PD-L1 recombinant expression plasmid

Taking the cDNA of PD-L1 (NM_001267706.1) as a reference, the DNA sequence of PD-L1 (amino acids 1-238) was synthesized, and the restriction sites EcoRI and BglII were introduced respectively, and the C-terminal was introduced into the tag of 6 His amino acids, wherein The cleavage site of EcoRI is located at the 5' end of the sequence, and the tag of 6 His amino acids and the cleavage site BglII are located at the 3' end of the sequence in turn. The synthetic PD-L1 DNA sequence was cloned into the expression vector pCAGGS (Addgene) using the restriction sites EcoRI and BglII to establish a recombinant eukaryotic expression plasmid of the PD-L1 full-length protein.

2. Expression and purification of PD-L1 recombinant protein

1) Transfection of HEK293T cells: HEK293T cells (ATCC) were transferred to a petri dish at a ratio of 1:3 for continued culture; 7.5 mL of DMEM (serum and antibiotic free) (GIBCO) was taken into a 50 mL tube, and 300 μL of polyetherimide (PEI) was added. ) 1.0 and mix well; add 75 μg PD-L1 recombinant plasmid DNA to the mixing solution, mix well and let stand for 30 minutes; respectively take 515 μL to each petri dish and incubate in a 37°C 5% CO ₂ incubator.

2) Collect supernatant: 48 hours after transfection, centrifuge at 4°C to collect supernatant.

3) Nickel affinity chromatography column purification: filter the supernatant after centrifugation with 0.45μM, 33mm PVDF membrane filter, and combine with nickel affinity chromatography column (GE Health) at 4°C for more than 4 hours; 20mM, 50mM, 100mM, 200mM, 300mM, 500mM and other eluents with different concentrations of imidazole were used for elution, and then the solution was changed to 20mM Tirs-HCl, 150mM NaCl buffer for use, and SDS-PAGE was used to identify the purity (Figure 1).

3. Expression and purification of PD-L1-mFc protein

Using the cDNA of PD-L1 (NM_001267706.1) as a reference, the DNA sequence of PD-L1 (amino acids 1-238) was synthesized, and the restriction sites EcoRI and BglII were introduced respectively, and the C-terminal was introduced into the tag of mouse Fc amino acid, wherein EcoRI The restriction site is located at the 5' end of the sequence, and the Fc tag and the restriction site BglII are located at the 3' end of the sequence. The synthetic PD-L1 DNA sequence was cloned into the expression vector pCAGGS (Addgene) using the restriction sites EcoRI and BglII to establish a recombinant eukaryotic expression plasmid of PD-L1-mFc protein.

PD-L1-mFc protein expression:

1) Transfection of HEK293T cells: HEK293T cells (ATCC) were transferred to a petri dish at a ratio of 1:3 for continued culture; 7.5 mL of DMEM (serum and antibiotic free) was taken into a 50 mL tube, and 300 μL of polyetherimide (PEI) 1.0 was added to mix. Homogenize; add 75 μg PD-L1-mFc recombinant plasmid DNA to the mixing solution, mix and let stand for 30 minutes; take 515 μL to each petri dish and incubate in a 37°C 5% CO ₂ incubator.

2) Collect supernatant: 48 hours after transfection, centrifuge at 4°C to collect supernatant.

3) Purification by Protein A affinity chromatography column: filter and centrifuge the supernatant with a 0.45 μM, 33 mm PVDF membrane filter, add an equal volume of 20 mM Na3PO4 (pH 7.0) to it after centrifugation to remove the precipitation, mix well, and then use 0.22 μm Filter membrane and bind to Protein A affinity chromatography column (GE Health) at 4°C for more than 4 hours; then use 0.1M GlypH3.0 to elute the antibody hanging on the column, add 1M Tris pH9.0 to about 0.8mL (The collection volume was 3.2 mL), and then the medium was changed to 20 mM Tirs-HCl and 150 mM NaCl buffer for later use.

4. Preparation and screening of PD-L1 mouse monoclonal antibody

According to the method of intraperitoneal injection of Freund's complete adjuvant, recombinantly produced and purified full-length PD-L1 recombinant protein (hereinafter referred to as PD-L1 antigen) was used to immunize B6/C57 mice (Beijing Weitong Lihua Laboratory Animal Technology). Ltd.) for immunization. The specific method is as follows:

1) Animal immunization: The purified PD-L1 antigen was emulsified with complete Freund's adjuvant (Sigma), and 6-8 week-old B6/C57 mice were immunized by intraperitoneal injection at a dose of 50 μg/mice, after an interval of two weeks. The second immunization was carried out, emulsified with incomplete Freund's adjuvant, and the immunization dose was 50 μg/only. After two immunizations, the tail blood was taken and the serum titer was determined by ELISA gradient dilution; according to the results, it was determined whether to boost the immunization, and the mouse with the highest antibody titer was selected for cell fusion.

2) Cell fusion: Myeloma cells use sp2/0 derived from BALB/c, and they are in logarithmic growth phase when fused; take the spleen of the immunized mouse to make a single-cell suspension of lymphocytes; mouse spleen lymphocytes and myeloma The cells were mixed at a ratio of 1:5-1:10, 1 mL of 50% PEG (pH 8.0) at 37°C was added dropwise, the incomplete medium and the rest of the stop solution were added, the supernatant was discarded by centrifugation, and the HAT medium was added to suspend and mix well. volume to 50mL, aliquoted into 3.5cm petri dishes, placed in a wet box, and cultured in a 37°C, 5% CO ₂ constant temperature incubator.

3) Screening and cloning: Select cell clones within 7-10 days of fusion, and use purified PD-L1 recombinant protein to perform ELISA to test the binding ability of hybridoma supernatant to PD-L1 protein. The PD-L1 recombinant protein was coated on an ELISA plate at 300 ng/well, incubated at 4°C overnight, washed three times with PBS buffer, added with 5% nonfat dry milk (Yili) blocking solution, and incubated at room temperature at 25°C for 2 hours; After washing with PBS buffer three times, the culture supernatant of the cell clone was added, and incubated at room temperature for 1 hour at 25°C; then washed three times with PBS buffer, and horseradish peroxidase-labeled goat anti-mouse IgG antibody was added at room temperature. Incubate at 25°C for 1 hour; then wash with PBS buffer 3 times, add TMB chromogenic solution, add stop solution after 15 min, and use a spectrophotometer to detect the absorption value of OD450. Cell clones with OD450>0.5 were screened for subsequent screening. Label the cell line number. The cells in the positive wells were subjected to limited dilution, and the ELISA value was determined 5-6 days after each limited dilution, and the monoclonal wells with a higher positive OD450 value detected by ELISA were selected for limited dilution until the results of the entire 96-well plate by ELISA were positive. Pick monoclonal clones with high positive values. Its corresponding fusion plate cell line is PD-L1.A.

4) Preparation and purification of the monoclonal antibody on the cells: The cell line PD-L1.A was cultured in a 10cm petri dish with DMEM medium containing 15% serum, and when the cells were expanded to about ⁴ ×107 cells, centrifuged at 800rpm for 5min , discard the supernatant and transfer the cells to a 2L spinner flask, add serum-free medium to make the cell density approximately 3×10 ⁵ cells/mL. After culturing for 1-2 weeks, when the cell death rate reaches 60%-70% (the cell density is about 1-2×10 ⁶ cells/mL), collect the cell suspension by high-speed centrifugation at 6000 rpm for 20 min, take the supernatant, The supernatant was purified by affinity chromatography, and the corresponding column material was selected according to the antibody profile, and protein G was used for purification. The concentration of purified monoclonal antibody was determined, packaged (100uL/tube, the concentration was 1mg/mL), and stored at 4-8°C.

5. Expression and purification of mouse PD-L1.A antibody

2 × 10 ⁶ cell line PD-L1.A cells were injected intraperitoneally into 6-8 week-old BALB/C mice (purchased from Viton Lever Company), and the ascites of the mice was collected after 2-3 weeks. The ascites fluid was first centrifuged to remove the precipitation and then added an equal volume of 20mM Na ₃ PO ₄ (pH 7.0) to mix well, and then filter the ascites fluid with a 0.22 μm filter, mainly to prevent other impurities in the ascites from causing damage to the column; filter After completion, it is ready for sample purification.

Connect Protein G (5mL) HP affinity column (GE company) to AKTA Purifier/Explorer/FPLC/START (GE company), and operate the following process on the machine: first flush out the 20% ethanol in the column with water, Then use 20mM Na ₃ PO ₄ , pH 7.0 buffer to equilibrate the column. After the conductivity is 4.5% on the instrument, the ascites is injected through a 5mL loop to combine with Protein G, and the flow rate is 1mL/min; , add about 0.8mL of 1M Tris pH 9.0 to the subsequent collection tube (collection volume is 3.2mL), then change the program to 100% 0.1M Gly pH 3.0 to elute the antibody hanging on the column, collect the elution The samples were prepared, identified by gel electrophoresis, and judged that the size of the gel band was correct, further purified by superdex200 molecular sieve chromatography, and identified its purity by SDS-PAGE (Figure 2). The method is to continuously dilute the concentrated antibody with PBS, repeatedly concentrate and dilute more than 100 times, and then divide the sample into packaging, use it directly or store it in a -80°C refrigerator.

Example 2. PD-L1 blocking antibody screening and affinity analysis

The mice were immunized with the PD-L1 protein expressed in vitro by 293T cells, and the obtained monoclonal antibody was subjected to the blocking experiment of PD-1 and PD-L1 to screen antibodies that could specifically block the interaction between PD-1 and PD-L1.

1. Preparation of 293T cells expressing full-length PD-1

In this example, PD-1-GFP-p plasmid (Clontech company) containing full-length PD-1 (pEGFP-N1 vector-GFP tag plasmid) was transfected into 293T cells (ATCC) to obtain PD-1 expression Full-length 293T cells. One day before transfection, 0.5-2×10 ⁵ cells per well were inoculated into a 24-well culture plate, and 500 μL of DMEM complete medium without antibiotics (GIBCO) was added to ensure that the cells were confluent at 70-80% during transfection . 1 μg of PD-1-GFP-p plasmid was diluted in 50 μL of serum- and antibiotic-free medium, and mixed gently. 2 gL PEI (Sigma) (4 mg/ml) was diluted in 50 μL serum and antibiotic free medium and mixed gently. After 5 minutes, 50 μL of PEI dilution was added dropwise to 50 μL of PD-1-GFP-p plasmid dilution, mixed gently, and incubated at room temperature for 20 minutes. 100 μL of PEI/PD-1-GFP-p plasmid complex was added dropwise to each well and mixed with fresh medium by gentle shaking. After the cells were incubated in an incubator for 4-6 hours, the serum-containing culture medium was replaced to remove the complex. After the cells were placed at 37°C and incubated in a CO ₂ incubator for 24 hours, the expression level of GFP was detected by flow cytometry (BDARIA II), and the expression level of PD-1 full-length expressing 293T cells was evaluated.

2. Antibody blocking experiment

The PD-L1.A antibody prepared in Example 1 and the PD-L1-mFc protein (obtained in Example 1) were mixed in a molar ratio of 2:1, incubated on ice for 1 hour, and then added to a solution containing 2×10 ⁵ PD-1 full-length expressing 293T cells were incubated on ice for 30 minutes. The Ebola virus GP protein-specific antibody 4G7 (Mapp Biopharmaceutical) was set as a negative control; after that, PBS was washed twice, APC-labeled anti-mouse IgG secondary antibody (BD) was added, and after incubation for 30 minutes, it was washed twice with PBS buffer. Flow cytometric analysis was performed after resuspending in 300 mL PBS solution. The results are shown in Figure 3. The results show that PD-L1-mFc can significantly bind to 293T cells expressing full-length PD-1, and the addition of PD-L1.A antibody can completely inhibit the interaction between PD-1 and PD-L1. Binding, thereby making PD-L1-mFc unable to bind to the PD-1 protein on the surface of 293T cells (Figure 3). Therefore, the PD-L1.A antibody can significantly inhibit the binding of PD-1 and PD-L1 at the cellular level.

Example 3. The ability of PD-L1 blocking antibody to activate T cells in vitro

One of the important applications of PD-L1 blocking antibody is its anti-tumor effect. In this example, peripheral blood lymphocytes from 3 healthy individuals were collected and isolated to evaluate the anti-tumor effect of the PD-L1 blocking antibody screened by the present invention at the cellular level in vitro potential.

1. Sample Enrollment Screening

The cases included in this example are healthy individuals.

2. PBMCs sorting

The lymphocytes used in the present invention are derived from the venous peripheral blood of an individual. After the selected individuals pass the physical examination of the clinician, the experimenter will inform the specific project process and the amount of blood required, and after the volunteers agree and sign the informed consent form, the clinical medical staff will collect blood from the volunteers. A 9 mL disposable vacuum blood collection tube (VACUETTE, Greiner, Austria) containing EDTA-K ₂ anticoagulation was used for blood collection. About 20-25 mL of blood was collected from each volunteer, which was inverted immediately after blood collection to prevent coagulation.

1) First, dilute the freshly collected peripheral blood with phosphate buffered saline (PBS, pH 7.4) after autoclaving and cooling at 121°C, and carefully add the diluted blood sample to the prepared 15mL lymphocyte separation solution ( Purchased from Tianjin Haoyang Biotechnology Co., Ltd.), be very careful when adding, add slowly to avoid interface confusion;

2) Centrifuge at 700g for 20 minutes with a horizontal centrifuge (SORVALL Stratos, Thermo, USA) at 25°C, and adjust the speed to the slowest when stopped;

The centrifuged sample is divided into four layers, the uppermost layer of plasma is aspirated with Pasteur pipette, and then the second layer of lymphocytes is carefully aspirated into a new sterile centrifuge tube, which is the crude pure PBMCs cells;

3) Dilute the crude PBMCs with an equal volume of phosphate buffered saline (PBS, pH 7.4), and then centrifuge at 800 g for 10 minutes at 25°C;

Discard the supernatant, resuspend with 7 mL of serum-free RPMI1640 (Hyclone brand from GE, USA), and centrifuge at 500g for 5 minutes at 25°C;

Discard the supernatant, resuspend, add 7 mL of RPMI-1640 medium containing 10% fetal bovine serum (FBS, Thermo Fisher, USA, sourced from Australia) for washing, and centrifuge at 500g for 5 minutes at 25°C;

4) After discarding the supernatant, resuspend with 3 mL of RPMI-1640 medium containing 10% FBS, take an appropriate amount of reselection solution to count cells on a hemocytometer, and use RPMI-1640 medium containing 10% serum to adjust to 2.5×10 ⁶ cells/mL density to obtain PBMCs cells for use;

5) ELISPOT detects the level of T cell activation of antibodies

a. ELISPOT detection of cell stimulation culture

The ELISPOT plate (Merck Millipore Corporation) was coated with anti-human interferon-gamma monoclonal antibody (BD Corporation) diluted in phosphate buffer (pH 7.4) more than 12 hours in advance, and placed horizontally at 4°C. The cells were blocked with 10% serum (HyClone) in RPIM-1640 medium for 1 hour at room temperature prior to the addition of stimulating antigen and cells.

The PD-L1.A antibody and negative control antibody (EBOLA virus GP protein antibody 4G7 (Mapp Biopharmaceutical)) were diluted with 10% serum (HyClone) in RPMI-1640 medium, and 100 μL was added to each well of the ELISPOT plate. Blank control wells were stimulated and phytohemagglutinin (PHA) (Sigma) stimulated positive control wells. The controls for this experimental setup include:

The added antibodies included: PD-L1.A antibody and 4G7 negative control antibody antibody were added to 96 wells at a final concentration of 10 μg/mL for stimulation culture.

The diluted PBMCs cells were added to each well of 100 μL and 100 μl of PBMCs dilution ELISPOT plate and incubated at 37° C. and 5% CO ₂ (carbon dioxide) for 18 hours.

b. ELISPOT plate washing and result acquisition

a) After the incubation, discard the cell fluid in the wells, quickly add 200 μL of deionized water at room temperature to each well to wash twice, and then wash three times with PBS (PBST) containing 5‰ Tween-20.

b) Remove the washing solution, dry it on absorbent paper, add 100 μL of diluted detection antibody (BD) to each well, and incubate at room temperature for 2 h.

c) Remove the detection antibody solution, wash 3 times with PBST, then add 100 μL of diluted streptavidin-HRP conjugate (BD) to each well, and incubate at room temperature for 1 h.

d) Color development: the streptavidin-HRP conjugate solution was removed, washed 3 times with PBST, and then washed 2 times with PBS. Dry on absorbent paper, add 100 μL of AEC substrate (BD) solution to each well, and incubate at room temperature for 15-30 minutes. When clear spots are seen, rinse with distilled water to stop the reaction.

e) Dry at 37°C or room temperature, then count the reaction spots in the ELISPOT wells with an automatic plate reader (C.T.L) on the ELISPOT plate, and then adjust the parameters and perform quality control to give the final reaction result.

3. Analysis of results

The effect of PD-L1 blocking antibodies on T cell activation was assessed by counting and analyzing the number of specific spots produced upon stimulation in 10 ⁵ cells.

The response of T cells to PD-L1 antibody was evaluated by the response of PBMCs of these three healthy individuals to PD-L1 antibody. The results showed (Figure 4) that the stimulation wells with PD-L1 antibody were able to generate stronger T cell immune responses than the negative control wells, and at the same time, they could generate a comparable level of T cell immune responses, while the 4G7 negative control antibody was stronger than the negative control. There were no significant differences in holes. Therefore, PD-L1 antibody can effectively activate T cells in healthy individuals under in vitro culture conditions and promote their production of IFN-γ, thereby enhancing their T cell immune function.

Example 4. Humanization of murine PD-L1.A antibody and detection of affinity between humanized antibody and PD-1

According to the sequence homology of the PD-L1.A antibody, the present invention obtains a humanized PD-L1.A antibody (hu- PD-L1.A).

SEQ ID NO.: 1: PD-L1.A murine PD-L1.A antibody heavy chain

SEQ ID NO.: 2: PD-L1.A murine PD-L1.A antibody light chain

SEQ ID NO.: 3: PD-L1.A heavy chain CDR1

SEQ ID NO.: 4: PD-L1.A heavy chain CDR2

SEQ ID NO.: 5: PD-L1.A heavy chain CDR3

SEQ ID NO.: 6: PD-L1.A light chain CDR1

SEQ ID NO.: 7: PD-L1.A light chain CDR2

SEQ ID NO.: 8: PD-L1.A light chain CDR3

SEQ ID NO.: 9: Humanized PD-L1.A heavy chain

SEQ ID NO.: 10: Humanized PD-L1.A light chain

SEQ ID NO: 11: Heavy chain variable region of murine PD-L1.A antibody

SEQ ID NO: 12: Light chain variable region of murine PD-L1.A antibody

SEQ ID NO: 13: Heavy chain variable region of humanized PD-L1.A antibody

SEQ ID NO: 14: Light chain variable region of humanized PD-L1.A antibody

SEQ ID NO: 15: Coding sequence of heavy chain variable region of murine PD-L1.A antibody

SEQ ID NO: 16: Coding sequence of light chain variable region of murine PD-L1.A antibody

SEQ ID NO: 17: Coding sequence of heavy chain variable region of humanized PD-L1.A antibody

SEQ ID NO: 18: Coding sequence of light chain variable region of humanized PD-L1.A antibody

SEQ ID NO: 19: Coding sequence of humanized PD-L1.A antibody heavy chain

SEQ ID NO: 20: Coding sequence of humanized PD-L1.A antibody light chain

1) Expression and purification of humanized PD-L1.A antibody:

Humanized PD-L1.A antibody was expressed by constructing pCAGGS containing polynucleotides (SEQ ID NO: 19 and SEQ ID NO: 20) encoding the amino acid sequences of SEQ ID NO.: 9 and SEQ ID NO.: 10, respectively vector (Addgene Company), and introduced the restriction sites EcoRI and BglII respectively, wherein the EcoRI restriction site is located at the 5' end of the sequence, and the restriction restriction site BglII is located at the 3' end of the sequence. The DNA sequence of the synthesized hu-PD-L1.A light or heavy chain was cloned into the expression vector pCAGGS (Addgene) using the restriction sites EcoRI and BglII to establish the hu-PD-L1.A antibody light chain and heavy chain recombinant eukaryotic expression plasmids. The expression plasmids of hu-PD-L1.A antibody light chain and heavy chain were co-transfected into 293T cells at a ratio of 2:1, and the expressed antibody was purified by Protein G affinity column chromatography. Specifically include:

a. 14-16h before transfection, divide the cells with higher cell density into a plate (for example, a 10cm dish with 100% confluence of cells is passaged at 1:3), after 14-16h, the cell density reaches above 70% ready for transfection.

b. Take 10cm culture dish to transfect adherent 293T cells as an example: the amount of plasmid required for transfection is 20μg/dish (light chain:heavy chain=1:1, mass ratio), diluted to 100μL/dish of HBS solution , and let stand after mixing; determine the amount of PEI (1 mg/mL) at the ratio of PEI (μL):plasmid mass (μg)=1:4, dilute into 100 μL/disk of HBS solution, and let stand after mixing . The above two solutions were separately left to stand and mixed for 5 minutes, then the two solutions were mixed and continued to stand for 20 minutes, and finally added to the cell culture medium to be transfected.

c. After 4-6 hours of transfection, change the medium for the transfected cells, rinse twice with 2-3 mL of PBS, and then change to fresh serum-free DMEM medium (added with Streptomyces penicillin at 1:1000). Element), cultured in a 37°C constant temperature, 5% _CO2 incubator for expression.

The transfected cell culture medium was collected after 3 days of culture, the medium was replaced with DMEM medium, and the supernatant was collected again on the seventh day. The supernatants collected twice were mixed to purify the target protein. The purification method used the purification method of Protein G mouse PD-L1.A antibody in the above step 5 of this example. The purity of the antibody after protein G column affinity chromatography reached more than 95% (as shown in Figure 5).

2) Affinity verification of humanized PD-L1.A antibody:

In this example, the PD-L1.A antibody and the humanized PD-L1.A antibody were subjected to affinity identification by surface plasmon resonance (SPR).

The PD-L1 protein and humanized PD-L1.A antibody prepared in Example 1 were exchanged into SPR buffer (10 mM HEPES-HCl, 150 mM Na-Cl, 0.005% Tween-20, pH 7.4). The antibody protein was diluted to 20 μg/mL and immobilized on a CM5 chip (GE Health), and then the serially diluted PD-L1 was passed through each channel of the CM5 chip, and the binding kinetic parameters were analyzed by BIAcore T100, and the affinity constant was calculated.

Through the detection of the affinity of murine PD-L1.A antibody and humanized PD-L1.A antibody to PD-L1, the results show that the affinity of murine PD-L1.A antibody to PD-L1 is 8.81nM, while The affinity of the humanized PD-L1.A antibody to PD-1 was 7.77 nM. Therefore, it can be seen from the SPR results that the affinity of the humanized PD-L1.A antibody is comparable to that of the murine PD-L1.A antibody, and the affinity of the order of nM is still maintained (Fig. 6).

Example 6. NCG immunodeficient mouse HCC827 tumor inhibition experiment of hu-PD-L1.A antibody

In this example, the tumor inhibitory effect of hu-PD-L1.A antibody was evaluated by using the NCG immunodeficient mouse HCC827 tumor model.

The experimental steps for tumor inhibition of hu-PD-L1.A antibody in NCG mice include:

1. Reconstruction of human immune system in NCG mice

NCG mice were obtained from Nanjing University-Nanjing Institute of Biomedicine. First, inoculate each NCG mouse with PBMCs cells derived from healthy individuals to establish mice with a human immune system in NCG mice:

i. Number of inoculated human PBMCs: 1×10 ⁷ cells/200μL/cell;

ii Inoculation site: tail vein;

2. HCC-827 cell line tumorigenesis

3 days after inoculation with PBMCs cells, each NCG mouse was inoculated with HCC-827 non-small cells (Cell Bank of Type Culture Collection, Chinese Academy of Sciences) lung cancer cell line:

a) Number of inoculated HCC-827 cells: 5×10 ⁷ cells/200 μL/cell;

b) Inoculation site: subcutaneous on the back;

3. Grouping and processing:

About 1 week after tumor cell injection, mice with relatively uniform tumor formation were selected for grouping, and then the antibody was injected intraperitoneally. In this example, the Ebola virus-specific antibody 13C6 (Mapp Biopharmaceutical) injection group was used as the negative control, and the hu-PD-L1.A antibody was used as the treatment group for parallel experiments, with 7 mice in each group.

grouping of mice Antibody injection and dosage number of mice Negative antibody control group 10mg/kg, 100μL 7 hu-PD-L1.A antibody panel 10mg/kg, 100μL 7

Antibody injection: After the tumor formation of mice was obvious (7 days), the antibody was injected in 4 times, the first time was 200 μg/mouse, and then the second time was 200 μg/mouse on the third day, and then every three days or four times. daily injection (both 200μg/only).

Tumor size was measured every three to four days after tumor formation and continued for two weeks after the last injection.

4. Observation of treatment effect:

1) Detection of tumor size:

a) Measure the diameter with a caliper after antibody injection, the unit is mm, and the calculation formula is: v=1/2×a×b×b (a is the long diameter, b is the short diameter);

b) After the last observation, the experiment was terminated, and the tumor tissue was separated and directly photographed for observation;

The results showed that the control group mice injected with 13C6 antibody all grew rapidly after 13C6 antibody injection. The tumor growth of the hu-PD-L1.A antibody injection group rapidly entered a plateau after antibody injection, and two mice accelerated their growth 10 days and 14 days after antibody injection, respectively, but were still significantly smaller than the 13C6 antibody control group. The results of this example show that the hu-PD-L1.A antibody can effectively inhibit tumor growth and has potential tumor therapeutic value (as shown in Figure 7).

Claims (16)

1. An anti-PD-L1 antibody or an antigen-binding fragment thereof that specifically binds to a PD-L1 molecule, comprising the heavy chain CDRs shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; and The light chain CDRs shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. 2. The anti-PD-L1 antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab') ₂ , Bis Antibodies and peptides comprising CDRs, the anti-PD-L1 antibodies or antigen-binding fragments thereof are capable of blocking the binding of PD-L1 to PD-1, wherein the diabodies are small antigen-binding fragments with two antigen-binding sites . 3. The anti-PD-L1 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the anti-PD-L1 antibody comprises the heavy chain sequence shown in SEQ ID NO:9 and the heavy chain sequence shown in SEQ ID NO:10 light chain sequence. 4. The anti-PD-L1 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the anti-PD-L1 antibody comprises the heavy chain sequence shown in SEQ ID NO: 1 and the heavy chain sequence shown in SEQ ID NO: 2 light chain sequence. 5. The anti-PD-L1 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the anti-PD-L1 antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 11 and SEQ ID NO: The light chain variable region sequence shown in 12, or, comprises the heavy chain variable region sequence shown in SEQ ID NO:13 and the light chain variable region sequence shown in SEQ ID NO:14. 6. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a murine or humanized anti-PD-L1 monoclonal antibody. 7. The anti-PD-L1 antibody or antigen-binding fragment thereof of claim 6, wherein the humanized anti-PD-L1 monoclonal antibody comprises a human Fc region. 8. A polynucleotide encoding the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-4. 9. The polynucleotide of claim 8, wherein the polynucleotide sequence is shown in SEQ ID NO: 15 or 16. 10. An expression vector comprising the polynucleotide of any one of claims 8-9. 11. A host cell comprising the expression vector of claim 10. 12. A method of preparing the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-6, the method comprising: 1) culturing the host cell as claimed in claim 11; 2) recovering the anti-PD-L1 antibody or its antigen-binding fragment from the host cell or culture medium. 13. A pharmaceutical composition comprising the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-6 and a pharmaceutically acceptable carrier. 14. Use of the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-6 in the manufacture of a medicament for increasing the level of IFN-γ secreted by T cells. 15. Use of the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-6 in the preparation of an anti-tumor drug for treating cancer. 16. The use of claim 15, wherein the cancer is non-small cell lung cancer.

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