CN115925937A - Antibody or fragment thereof and pharmaceutical application thereof - Google Patents

Abstract

The present invention relates to an antibody or fragment thereof comprising: the antibody or fragment thereof comprises a heavy chain variable region (VH) comprising the H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37 and a light chain variable region (VL) comprising the L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40. The invention provides an antibody which is combined with PD-L1, especially human PD-L1, with high affinity, and the antibodies screened by the invention are improved in the aspects of affinity and dissociation rate to PD-L1; meanwhile, based on the mouse antibody obtained by initial screening, the immunogenicity of the antibody is reduced by performing humanization transformation to the maximum extent; in addition, pharmacodynamic experiments prove that the antibody provided by the invention has better tumor inhibition effect.

Description

Antibody or fragment thereof and pharmaceutical application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an antibody or a fragment thereof, and also relates to application of the antibody or the fragment thereof.

Background

PD-L1, the gene encoding it, also known as CD274, is a ligand molecule for the programmed death factor PD1, often expressed on hematopoietic and non-hematopoietic cells such as T-cells and B-cells, as well as on various types of tumor cells. PD-L1 is assigned to type 1 transmembrane proteins, which have immunoglobulin V-like and C-like domains. When PD-L1 interacts as a ligand with its receptor molecule PD-1, it inhibits T cell activation and cytokine production. This interaction is important to prevent autoimmunity by maintaining the homeostasis of the immune response during infection or inflammation of normal tissues. At the same time, this interaction plays a crucial role in the induction and maintenance of immune tolerance to itself, which in the tumor microenvironment usually leads to the inactivation of toxic T cells, thus providing immune escape for the tumor cells. The expression of the gene in tumor cells is considered to be one of the criteria for whether many types of human malignant tumors can use PD-1 or PD-L1 antibody drugs, and the malignant tumors comprise melanoma, non-small cell lung cancer and the like.

Several PD-L1 monoclonal antibody drugs are currently on the market, such as the first PD-L1 monoclonal antibody drug Atezolizumab (trade name Tecntriq) developed and produced by GENECECH under Roche, and used for treating advanced bladder cancer with failed or advanced platinum chemotherapy. In 2017, the U.S. Food and Drug Administration (FDA) accelerated the approval of a new drug Bavencio for the treatment of Merkel Cell Carcinoma (MCC) in adults and 12 year old children. The medicine mainly contains Aveluab, and has sustained remission effect on metastatic Merkel cell carcinoma (mMCC). In europe, the approval application for PD-L1 antibody biologics for the treatment of metastatic urothelial cancer is also under FDA priority review. Astrikon, 3 months 2018, also published that its immunotherapy drug, imfinzi (Durvalumab), has formally received us Food and Drug Administration (FDA) approval for the treatment of non-small cell lung cancer stage III (NSCLC) patients who cannot be surgically resected.

At present, the monoclonal antibody drug aiming at PDL1 has a space for improving affinity and dissociation speed, and clinical test data shows that the immunogenicity of the Atezolizumab drug is higher, so that more anti-antibody is generated, and the treatment effect of the drug is greatly influenced.

Disclosure of Invention

Therefore, the invention provides an antibody or a fragment thereof, and the antibody or the fragment thereof can be used for obtaining the antibody which specifically binds to the human PD-L1, particularly has high affinity to the human PD-L1 through hybridoma screening and humanization technology. In view of the above technical problems, it is an object of the present invention to provide an anti-PD-L1 antibody or a fragment thereof, and uses thereof based on the antibody or the fragment thereof. "fragments" of the antibody molecules of the invention encompass various functional fragments of an antibody, for example, antigen-binding portions thereof, such as Fab, F (ab') 2 or scFv fragments.

To achieve the above object, the present invention provides an antibody or a fragment thereof, comprising:

the antibody or fragment thereof comprises a heavy chain variable region (VH) comprising the H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37 and a light chain variable region (VL) comprising the L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40.

Further, the heavy chain variable region comprises a sequence selected from: an amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 9; and/or, the light chain variable region comprises a sequence selected from: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID No. 10.

Further, the antibody or fragment thereof is an anti-PD-L1 antibody or fragment thereof.

Further, the antibody includes any form of monoclonal antibody, single chain antibody, diabody, single domain antibody, nanobody, fully or partially humanized antibody, and chimeric antibody, or the antigen binding fragment is a half-antibody or an antigen binding fragment of an antibody or a half-antibody, and the antibody of the antigen binding fragment includes scFv, bsFv, dsFv, (dsFv) ₂ 、Fab、Fab'、F(ab') ₂ Fv, and IgG.

Further, the antibody or fragment thereof further comprises a constant region of human or murine origin comprising a heavy chain constant region (CH) of human or murine origin and/or a light chain constant region (CL), wherein the heavy chain constant region comprises an antibody or fragment thereof of IgG, igA, igM, igD or IgE and the light chain constant region comprises a kappa or lambda type.

Further, the antibody comprises a monoclonal antibody which is a monoclonal antibody, a mouse monoclonal antibody, a chimeric monoclonal antibody or a humanized monoclonal antibody, wherein the heavy chain constant region of the monoclonal antibody is of an IgG1 or IgG4 subtype, and the light chain constant region of the monoclonal antibody is of a kappa type;

the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence with at least 75% identity to the amino acid sequence shown in SEQ ID NO. 1;

the light chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO. 2 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO. 2.

The nucleic acid molecules of the invention may be cloned into vectors for transformation or transfection of host cells. Thus in a further aspect, the invention also provides a vector comprising a nucleic acid molecule of the invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector and the like. The vectors or nucleic acid molecules of the invention may be used to transform or transfect host cells for purposes such as preservation or antibody expression. Thus, in a further aspect, the present invention provides a host cell comprising or transformed or transfected with a nucleic acid molecule and/or vector of the invention. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

The antibodies or fragments thereof provided by the present invention can be obtained using any method known in the art. For example, the heavy chain variable region and/or the light chain variable region of the antibody may be obtained from the nucleic acid molecule provided by the present invention, or the heavy chain and/or the light chain of the antibody may be obtained and then assembled into an antibody with optional other domains of the antibody; alternatively, the host cell provided by the present invention is cultured under conditions that allow the host cell to express the heavy chain variable region and/or the light chain variable region of the antibody or the heavy chain and/or the light chain of the antibody to assemble into the antibody. Optionally, the method further comprises the step of recovering the produced antibody.

The antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention may be comprised in a pharmaceutical composition, more particularly in a pharmaceutical preparation, for use for various purposes according to the actual needs. Thus, in a further aspect, the invention also provides a pharmaceutical composition comprising an antibody or fragment thereof, a nucleic acid molecule, a vector and/or a host cell according to the invention, and optionally a pharmaceutically acceptable excipient.

In yet another aspect, the present invention also provides the use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition for the manufacture of a medicament for the prevention or treatment of a disease including a tumor or cancer, such as non-small cell lung cancer, melanoma, bladder cancer, merkel lymphoma, cutaneous squamous cell carcinoma, lung cancer, hodgkin lymphoma, kidney cancer, liver cancer, esophageal cancer, non-hodgkin lymphoma, breast cancer, thyroid cancer, stomach cancer, intestinal cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, leukemia, laryngeal cancer, oral cancer, ear-eye tumor, biliary tract cancer, gall bladder cancer, adrenal cancer, reproductive system tumor, multiple myeloma, nervous system tumor, urothelial cell cancer.

Accordingly, the present invention also provides a method of preventing or treating a disease comprising a tumor or cancer, such as non-small cell lung cancer, melanoma, bladder cancer, mercker's lymphoma, cutaneous squamous cell carcinoma, lung cancer, hodgkin's lymphoma, kidney cancer, liver cancer, esophageal cancer, non-hodgkin's lymphoma, breast cancer, thyroid cancer, stomach cancer, intestinal cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, leukemia, laryngeal cancer, oral cancer, ear and eye tumor, biliary tract cancer, gallbladder cancer, adrenal cancer, reproductive system tumor, multiple myeloma, nervous system tumor, urothelial cell cancer, comprising administering the antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition of the invention to a subject in need thereof. Preferably, the subject is a mammal; more preferably, the subject is a human.

In yet another aspect, the invention provides a kit comprising an antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell and/or a pharmaceutical composition of the invention. The kit may be for therapeutic or diagnostic use.

Compared with the prior art, the invention has the beneficial effects that the invention provides a novel antibody capable of binding PD-L1, especially human PD-L1 with high affinity. Compared with the existing anti-PD-L1 antibody, the antibody has the following characteristics: compared with the existing antibody (taking Atezolizumab (Tecnriq) of Genetech as an example), the antibody screened by the method is improved in the aspects of affinity and dissociation rate to PD-L1; meanwhile, based on the mouse antibody obtained by initial screening, the immunogenicity of the antibody is reduced by performing humanization transformation to the maximum extent; in addition, pharmacodynamic experiments prove that the antibody provided by the invention has better tumor inhibition effect.

Drawings

FIG. 1 shows the results of FACS detection of the binding of the culture supernatant of hybridoma cell lines of the present invention to antigen human PD-L1;

FIG. 2 shows the FACS detection result of the binding of the culture supernatant of the hybridoma cell line of the present invention to the antigen monkey PD-L1;

FIG. 3 shows the result of FACS detection of blocking the binding of PD-L1 to cells by culture supernatant of hybridoma cells according to an embodiment of the present invention;

FIG. 4 shows the results of FACS detection of the binding of the humanized antibody to the antigen human PD-L1 according to the embodiment of the present invention;

FIG. 5 shows the results of FACS detection of the binding of the humanized antibody of the present invention to the antigen monkey PD-L1;

FIG. 6 shows the results of FACS detection of the humanized antibody of the present invention blocking the binding of PD-L1 to cells;

wherein, the abscissa in fig. 1-6 is the parameter after the logarithm of the concentration is taken.

Detailed Description

In order that the objects and advantages of the invention will be more clearly understood, the invention is further described below with reference to examples; it should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only for explaining the technical principle of the present invention, and do not limit the scope of the present invention.

It should be noted that in the description of the present invention, the terms of direction or positional relationship indicated by the terms "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, which are only for convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.

Furthermore, it should be noted that, in the description of the present invention, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

The experimental procedures in the following examples are all conventional ones unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.

Atezolizumab (Reference): manufactured by Genetech under the trade name Tecntriq, disclosed in US2016/0319022A1, and see SEQ ID NO 6 and SEQ ID NO 7 for the light and heavy chains, respectively.

EXAMPLE 1 preparation of hybridoma cells according to the examples of the invention

The PD-L1 protein (KP 1008 PDL1-mFc kyinno) is used as immunogen to immunize mice, 5 mice are selected to immunize subcutaneously, 5 mice are immunized intramuscularly, the adjuvant adopts quinkanody 5W water-soluble adjuvant, the titer is measured 2 weeks after the boosting immunization, two mice with high titer are selected to carry out immune shock, and cell fusion is carried out after 3 days.

The cell fusion method specifically comprises the steps of taking serum of 2 mice to be fused, dissecting and taking spleens, separating splenocytes, fusing the splenocytes with cultured myeloma cells, laying the fused cells on a 96-well plate, adding a selective culture medium for screening, changing the culture medium after 7 days, performing ELISA detection after 10 days, and selecting cells with OD (optical density) values more than 10 times of that of a negative control for flow cytometry detection.

Selecting double positive cells, performing subclone plating by a cell limiting dilution method, and selecting monoclonal cells. And (3) taking the selected monoclonal cells, taking culture supernatant, performing ELISA detection and flow cytometry detection, and selecting the cells of double positive for amplification culture.

EXAMPLE 2 FACS detection of binding of PD-L1 to culture supernatants of hybridoma cells of examples of the present invention

293T-PDL1 (KC-0205 293T-PDL1 kyinno) cells were prepared into a cell suspension at a cell concentration of 1X 107 cells/ml in PBS containing 2% FBS.

50 μ l of cell suspension was added to each flow tube (sample tube), while the antibody to be detected (purified hybridoma antibody) and a blank (PBS) were added to each well, starting at 20ug/mL, diluted 3.16-fold, diluted 8 doses-graded, and incubated at 4 ℃ for 60 minutes. To each flow tube, 1ml of flow buffer was added, centrifuged at 1200rpm for 5 minutes, the supernatant was discarded, and the washing was repeated three times. Control tubes 1 (without addition of culture supernatant and secondary antibody below, only cell suspension) and 2 (without addition of culture supernatant, only cell suspension and secondary antibody below) were set simultaneously.

Then 100. Mu.l of flow buffer was added to each flow tube for resuspension, 5. Mu.l of PE-labeled anti-mouse Fc-labeled secondary antibody (Biolegend, cat. No. 409304) was added according to the experimental requirements, incubated at 4 ℃ for 30 minutes in the dark, then 1ml of flow buffer was added, centrifuged at 1200rpm for 5 minutes at room temperature, the supernatant was discarded, and the washing was repeated three times.

Add 250. Mu.l of flow buffer to each flow tube again, resuspend and mix well, and test on the machine.

The results of the FACS binding measurements are shown in table 1 and figure 1.

TABLE 1 FACS detection of the binding of culture supernatants of hybridoma cells to PD-L1

Numbering	Name (R)	EC50(μg/mL)	Fluorescence intensity max
				PL-29	26G9-5	0.2189	12113
PL-73	54F9-4	0.3098	10474
				PL-80	60A12-1	0.3784	12215
PL-82	48B10-6	0.4460	10728

EXAMPLE 3 FACS detection of binding of culture supernatants of hybridoma cells of the examples of the present invention to cynoPD-L1

293T-cynoPDL1 (KC-1001, kyinno) cells were prepared into a cell suspension at a cell concentration of 1X 107 cells/ml in PBS containing 2% FBS.

To each flow tube (sample tube) was placed 50 μ l of cell suspension, while to each well was added the antibody to be detected (purified hybridoma antibody) and a blank control (PBS), starting at 20ug/mL, diluted 3.16 fold, diluted 8doses gradient, and incubated at 4 ℃ for 60 minutes. To each flow tube, 1ml of flow buffer was added, centrifuged at 1200rpm for 5 minutes, the supernatant was discarded, and the washing was repeated three times. Control tubes 1 (without addition of culture supernatant and secondary antibody below, only cell suspension) and 2 (without addition of culture supernatant, only cell suspension and secondary antibody below) were set simultaneously.

Then 100. Mu.l of flow buffer was added to each flow tube for resuspension, 5. Mu.l of PE-labeled anti-mouse Fc-labeled secondary antibody (Biolegend, cat. No. 409304) was added according to the experimental requirements, incubated at 4 ℃ for 30 minutes in the dark, then 1ml of flow buffer was added, centrifuged at 1200rpm for 5 minutes at room temperature, the supernatant was discarded, and the washing was repeated three times.

Add 250. Mu.l of flow buffer to each flow tube again, resuspend and mix well, and test on the machine.

The results of the FACS binding measurements are shown in table 2 and figure 2.

TABLE 2 FACS detection of hybridoma cell culture supernatants associated with cynoPD-L1

Numbering	Name (R)	EC50(μg/mL)	Fluorescence intensity max
				PL-29	26G9-5	0.3073	15356
PL-73	54F9-4	0.3636	12176
				PL-80	60A12-1	0.3710	12581
PL-82	48B10-6	0.2974	5278

EXAMPLE 4 FACS detection of blocking binding of PD-L1 to cells by culture supernatants of hybridoma cells according to the examples of the invention

An extracellular region (from position 24 to position 170 of SEQ ID NO: 8) gene fragment of human PD-1 protein (SEQ ID NO: 8) was synthesized, constructed into PLVX-IRES-PURO (Clontech), transformed into DH 5. Alpha. Competent cells, plated, and cultured overnight at 37 ℃. Selecting a monoclonal colony, shaking bacteria, carrying out enzyme digestion identification, extracting plasmids, selecting correct clones to extract plasmids through sequencing verification, and storing for later use.

Recovering 293T cells, continuously passaging for 2 times, plating according to 2 multiplied by 106 cells/10 ml/dish, after 16-24 h, when the cell density grows to 70-80%, transfecting the cells with the eukaryotic expression plasmid, and culturing in a 5% CO2 incubator at 37 ℃. Culturing for 12-16 h, removing the culture medium, changing the culture medium, and adding a corresponding amount of Puromycin (the final concentration is 10 mug/ml) after 48-56 h. The cells were cultured continuously for 10 days in DMEM + 10. Mu.g/ml Puromycin containing 10% FBS, if the cell growth state was good. And (5) initially judging to take the polyclonal cell and carrying out flow detection.

Taking positive polyclonal cells, plating each well according to 0.5 cell, picking out monoclonal cells after 7 days, and carrying out expanded culture. And performing FACS detection on the selected monoclonal, selecting positive cells, namely 293T cells stably expressing h-PD1, and naming the cells as the 293T-h-PD1 cells.

The cells were washed once with FACS buffer, plated in 96-deep well plates at 2-5 × 105 cells/well, while the antibody to be tested (hybridoma antibody after purification) and a blank control (PBS) were added to each well, and incubated for 2 hours starting at 20ug/mL, 3.16 fold dilution, 8doses gradient dilution, and 500ng of a pre-mix of the fusion protein PD-L1 (hFC tag) of example 2. Then, 400. Mu.l of FACS buffer was added to each well and washed twice, a PE anti-human IgG Fc secondary antibody was added thereto and incubated for 1 hour, and then 400. Mu.l of FACS buffer was added to each well and washed twice. And (6) performing detection on the machine.

The results of the FACS blockade detection are shown in table 3 and fig. 3.

TABLE 3 FACS detection of hybridoma cell culture supernatants for blocking the binding of PD-L1 to the cells

Numbering	Name (R)	EC50(μg/mL)	Max/% of blocking rate
				PL-29	26G9-5	0.3933	99.586
PL-73	54F9-4	0.4892	99.205
				PL-80	60A12-1	0.4251	99.615
PL-82	48B10-6	0.2519	99.764

Example 5 affinity assay for binding of culture supernatants of hybridoma cells of the examples of the invention to PD-L1

The experimental method comprises the following steps:

step 1, selecting Capture Surface Dip of the same species.

Step 2. Take 96 well plates, add 200. Mu.l SD buffer (1 XPBS +0.02% Tween20+0.1% BSA) per well, put in ForteBio Octet, pre-cycle.

And 3, fixing the antibody, wherein the antibody concentration of the purified supernatant adopts 10 mu g/ml.

Step 4. Extracellular regions of human PD-L1 protein (position 19 to position 238 of SEQ ID NO: 3) were diluted with 6 gradients of 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, respectively, and added to the corresponding wells.

And 5, detecting on the computer. The results are shown in Table 4.

TABLE 4 affinity assay of the binding of culture supernatants of hybridoma cells to PD-L1

And selecting corresponding hybridoma cell strains based on the experiment, extracting RNA from the monoclonal cells, performing reverse transcription to form cDNA, and connecting the cDNA into a sequencing vector for sequencing analysis.

The sequences of exemplary murine antibodies are as follows, with the CDRs (obtained according to the AbM definition method) underlined. The nomenclature of the murine antibody depends on the hybridoma cell line from which it is derived.

A1 (murine antibody 48B 10)

>48B10-VH

QVQLKESGGGLVKPGGSLKLSCAASGFTFSSYAMSWIRQTPEKRLEWVATITSDGRYSYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTATYYCARRENWFTYWGQGTLVTVSA

>48B10-VL

DIVITQSPALMSASPGEKVTMTCSASSSVTYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSFSLTISNMEAEDAATYYCQQWSSGPTFGAGTKLELK

Example 6 humanization of antibodies of the present examples

The A1 antibody (murine antibody 48B 10) was selected for humanization of the antibody. Comprehensively considers the sequence similarity, expression quantity and whether the light and heavy chain combination of the antibody and different humanized templates is used by the antibody which is already prepared, and the like. According to the factors that the similarity value of the light chain and the heavy chain is more than 200, the expression amount is more than 50mg/ml, the antibody of the light chain and the heavy chain is used in combination with the drug and the like, finally, the humanized sequences are respectively selected as templates of the heavy chain and the light chain: IGHV1-46 x 01 (SEQ ID NO: 4) and IGKV1-5 x 01 (SEQ ID NO: 5). And carrying out homologous modeling on the A1 murine monoclonal antibody, and carrying out structural simulation of the Fab region. And finally obtaining the predicted Fab structure of the A1 antibody through homologous modeling calculation.

Through the comparison analysis of the predicted Fab structure and heavy chain with IGHV1-46 x 01 sequence, all CDR regions are replaced by human-derived template, and then structure simulation and kinetic calculation are carried out. Through structural simulation and kinetic analysis, several amino acids affecting the CDR structure are retained, and the final heavy chain version except the CDR region and 68A, 70L, etc. are the original murine amino acids and the other murine amino acids are all replaced with corresponding IGHV1-46 x 01 template human amino acids.

Through the comparison analysis of the predicted Fab structure and light chain with IGKV1-5 x 01 sequence, CDR area is replaced with adult template, and then structure simulation and kinetic calculation are carried out. Through structural simulation and kinetic analysis, several amino acids affecting the CDR structure are retained, and the final light chain version except the CDR area retains the original murine amino acids is replaced with the corresponding IGKV1-5 × 05 template human amino acids.

The sequence of the humanized antibody of the A1 antibody is as follows:

VH humanized sequence 48B10-VH-hz

QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWIRQAPGKGLEWVATITSDGRYSYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRENWFTYWGQGTTVTVSS

' VL humanized sequence 48B10-VL-hz

DIQITQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGSSPKPWIYLTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSGPTFGQGTRLEIK

The sequence shown in SEQ ID NO. 1 is used as a heavy chain constant region, the sequence shown in SEQ ID NO. 2 is used as a light chain constant region, a primer is redesigned for the DNA sequence of the antibody aiming at the PD-L1 target obtained by sequencing, the corresponding genes of the chimeric antibody and the humanized antibody are synthesized and connected into a eukaryotic expression vector, DH5alpha competent cells are transformed, the cells are cultured in a constant temperature incubator at 37 ℃ for overnight, and monoclonal strains are selected for sequencing and identification. The correct strain was selected, shaken, and then transfected into mammalian expression cells 293F, which were then cultured at 37 ℃ in a 5% CO2 incubator for 7 days.

Collecting the expression supernatant, centrifuging, filtering, selecting protein G affinity chromatography column, purifying, detecting the purity of the purified antibody by SDS-PAGE electrophoresis, detecting the antibody concentration by using BCA protein detection kit, subpackaging, and storing in a refrigerator at-80 ℃ for later use. Among them, the chimeric antibody was named "murine antibody abbreviated (chi)", and the humanized antibody was named "murine antibody abbreviated (hz)".

Example 7 FACS detection of the binding of the antibodies of the invention to PD-L1

The experimental procedure was as in example 3, except that after 50. Mu.l of cell suspension was put into each flow tube, the test antibody, the positive control antibody (Reference) or the negative Reference antibody (hIgG 1) was added at the concentration shown in FIG. 3. Control tubes 1 (no antibody and secondary antibody added, only cell suspension added) and 2 (no antibody added, only cell suspension and secondary antibody added) were set simultaneously.

The results of the FACS binding measurements are shown in table 5 and figure 4.

TABLE 5 FACS detection of antibody binding to PD-L1

Name (R)	EC50(μg/mL)	Fluorescence intensity max
			Atezolizumab	0.4584	1499
48B10-4(hz)	0.377	3850

EXAMPLE 8 FACS detection of binding of the antibodies of the invention to cynoPD-L1

Antibody binding method to cynoPD-L1 Reference example 3, except that after 50. Mu.l of cell suspension was put into each flow tube, the test antibody, positive control antibody (Reference) or negative Reference antibody (hIgG 1) was added. Control tubes 1 (no antibody and secondary antibody added, only cell suspension added) and 2 (no antibody added, only cell suspension and secondary antibody added) were set simultaneously.

The results of the FACS binding measurements are shown in table 6 and fig. 5.

TABLE 6 FACS detection of antibody binding to PD-L1

Name(s)	EC50(μg/mL)	Fluorescence intensity max
			Atezolizumab	0.5913	5034
48B10-4(hz)	0.3417	8668

Example 9 FACS detection of blocking of PD-L1 binding to cells by antibodies of the invention

The experimental procedure was as in example 4, except that the test antibody, the positive control antibody Atezolizumab (Reference) or the negative Reference antibody (hIgG 1) was added to each well.

The results of the FACS blockade detection are shown in table 7 and fig. 6.

TABLE 7 FACS detection of antibody blocking PD-L1 binding to cells

Name (R)	EC50(μg/mL)	Max/% of blocking rate
			Atezolizumab	0.2502	99.87
48B10-4(hz)	0.5245	99.83

Example 10 affinity assay for binding of antibodies of the examples of the invention to PD-L1

Extracellular domains of human PD-L1 protein (positions 19 to 238 of SEQ ID NO: 3) were prepared in PBS buffer at a maximum concentration of 30nM and 3-fold diluted to 6 concentrations with a 0 concentration control. The murine, chimeric and humanized antibodies of the invention were prepared as 20nM solutions in PBS.

The affinity of the antibody and antigen was determined using ForteBio Octet as an affinity detection instrument. The specific procedure is as in example 5.

The results are shown in Table 8.

TABLE 8 result of affinity assay of antibody binding to antigen PD-L1

Antibodies	Antigens	KD(M)	kon(1/Ms)	kdis(1/s)
					Atezolizumab	Human PD-L1-his	9.86E-10	4.77E+05	4.71E-04
48B10-4(hz)	Human PD-L1-his	5.95E-10	4.26E+05	2.54E-04

So far, the technical solutions of the present invention have been described in connection with the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of the present invention is obviously not limited to these specific embodiments. Equivalent changes or substitutions of related technical features can be made by those skilled in the art without departing from the principle of the invention, and the technical scheme after the changes or substitutions can fall into the protection scope of the invention.

Claims (12)

1. An antibody or fragment thereof, comprising:

the antibody or fragment thereof comprises a heavy chain variable region (VH) comprising the H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37 and a light chain variable region (VL) comprising the L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40.

2. The antibody or fragment thereof of claim 1, wherein the heavy chain variable region comprises a sequence selected from the group consisting of: an amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 9; and/or, the light chain variable region comprises a sequence selected from: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID No. 10.

3. The antibody or fragment thereof of claim 2, wherein the antibody or fragment thereof is an anti-PD-L1 antibody or fragment thereof.

4. The antibody or fragment thereof of claim 3, wherein the antibody comprises any of a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody, and a chimeric antibody, or the antigen-binding fragment is a half-antibody or an antigen-binding fragment of an antibody or a half-antibody, and the antibody of the antigen-binding fragment comprises scFv, bsFv, dsFv, (dsFv) ₂ 、Fab、Fab'、F(ab') ₂ Fv, and IgG.

5. The antibody or fragment thereof of claim 4, wherein the antibody or fragment thereof further comprises a constant region of human or murine origin comprising a heavy chain constant region (CH) of human or murine origin and/or a light chain constant region (CL), wherein the heavy chain constant region comprises an antibody or fragment thereof of IgG, igA, igM, igD or IgE and the light chain constant region comprises a kappa or lambda type.

6. The antibody or fragment thereof of claim 5, wherein the antibody comprises a monoclonal antibody that is murine, chimeric or humanized, wherein the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type;

the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence with at least 75% identity to the amino acid sequence shown in SEQ ID NO. 1;

the light chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO. 2 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO. 2.

7. A nucleic acid molecule encoding the antibody or fragment thereof of any one of claims 1 to 6, wherein the antibody or fragment thereof encodes or a heavy chain CDR, a light chain variable region, a heavy chain or a light chain comprised in the antibody or fragment thereof.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising or transformed or transfected with the nucleic acid molecule of claim 7 and/or the vector of claim 8.

10. A pharmaceutical composition comprising the antibody or fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, or the host cell of claim 9, and any pharmaceutically acceptable excipient.

11. Use of the antibody or fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, or the pharmaceutical composition of claim 10 in the manufacture of a medicament for the prevention or treatment of a disease comprising a tumor or cancer, including non-small cell lung cancer, melanoma, bladder cancer, merkel lymphoma, cutaneous squamous cell carcinoma, lung cancer, hodgkin lymphoma, kidney cancer, liver cancer, esophageal cancer, non-hodgkin lymphoma, breast cancer, thyroid cancer, gastric cancer, intestinal cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, leukemia, laryngeal cancer, oral cancer, otic eye tumor, biliary tract cancer, gallbladder cancer, adrenal cancer, reproductive system tumor, multiple myeloma, nervous system tumor, urothelial cell cancer.

12. A kit comprising the antibody or fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, or the pharmaceutical composition of claim 10.

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