Pharmaceutical composition of PD-L1 antibody, method for preparing said composition and its use.

Abstract

The present invention provides a PD-L1 antibody pharmaceutical composition and its use. In particular, the present invention provides a pharmaceutical composition comprising PD-L1 antibody or an antigen-binding fragment thereof in a succinate buffer. In addition, the pharmaceutical composition may also contain a sugar and a non-ionic surfactant.

Description

1 / 59 Pharmaceutical composition of PD-L1 antibody, method for preparing said composition and its use.

[001] This application claims priority from Chinese Patent Application No. CN201710341680.7 filed on May 16, 2017, the content of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[002] The present invention relates to the field of pharmaceutical preparations, in particular to a pharmaceutical composition comprising an anti-PD-L1 antibody or antigen-binding fragment thereof, and its use as an anticancer drug. BACKGROUND OF THE INVENTION

[003] Programmed death 1 (PD-1), a protein receptor expressed on the surface of T cells and discovered in 1992, is involved in the process of apoptosis. PD-1 has two ligands, namely PD-L1 and PD-L2. PD-L1 is expressed primarily on T cells, B cells, macrophages, and dendritic cells (DCs), and expression on activated cells can be upregulated. PD-L2 expression is relatively limited, being expressed primarily on antigen-presenting cells such as activated macrophages and dendritic cells.

[004] PD-L1 inhibits the immune system by binding to PD-1 and B7-1, and many tumor cells in the tumor microenvironment express PD-L1. Recent studies have found that high expression of the PD-L1 protein in human tumor tissues, such as breast cancer, lung cancer, stomach cancer, bowel cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, liver cancer, and others, and the level of PD-L1 expression is closely related to the clinical condition and prognosis of patients. Because PD-L1 acts as a second signaling pathway to inhibit the Petition 870240084342, dated 02 / 10 / 2024, page 23 / 104 2 / 59 T cell proliferation, blocking the PD-L1 / PD-1 binding has become a very promising emerging target in the field of tumor immunotherapy.

[005] Compared with other chemical drugs, antibody drugs become unstable due to their higher molecular weight, more complicated structure, and ease of degradation, polymerization, or undesirable chemical modification. In order to make antibody molecules suitable for administration and maintain stability during storage and subsequent use, and to achieve better effects, studies on the preparation of antibody drugs are particularly important.

[006] Currently, several multinational pharmaceutical companies are developing pharmaceutical compositions containing PD-L1 / PD-1 antibodies, such as CN105793288A, CN103429264A and CN105960415A.

[007] The present invention provides a pharmaceutical composition comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof that is sufficiently stable and suitable for administration. SUMMARY OF THE INVENTION

[008] The present invention provides a pharmaceutical composition comprising an anti-PD-L1 antibody or antigen-binding fragment thereof, and a buffer, wherein the buffer is preferably a succinate buffer or an acetate buffer, more preferably a succinate buffer.

[009] In embodiments of the present invention, the buffer concentration is from about 5 mM to 50 mM, preferably from about 10 mM to 30 mM, more preferably from 10 mM to 20 mM; non-limiting embodiments of the buffer concentration include 10 mM, 12 mM, 14 mM, 16 mM, 18 mM and 20 mM.

[010] In embodiments of the present invention, the pH of the composition Petition 870240084342, dated 02 / 10 / 2024, page 24 / 104 3 / 59 pharmaceutical is approximately 4.5 to 6.0, preferably approximately 4.8 to 5.7, more preferably 5.0 to 5.5, and may be 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5.

[011] In embodiments of the present invention, the antibody concentration in the pharmaceutical composition is from about 30 mg / mL to about 80 mg / mL, preferably from about 40 mg / mL to about 60 mg / mL, more preferably from 45 mg / mL to about 55 mg / mL; non-limiting embodiments of the antibody concentration include 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL and 55 mg / mL.

[012] In addition, the pharmaceutical composition of the present invention also comprises a saccharide. The saccharide of the present invention comprises the conventional composition (CH₂O)ₙ and derivatives thereof, including monosaccharide, disaccharide, trisaccharide, polysaccharide, polyol, reducing sugar, non-reducing sugar and the like, which may be selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, melitriose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, isomaltulose and the like. The saccharide is preferably a non-reducing disaccharide, more preferably trehalose or sucrose.

[013] In embodiments of the present invention, the concentration of the saccharide in the pharmaceutical composition is from about 30 mg / mL to about 90 mg / mL, preferably from about 50 mg / mL to about 70 mg / mL, more preferably from 55 mg / mL to about 65 mg / mL; non-limiting embodiments of the saccharide concentration include 55 mg / mL, 57 mg / mL, 59 mg / mL, 60 mg / mL, 61 mg / mL, 63 mg / mL and 65 mg / mL.

[014] In addition, the pharmaceutical composition also comprises a Petition 870240084342, dated 02 / 10 / 2024, page 25 / 104 4 / 59 surfactant, which may be selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer, Triton, sodium dodecyl sulfate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine or stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauryl amidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitoylamidopropyl betaine, or isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitoylamidopropyl dimethylamine, isostearamidopropyl dimethylamine, Sodium methyl cocoyl taurate, or disodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, and ethylene-propylene glycol copolymer, etc. The surfactant is preferably polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[015] In embodiments of the present invention, the surfactant concentration in the pharmaceutical composition is from about 0.1 mg / mL to 1.0 mg / mL, preferably 0.2 mg / mL to 0.8 mg / mL, more preferably 0.4 mg / mL to 0.8 mg / mL; non-limiting embodiments of surfactant concentration include 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL and 0.8 mg / mL.

[016] In embodiments of the present invention, wherein the antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises any of the CDR region sequences or mutant sequences selected from the group consisting of: antibody heavy chain variable region HCDR sequence: SEQ ID NO: 1 to 3, SEQ ID NO: 7 to 9; and / or antibody light chain variable region LCDR sequences: SEQ ID NO: 4 to 6, SEQ ID NO: 10 to 12; specifically as follows: Petition 870240084342, dated 02 / 10 / 2024, page 26 / 104 5 / 59 HCDR1 is selected from: NDYWXi SEQ ID NO: 1 or SYWMH SEQ ID NO: 7, HCDR2 is selected from: YISYTGSTYYNPSLKS SEQ ID NO: 2 or RIX4PNSG X5TSYNEKFKN SEQ ID NO: 8, and / or HCDR3 is selected from: SGGWLAPFDY SEQ ID NO: 3 or GGSSYDYFDY SEQ ID NO: 9; and / or LCDR1 is selected from: KSSQSLFY X2 SNQK X3SLA SEQ ID NO: 4 or RASESVSIHGTHLMH SEQ ID NO: 10, LCDR2 is selected from: GASTRES or AASNLES LCDR3 is selected from: QQYYGYPYT or QQSFEDPLT SEQ ID NO: 5 SEQ ID NO: 11, and / or SEQ ID NO: 6 SEQ ID NO: 12; where X1 is selected from N or T, X2 is selected from R or H, X3 is selected from N or H, X4 is selected from H or G, and X5 is selected from G or F.

[017] In embodiments of the present invention, preferably, the antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises a variable region CDR sequence of the light chain selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or a mutated sequence thereof, and a variable region CDR sequence of the heavy chain selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or a mutant sequence thereof; more preferably, the antibody or antigen-binding fragment thereof comprises the sequences LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and sequences HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; Petition 870240084342, dated 02 / 10 / 2024, p. 27 / 104 6 / 59 or, preferably, the antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises a variable region CDR sequence of the heavy chain selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or a mutated sequence thereof, and a variable region CDR sequence of the light chain selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or a mutant sequence thereof; more preferably, the antibody or antigen-binding fragment thereof comprises the sequences HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and sequences LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively;

[018] In embodiments of the present invention, the antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises a variable region CDR light chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and a variable region CDR heavy chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity sequence with the amino acid sequences, as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[019] In embodiments of the present invention, the antibody or antigen-binding fragment thereof in the pharmaceutical composition may be selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody and a human antibody, preferably a humanized antibody.

[020] In embodiments of the present invention, the antibody or fragment of Petition 870240084342, dated 02 / 10 / 2024, p. 28 / 104 7 / 59 The antigen linkage in the pharmaceutical composition comprises a variable region heavy chain sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence, as shown in SEQ ID NO: 13, and a variable region light chain sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence, as shown in SEQ ID NO: 14.

[021] In embodiments of the present invention, the antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises a variable region heavy chain sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence, as shown in SEQ ID NO: 15, and a variable region light chain sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence, as shown in SEQ ID NO: 17.

[022] The present invention further provides a pharmaceutical composition comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof with a concentration of about 30 mg / mL to about 80 mg / mL, a succinate buffer at pH 5.0-6.0 with a concentration of about 5 mM to about 50 mM, a disaccharide with a concentration of about 30 mg / mL to about 90 mg / mL and a polysorbate 80 with a concentration of about 0.1 mg / mL to about 1.0 mg / mL.

[023] The present invention further provides a pharmaceutical composition comprising 40 mg / mL to 60 mg / mL of anti-PDL1 antibody or antigen-binding fragment thereof, 10 mM to 30 mM of Petition 870240084342, dated 02 / 10 / 2024, page 29 / 104 8 / 59 succinate buffer at pH 5.0-5.5, 40 mg / mL to 80 mg / mL of sucrose and 0.4 mg / mL to 0.8 mg / mL of polysorbate 80.

[024] The present invention further provides a pharmaceutical composition comprising 45 mg / mL to 55 mg / mL of PD-L1 antibody or antigen-binding fragment thereof, 10 mM to 20 mM of succinate buffer at pH 5.0-5.5, 55 mg / mL to 65 mg / mL of sucrose and 0.5 mg / mL to 0.7 mg / mL of polysorbate 80.

[025] The present invention further provides a pharmaceutical composition comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof with a concentration of about 30 mg / mL to about 80 mg / mL, an acetate buffer at pH 5.0 to 6.0 with a concentration of about 5 mM to about 50 mM, a disaccharide with a concentration of about 30 mg / mL to about 90 mg / mL and a polysorbate 80 with a concentration of about 0.1 mg / mL to about 1.0 mg / mL.

[026] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 10 mM succinate buffer at pH 5.3 and 60 mg / mL of sucrose.

[027] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 10 mM acetate buffer at pH 5 and 90 mg / mL of sucrose.

[028] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 30 mM acetate buffer at pH 5 and 60 mg / mL of trehalose.

[029] The present invention further provides a composition Petition 870240084342, dated 02 / 10 / 2024, page 30 / 104 9 / 59 pharmaceutical comprising 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 30 mM acetate buffer at pH 5 and 60 mg / mL of trehalose.

[030] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 30 mM acetate buffer at pH 5.6 and 90 mg / mL of sucrose.

[031] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 10 mM succinate buffer at pH 5.0 to 5.5, 60 mg / mL of sucrose and 0.2 mg / mL of polysorbate 20.

[032] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 10 mM to 20 mM succinate buffer at pH 5.2, 60 mg / mL of sucrose and 0.2 mg / mL of polysorbate 20.

[033] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of PD-L1 antibody or antigen-binding fragment thereof, 20 mM acetate buffer at pH 5.2, 60 mg / mL of sucrose and 0.1 to 0.3 mg / mL of polysorbate 20.

[034] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of PD-L1 antibody or antigen-binding fragment thereof, 20 mM acetate buffer at pH 5.2, 60 mg / mL of sucrose and 0.1 mg / mL to 0.3 mg / mL of polysorbate 80.

[035] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.2, 60 mg / mL of sucrose and 0.2 mg / mL to 0.6 mg / mL of polysorbate 20. Petition 870240084342, dated 02 / 10 / 2024, page 31 / 104 10 / 59

[036] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.2, 60 mg / mL of sucrose and 0.4 mg / mL to 0.8 mg / mL of polysorbate 80.

[037] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.2, 60 mg / mL of sucrose and 0.4 mg / mL of polysorbate 80.

[038] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.2, 60 mg / mL of sucrose and 0.6 mg / mL of polysorbate 80.

[039] The present invention further provides a pharmaceutical composition comprising 50 mg / mL of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.2, 60 mg / mL of sucrose and 0.8 mg / mL of polysorbate 80.

[040] In some embodiments, the concentration of succinate buffer in the pharmaceutical composition is about 5 mM to 50 mM. In some embodiments, the concentration of succinate buffer in the pharmaceutical composition is about 10 mM to 30 mM. In some embodiments, the concentration of succinate buffer in the pharmaceutical composition is about 20 mM.

[041] In some embodiments, the concentration of acetate buffer in the pharmaceutical composition is about 5 mM to 50 mM. In some embodiments, the concentration of acetate buffer in the pharmaceutical composition is about 10 mM to 30 mM. In some embodiments, the concentration of acetate buffer in the pharmaceutical composition is about 20 mM.

[042] In some forms, the pH of the pharmaceutical composition is Petition 870240084342, dated 02 / 10 / 2024, p. 32 / 104 11 / 59 around 5.0 to 6.0. In some embodiments, the pH of the pharmaceutical composition is around 5.0 to 5.5. In some embodiments, the pH of the pharmaceutical composition is around 5.2 to 5.5.

[043] In some embodiments, the antibody concentration in the pharmaceutical composition is from about 30 mg / mL to about 80 mg / mL. In some embodiments, the antibody concentration in the pharmaceutical composition is from about 40 mg / mL to about 60 mg / mL. In some embodiments, the antibody concentration in the pharmaceutical composition is about 50 mg / mL.

[044] In some embodiments, the concentration of the disaccharide in the pharmaceutical composition is from about 30 mg / mL to about 90 mg / mL. In some embodiments, the concentration of the disaccharide in the pharmaceutical composition is from about 40 mg / mL to about 80 mg / mL. In some embodiments, the concentration of the disaccharide in the pharmaceutical composition is about 60 mg / mL.

[045] In some embodiments, the polysorbate in the pharmaceutical composition is polysorbate 20 or polysorbate 80. In some embodiments, the polysorbate in the pharmaceutical composition is polysorbate 80. In some embodiments, the concentration of polysorbate in the pharmaceutical composition is about 0.1 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of polysorbate in the pharmaceutical composition is about 0.4 mg / mL to 0.8 mg / mL. In some embodiments, the concentration of polysorbate in the pharmaceutical composition is about 0.6 mg / mL.

[046] In some forms, the preparation is stable at 2 to 8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months or at least 24 months. In some forms, the preparation is stable at 40°C for at least 7 days, at least 14 days or at least 28 days.

[047] The present invention further provides an article or kit that Petition 870240084342, dated 02 / 10 / 2024, p. 33 / 104 12 / 59 comprises a container containing any of the stable pharmaceutical compositions described herein. In some embodiments, the container is a glass vial and the glass vial is a neutral borosilicate glass vial for injection.

[048] The present invention further provides a method for preparing the pharmaceutical compositions described above, comprising mixing an anti-PD-L1 antibody or an antigen-binding fragment thereof with a pharmaceutically acceptable excipient.

[049] The present invention further provides a use of the pharmaceutical composition described above in the manufacture of a medicament for the treatment of a disease or condition mediated by PD-L1, wherein the disease or condition is preferably a cancer; more preferably a cancer that expresses PD-L1; more preferably breast cancer, lung cancer, stomach cancer, bowel cancer, kidney cancer, melanoma or non-small cell lung cancer; even more preferably non-small cell lung cancer, melanoma, bladder cancer or kidney cancer.

[050] The present invention provides a method for treating or preventing a disease or condition mediated by PD-L1, comprising administering a therapeutically effective amount of the pharmaceutical composition comprising an anti-PD-L1 antibody or antigen-binding fragment thereof to an individual in need thereof; wherein the disease is preferably a cancer; more preferably a cancer expressing PD-L1; the cancer is more preferably breast cancer, lung cancer, stomach cancer, bowel cancer, kidney cancer, melanoma, non-small cell lung cancer or bladder cancer; more preferably non-small cell lung cancer, melanoma, Petition 870240084342, dated 02 / 10 / 2024, page 34 / 104 13 / 59 bladder cancer or kidney cancer.

[051] It is appreciated that one, some or all of the features of the various embodiments described in the present invention may be further combined to obtain other embodiments of the present invention. Other embodiments obtained by combining the above embodiments of the present invention are further described in the detailed description below. BRIEF DESCRIPTION OF THE FIGURES

[052] Figure 1: Schematic diagram of primer design during humanized clone construction.

[053] Figure 2: Schematic diagram of the vector construction during the construction of the humanized clone.

[054] Figure 3: Main effect plot for the Tm factors (including buffer system, buffer concentration, pH of the pharmaceutical composition, saccharide concentration and saccharide type). DETAILED DESCRIPTION OF THE INVENTION TERMS

[055] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless specifically defined otherwise in the present invention, all other technical and scientific terms used in the present invention should be considered as having the same meaning as that commonly understood by one skilled in the art to which this invention pertains.

[056] Buffer refers to a buffer resistant to changes in pH due to its conjugate acid-base component. The pH value of the buffer of the present invention is about 4.5 to 6.0, preferably about 5.0 to 6.0, more preferably about 5.0 to 5.5, and even more preferably 5.2. Examples of buffers controlling pH in such a range Petition 870240084342, dated 02 / 10 / 2024, page 35 / 104 14 / 59 include acetate buffer, succinate buffer, gluconate buffer, histidine buffer, oxalate buffer, lactate buffer, phosphate buffer, citrate buffer, tartrate buffer, fumarate buffer, glycylglycine and other organic acid buffers. The buffer of the present invention is preferably succinate buffer or acetate buffer, more preferably succinate buffer.

[057] Succinate buffer refers to a buffer that includes succinate ions. Examples of succinate buffers include sodium succinate-succinate, histidine succinate, potassium succinate-succinate, calcium succinate-succinate, and the like. The succinate buffer of the present invention is preferably sodium succinate-succinate.

[058] Acetate buffer refers to a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine acetic acid, acetic acid-potassium acetate, calcium acetate, acetic acid-magnesium acetate, and the like. The preferred acetate buffer of the present invention is acetic acid-sodium acetate.

[059] Pharmaceutical composition refers to a mixture comprising one or more of the compounds described in the present invention or the physiologically / pharmaceutically acceptable salt thereof or prodrug thereof with other chemical components. Said other chemical components are, for example, physiologically / pharmaceutically acceptable vehicles and excipients. The objective of the pharmaceutical composition is to promote administration within the organism, which facilitates the absorption of the active ingredient, thus exerting biological activity.

[060] The pharmaceutical composition of the present invention is capable of achieving a stable effect: the antibody contained therein can substantially retain its physical stability and / or chemical stability and / or biological activity after storage; preferably, the pharmaceutical composition retains Petition 870240084342, dated 02 / 10 / 2024, page 36 / 104 15 / 59 substantially its physical stability, chemical stability, and biological activity after storage. Shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. Currently, there are several analytical techniques for measuring protein stability that measure stability after storage for a selected period at a selected temperature.

[061] A stable antibody pharmaceutical preparation is one in which no significant alteration is observed under the following conditions: storage at a refrigerated temperature (2 to 8°C) for at least 3 months, preferably for 6 months, more preferably for 1 year, and even more preferably for up to 2 years. In addition, a stable liquid preparation includes a liquid preparation that exhibits a desired characteristic during storage at temperatures including 25°C and 40°C for periods including 1 month, 3 months, and 6 months. Typical acceptable criteria for stability are as follows: typically no more than about 10%, preferably no more than about 5% of the antibody monomer is degraded, as assessed by SEC-HPLC. The liquid pharmaceutical preparation is colorless or clear to slightly opalescent white by visual analysis. The concentration, pH, and osmolarity of the preparation do not vary by more than ±10%.Generally, no more than 10%, preferably 5%, cleavage is observed. Generally, no more than 10%, preferably 5%, aggregation is observed.

[062] An antibody retains its physical stability in a pharmaceutical preparation if it does not exhibit significant increase in aggregation, precipitation and / or denaturation after visual examination of color and / or transparency, or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS). Changes in Petition 870240084342, dated 02 / 10 / 2024, page 37 / 104 16 / 59 Protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[063] An antibody retains its chemical stability in a pharmaceutical preparation if it does not show significant chemical alteration. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of the protein include hydrolysis or cleavage (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping in conjunction with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement) and isomerization (evaluated by measuring isoaspartic acid content, peptide mapping, etc.).

[064] An antibody retains its biological activity in a pharmaceutical preparation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical preparation was prepared. The biological activity of an antibody can be determined, for example, by an antigen binding assay.

[065] The three-letter and single-letter codes for amino acid residues used herein are described in J. Biol. Chem. 243, p. 3558 (1968).

[066] The antibody, as used in the present invention, refers to an immunoglobulin, which is a tetrapeptide chain structure linked by disulfide bonds between two identical heavy chains and two identical light chains. The difference lies in the amino acid composition and the order of the region Petition 870240084342, dated 02 / 10 / 2024, p. 38 / 104 The constant in the heavy chain (17 / 59) results in differences in the antigenicity of immunoglobulins. Therefore, immunoglobulins can be classified into five classes, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are: μ chain, δ chain, γ chain, α chain, and ε chain, respectively. According to their amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same Ig isotype can be divided into different subclasses; for example, IgG can be classified as IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ chain or λ chain according to the difference in the constant region. Each of the five Ig classes can have either a κ chain or a λ chain.

[067] In the present invention, the antibody light chain of the present invention may further comprise a constant light chain region comprising a human or murine κ or λ chain or a variant thereof.

[068] In the present invention, the antibody heavy chain of the present invention may further comprise a constant heavy chain region comprising human or murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[069] Approximately 110 amino acid sequences adjacent to the N-terminus of the antibody heavy and light chains are highly variable, known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, known as constant regions. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions that determine antibody specificity are also known as the complementarity-determining region (CDR). Each Petition 870240084342, dated 02 / 10 / 2024, page 39 / 104 Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, sequentially ordered from the amino terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three light chain CDR regions are referred to as LCDR1, LCDR2, and LCDR3, and the three heavy chain CDR regions are referred to as HCDR1, HCDR2, and HCDR3. The number and position of the amino acid residues of the CDR region in the LCVR and HCVR regions of the antibody or antigen-binding fragments described in the present invention comply with the known Kabat numbering criteria (LCDR1-3, HCDE2-3) or the Kabat and Chothia numbering criteria (HCDR1).

[070] The antibody of the present invention includes a murine antibody, a chimeric antibody and a humanized antibody, preferably a humanized antibody.

[071] The term murine antibody in the present invention refers to a monoclonal antibody against human PD-L1 prepared in accordance with the knowledge and skills of the field. During preparation, PD-L1 antigen was injected into a test subject and then the hybridoma expressing the antibody with the desired sequence or functional properties was separated. In a preferred embodiment of the present invention, the murine PD-L1 antibody or antigen-binding fragment thereof may further comprise a constant region of murine κ or λ chain light chain or a variant thereof, or even comprise a constant region of murine IgG1, IgG2, IgG3 heavy chain or a variant thereof.

[072] The term chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, and the chimeric antibody can alleviate the immune response induced by the murine antibody. To construct a chimeric antibody, Petition 870240084342, dated 02 / 10 / 2024, p. 40 / 104 19 / 59 First, a hybridoma that secretes a specific murine monoclonal antibody is constructed; a gene from the variable region is cloned from mouse hybridoma cells. Subsequently, a gene from the constant region of a human antibody is cloned as desired; the mouse variable region gene is linked to the human constant region gene to form a chimeric gene that can be introduced into a human vector; and finally, a chimeric antibody molecule is expressed in the eukaryotic or prokaryotic industrial system. In a preferred embodiment of the present invention, the light chain of the chimeric antibody PD-L1 further comprises the constant light chain regions of the human κ or λ chain or a variant thereof. The heavy chain of the chimeric antibody PD-L1 further comprises the constant heavy chain regions of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof.The constant region of a human antibody can be selected from the constant region of the heavy chain of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising the constant region of the heavy chain of human IgG2 or IgG4 or IgG4 without ADCC (antibody-dependent cell-mediated cytotoxicity) after amino acid mutation.

[073] The term humanized antibody, also known as CDR-grafted antibody, refers to an antibody generated by grafting murine CDR sequences onto a variable region framework of a human antibody, namely, an antibody produced from a different type of human germline antibody framework sequence. A humanized antibody overcomes the disadvantage of the strong antibody response induced by the chimeric antibody, which carries a large number of murine protein components. Such framework sequences can be obtained from a public DNA database covering germline antibody gene sequences. Petition 870240084342, dated 02 / 10 / 2024, page 41 / 104 20 / 59 germline or published references. For example, germline DNA sequences of the human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available at www.mrccpe.com.ac.uk / vbase), and can also be found in Kabat, EA, et al., 1991 Sequences of Proteins of Immunological Interest, 55th Ed. To avoid decreased activity during decreased immunogenicity, the framework sequences in the variable region of the human antibody are subjected to minimal mutations or backmutations to maintain activity. The humanized antibody of the present invention also comprises a humanized antibody to which CDR affinity maturation is performed by display in phages.

[074] Antigen-binding fragment in the present invention refers to a Fab fragment, a Fab' fragment, or an F(ab')2 fragment with antigen-binding activity, as well as an Fv or scFv fragment that binds to human PD-L1; it comprises one or more CDR regions of the antibodies described in the present invention, selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12. An Fv fragment comprises the variable region of the heavy chain and the variable region of the light chain, without a constant region, and is a minimal antibody fragment possessing all antigen-binding sites. Generally, an Fv antibody further comprises a polypeptide linker between the VH and VL domains and is capable of forming a structure necessary for antigen binding. In addition, different linkers can be used to connect the variable regions of two antibodies to form a polypeptide chain, referred to as a single-chain antibody or single-chain Fv (scFv).The term PD-L1 binding in the present invention means that it is capable of interacting with human PD-L1. The term antigen-binding sites in the present invention refers to the discontinuous three-dimensional sites on the... Petition 870240084342, dated 02 / 10 / 2024, p. 42 / 104 21 / 59 antigen, recognized by the antibody or antigen-binding fragment of the present invention.

[075] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the state of the art and can be found, for example, in the Antibody Experimental Technology Guide of Cold Spring Harbor, chapters 5 to 8 and 15. For example, a mouse can be immunized with human PD-L1 or a fragment thereof, and the antibody obtained can be renatured, purified, and subjected to amino acid sequencing by a conventional method. The antigen-binding fragment can also be prepared by a conventional method. The antibody or antigen-binding fragments of the present invention are genetically engineered to introduce one or more human framework regions (FRs) into a non-human derived CDR region. Human FR germline sequences can be obtained with ImMunoGeneTics (imgT), at http: / / imgt.cines.fr, or in The Immunoglobulin Facts Book, 2001 ISBN 012441351.

[076] The manipulated antibody or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. For example, cDNA sequences encoding a heavy chain and a light chain can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can then be stably transfected into CHO cells. As a more recommended and well-known method in the state of the art, mammalian expression systems will result in antibody glycosylation, typically at the highly conserved N-terminus in the Fc region. Stable clones can be obtained by expressing an antibody that specifically binds to human PD-L1. Positive clones can be expanded in serum-free culture medium for antibody production in bioreactors. The culture medium in which a Petition 870240084342, dated 02 / 10 / 2024, page 43 / 104 22 / 59 The secreted antibody can be purified by conventional techniques. For example, the medium can be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with adjusted buffer. The column is washed to remove non-specific binding components. The bound antibody is eluted by pH gradient and the antibody fragments are detected by SDS-PAGE and then pooled. The antibody can be filtered and concentrated using common techniques. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion or ion exchange. The product obtained can be immediately cryopreserved, for example, at -70°C, or it can be lyophilized.

[077] Conservative modifications or conservative substitutions refer to the replacement of amino acids in a protein with other amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, conformation and rigidity of the main chain, etc.), such that the changes can be made frequently without altering the biological activity of the protein. Those skilled in the art recognize that, in general, individual amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). Furthermore, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity.

[078] Identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base subunit or amino acid monomer, for example, if a position in each of the two DNA molecules is occupied by adenine, the molecules are identical in that respect. Petition 870240084342, dated 02 / 10 / 2024, page 44 / 104 23 / 59 position. The percentage of identity between the two sequences is a function of the number of matched or consistent positions shared by the two sequences divided by the number of positions compared x 100. For example, in the ideal alignment of sequences, if there are 6 matches or consistencies in 10 positions of the two sequences, the two sequences will have 60% identity. In general, comparisons are made when the two sequences are aligned to obtain the highest percentage of identity.

[079] Administration and treatment, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact of a pharmaceutical, therapeutic, diagnostic agent, or exogenous composition with the animal, human, individual, cell, tissue, organ, or biological fluid. Administration and treatment may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses the contact of a reagent with the cell, as well as the contact of a reagent with a fluid, where the fluid is in contact with the cell. Administration and treatment also mean in vitro and ex vivo treatments, for example, of a cell, by a reagent, diagnostic, binding compound, or by another cell. Treatment, as applied to humans, veterinarians, or a research subject, refers to therapeutic treatment, prophylactic or preventive measures, research applications, and diagnostics.

[080] Treating means administering a therapeutic agent, such as a composition comprising any of the binding compounds of the present invention, internally or externally to a patient with one or more disease symptoms for which the agent has known therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated patient or population, so as to Petition 870240084342, dated 02 / 10 / 2024, page 45 / 104 24 / 59 induce regression or inhibit the progression of such symptoms to any clinically measurable degree. The amount of a therapeutic agent that is effective in relieving any specific symptom of the disease (also referred to as the therapeutically effective amount) may vary depending on factors such as disease status, age and weight of the patient, and the ability of the drug to elicit a desired response in the patient. Whether a symptom of the disease has been relieved can be assessed by any clinical measure normally used by physicians or other specialized healthcare professionals to evaluate the severity or progression status of that symptom.Although an embodiment of the present invention (for example, a treatment method or article of manufacture) may not be effective in relieving the symptoms of the disease of interest in all patients, it should relieve the symptoms of the target disease of interest in a statistically significant number of patients as determined by any statistical test known in the art, such as the Student's t-test, the chi-square test, the Mann-Whitney U test, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[081] Effective amount encompasses an amount sufficient to alleviate or prevent a symptom or sign of a medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a given patient or individual veterinarian may vary depending on factors such as the condition being treated, the patient's overall health, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing protocol that avoids significant side effects or toxic effects.

[082] The Tm value refers to the midpoint of thermal denaturation of the protein, namely, the temperature at which half of the protein is unfolded and the Petition 870240084342, dated 02 / 10 / 2024, page 46 / 104 25 / 59 The spatial structure of the protein is destroyed. Therefore, the higher the Tm value, the greater the thermal stability of the protein.

[083] The invention is further described with the following embodiments, which are not intended to limit the scope of the invention. The experimental methods in the embodiments of the present invention that do not specify specific conditions are generally carried out in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer of the raw material or product. Reagents without a specific source are commercially available routine reagents.

[084] In the modalities, the AgilentHPLC 1260 high-pressure liquid chromatograph (Waters Xbridge® BEH 200A SEC 3.5μ^ι column 7.8x300mm and Thermo ProPac™ WCX-10 BioLC™, column 250x4mm) was used to measure SE-HPLC and IEC-HPLC. The Beckman PA800 plus capillary electrophoresis apparatus (MW SDS-Gel Analysis Kit) was used to measure reducing CE-SDS and non-reducing CE-SDS. The GE MicroCal VP-Capillary DSC differential scanning calorimeter was used to measure the midpoint of the thermal denaturation temperature of the protein (Tm). The Nano ZS Malvern Zetasizer nanoparticle size potentiometer was used to measure the average particle size of DLS (Dynamic Light Scattering). MODE 1: PREPARATION OF PD-L1 ANTIBODY (1) Preparation of PD-L1 antigen and the protein used for detection

[085] The complete human PD-L1 gene (Sino Biological Inc., HG10084-M) of Programmed Cell Death Ligand 1 UniProt (PD-L1) isoform 1 (SEQ ID NO: 19) was used as a template for the PD-L1 of the present invention. A genetic sequence encoding the antigen of the present invention and the protein used for detection was obtained, optionally recombined with the Fc heavy chain fragment of the antibody (such as human IgG1), cloned into a vector. Petition 870240084342, dated 02 / 10 / 2024, page 47 / 104 26 / 59 pTT5 (Biovector, Cat #: 102762) or a pTargeT vector (promega, A1410), subjected to transient expression in 293F cells (Invitrogen, R79007) or stable expression in CHO-S cells (Invitrogen, k9000-20), and purified to obtain the antigen and detection protein of the present invention. The human PD-1 gene was acquired from ORIGENE, Art. NO SC117011, NCBI Reference sequence: NM_005018.1. 1. Complete amino acid sequence of human PD-L1 MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGAILLC .LGVALTFIFr lrkgrmmdvk kcgiqdtnsk kqsdthleet

[086] SEQ ID NO: 19

[087] Note: The double underlined portion is the signal peptide (1 to 18); the single underlined portion is the extracellular domain (19 to 238) of PD-L1, where 19 to 127 is a V-type domain of the Ig analog, 133 to 225 is a C2-type domain of the Ig type; the dotted underlined portion is the transmembrane domain (239 to 259); and the italicized portion is the cytoplasmic domain (260 to 290). 2. Immunogen: PD-L1 with His and PADRE tags: PD-L1 (Extra Cellular Domain, Abbreviated ECD) -PADRE-His6 FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERGS GAKFVAAWTL KAAA HHHHHH Petition 870240084342, dated 02 / 10 / 2024, page 48 / 104 27 / 59

[088] SEQ ID NO: 20

[089] Note: The single underlined part is the extracellular domain of PDL1; the dotted underlined part is the PADRE tag; and the italicized part is the His6 tag. 3. PD-L1 with FLAG and HIS labels was obtained; PD-L1 (ECD)-Flag-His6 was used to test the performance of the antibodies of the present invention. FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERDY KDDDDKHHHH HH

[090] SEQ ID NO: 21

[091] Note: The single underlined part is the extracellular domain of PDL1; the dotted underlined part is the FLAG-Tag label; and the italicized part is the His6 tag. 4. PD-L1 Fc Fusion Protein: PD-L1 (ECD)-Fc was used as the immunological antigen or detection reagent of the present invention.

[092] VKL-PD-L1 (ECD)-Fc (human IgG1) FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHG EEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGA DYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQ VLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPE LPLAHPPNER DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPS Petition 870240084342, dated 02 / 10 / 2024, page 49 / 104 28 / 59 DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

[093] SEQ ID NO: 22

[094] Note: The single underlined portion is the extracellular domain of PDL1; and the italicized portion is the Fc portion of human IgG1. 5. PD-L1 Fc Fusion Protein: PD-L1 (ECD)-Fc-His6 was used to test the performance of the antibodies of the present invention. PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKL AAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESL RAELRVTERRAEVPTAHPSPSPRPAGQFQTLV EPKSSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK

[095] SEQ ID NO: 23

[096] Note: The single underlined portion is the extracellular domain (ECD) of PD-L1; and the italicized portion is the hFc portion (human IgG1). (2) Purification of PD-L1, recombinant PD-1 protein, as well as hybridoma antibody and recombinant antibody 1. Purification of PD-L1 with His and PADRE label: Recombinant PD-L1 protein (ECD)-PADRE-His6 (SEQ ID NO: 20)

[097] The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the buffer was displaced with PBS, followed by the addition of imidazole to a final concentration of 5 mM. The nickel column was equilibrated with PBS solution containing 5 mM imidazole and washed with 2 to 5 column volumes. The supernatant sample was loaded onto a Ni column (GE, 17-5318-01). The column was washed with PBS containing imidazole at Petition 870240084342, dated 02 / 10 / 2024, pp. 50 / 104 The column was then rinsed with 10 mM PBS plus imidazole to remove impure non-specific binding protein, and the effluent was collected. The target protein was eluted with 300 mM PBS containing imidazole, and the elution peak was collected. The collected eluent was concentrated and subsequently purified by Superdex 200 gel chromatography (GE) with PBS as the mobile phase. Polymer peaks were discarded, and elution peaks were collected. The obtained protein was confirmed by identification electrophoresis, peptide mapping (Agilent, 6530 Q-TOF), and LC-MS (Agilent, 6530 Q-TOF), and divided into aliquots for further use. PD-L1 with His and PADRE label: PD-L1 (ECD)-PADRE-His6 (SEQ ID NO: 2) was obtained and used as an immunogen for the antibody of the present invention. 2. Purification of recombinant PD-L1 (ECD)-Flag-His6 protein (SEQ ID NO: 21) with His tag and Flag tag

[098] The sample was centrifuged at high speed to remove impurities and concentrated to a desired volume. The protein peak eluted from the IMAC column, as above, was loaded onto a signaling affinity column equilibrated with 0.5xPBS (Sigma, A2220) and washed with 2-5 column volumes. The cell expression supernatant sample was loaded onto the column after impurity removal. The column was washed with 0.5xPBS until the A280 reading dropped to baseline. The column was washed with 0.3M NaCl-containing PBS, and the impure protein was eluted and collected. The target protein was eluted with 0.1M acetic acid (pH 3.5-4.0) and collected, followed by pH adjustment to neutral. The collected elute was concentrated and subsequently purified by Superdex 200 gel chromatography (GE) with PBS as the mobile phase. The polymer peaks were discarded and the elution peaks were collected.The collected sample was confirmed by electrophoresis identification, peptide mapping, and LC-MS, and divided into aliquots for later use. Petition 870240084342, dated 02 / 10 / 2024, page 51 / 104 30 / 59 PD-L1 with FLAG label and HIS label, for example, PD-L1 (ECD)-Flag-His6 (SEQ ID NO: 3) was obtained for testing the performance of the antibody of the present invention. 3. Purification of the Fc fusion protein from PD-L1 and PD-1

[099] The cell expression supernatant sample was centrifuged at high speed to remove impurities, concentrated to a desired volume, and loaded onto a protein A column (GE, 17-5438-01). The column was washed with PBS until the A280 reading dropped to baseline. The protein of interest was eluted with 100 mM sodium acetate at pH 3.0. The protein neutralized by 1M TrisHCl was further purified by balanced gel chromatography with PBS Superdex 200 (GE). Polymer peaks were discarded, and elution peaks were collected and aliquoted for later use. This method was used to purify PD-L1 (ECD)-Fc (SEQ ID NO: 4) and PD-1 (ECD)-Fc (SEQ ID NO: 5). PD-L1 (ECD)-Fc can be used as an immunizing antigen or detection reagent of the present invention, and PD-1 (ECD)-Fc can be used to test the performance of the antibody of the present invention. (3) Preparation of human anti-PD-L1 hybridoma monoclonal antibody 1. Immunization

[0100] Human anti-PD-L1 monoclonal antibody was produced from the immunization of a mouse, in which 6-week-old female white SJL mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001) were used. Feeding environment: SPF level. After the mice were purchased, they were reared in a laboratory environment (12 / 12 hour light / dark cycle adjustment, temperature 20 to 25°C; humidity 40 to 60%) for 1 week. Mice that adapted to the environment were immunized in two schemes (Scheme A and Scheme B), 6 to 10 per group. The antigen Petition 870240084342, dated 02 / 10 / 2024, p. 52 / 104 31 / 59 immunizing agent was PD-L1 with the His and PADRE label: PD-L1 (ECD)-PADRE-His6 (SEQ ID NO: 20)

[0101] In Scheme A, Freund's adjuvant (sigma Lot No.: F5881 / F5506) was used for emulsification: complete Freund's adjuvant (CFA) was used for primary immunization and incomplete Freund's adjuvant (IFA) was used for the resting booster immunization. The antigen-to-adjuvant ratio was 1:1, 100 pg / mouse (primary immunization), 50 pg / mouse (booster immunization). The emulsified antigen was injected intraperitoneally at 100 μg / mouse on day 0 and injected once every two weeks after primary immunization for a total of 6 to 8 weeks.

[0102] In Scheme B, Titermax (sigma Lot Num: T2684) and Alum (Thremo Lot Num: 77161) were used for cross-immunization. The antigen-to-adjuvant ratio (Titermax) was 1:1, and the antigen-to-adjuvant ratio (Alum) was 3:1, 10 to 20 pg / mouse (primary immunization), and 5 pg / mouse (booster immunization). The emulsified antigen was injected intraperitoneally at 20 / 10 μg / mouse on day 0 and injected once weekly after primary immunization. Titermax was used interchangeably with Alum for 6 to 11 weeks. After four weeks of immunization, the antigen was administered by injection in the back or intraperitoneally, based on the condition of the back nodule and abdominal swelling. 2. Cell fusion

[0103] Mice with high serum antibody levels tending to plateau were selected for splenocyte fusion. Shock immunization was performed by intraperitoneal injection 72 hours before splenocyte fusion. Hybridoma cells were obtained by fusing splenic lymphocytes with Sp2 / 0 myeloma cells (ATCC® CRL-8287TM). Petition 870240084342, dated 02 / 10 / 2024, pp. 53 / 104 32 / 59 through an optimized PEG-mediated fusion procedure. Hybridoma cells were resuspended in complete HAT medium (RPMI-1640 medium containing 20% ​​FBS, 1xHAT and 1xQPI) and divided into aliquots in 96-well cell culture plates (1x10⁵ / 150 μL / well), followed by incubation at 37°C under 5% CQ2. On the 5th day after fusion, complete HAT medium was added, 50 μL / well, and incubated at 37°C under 5% CO₂. From day 7 to day 8 after fusion, according to cell growth density, the entire medium was changed to complete HT medium (RPMI-1640 medium containing 20% ​​FBS, 1xHT and 1xQPI) at 200 μL / well, and incubated at 37°C under 5% CO2. 3. Hybridoma cell screening

[0104] On day 10 to day 11 after fusion, the PD-L1 binding ELISA method was performed according to cell growth density. Positive well cells detected in the ELISA were subjected to PD-L1 / PD-1 binding blocking ELISA. The Q medium in the positive wells was altered, and the positive cells were expanded into 24-well plates according to cell density. Cells transferred to the 24-well plate were subjected to a new test and then to cell preservation and initial subcloning. Q positive cells screened after initial subcloning were subjected to cell preservation, followed by a second subcloning. Q positive cells screened after initial subcloning were subjected to cell preservation, followed by protein expression. Hybridoma cells that blocked PD-L1 and PD-1 binding were obtained by multiple fusions.

[0105] Hybridoma clone cells 1 and 2 were screened by blocking assay and binding assay. The antibodies were additionally prepared by the ascites method or by the serum-free cell culture method, and the antibodies were purified according to the purification modes for use in the test modes. Petition 870240084342, dated 02 / 10 / 2024, pp. 54 / 104 33 / 59

[0106] The variable region sequences of the murine antibody were obtained by sequencing, where the variable region CDR sequences of the hybridoma clones are shown in Table 1 below: Table 1 Heavy chain Light chain 1 HCDR1 NDYWX1 SEQ ID NO: 1 LCDR1 KSSQSLFYX2SNQKX3SLA SEQ ID NO: 4 HCDR2 YISYTGSTYYNPSLKS SEQ ID NO: 2 LCDR2 GASTRES SEQ ID NO: 5 HCDR3 SGGWLAPFDY SEQ ID NO: 3 LCDR3 QQYYGYPYT SEQ ID NO: 6 2 HCDR1 SYWMH SEQ ID NO: 7 LCDR1 RASESVSIHGTHLMH SEQ ID NO: 10 HCDR2 RIX4PNSGX5TSYNEKFKN SEQ ID NO: 8 LCDR2 AASNLES SEQ ID NO: 11 HCDR3 GGSSYDYFDY SEQ ID NO: 9 LCDR3 QQSFEDPLT SEQ ID NO: 12

[0107] Where Xi is selected from N or T, X2 is selected from R or H, X3 is selected from N or H, X4 is selected from H or G, and X5 is selected from G or F. (4) Humanization of the human anti-PD-L1 hybridoma monoclonal antibody

[0108] Germline genes with high homologous regions in the variable heavy and light chain regions were selected as models, aligning with the germline genetic database of the human antibody variable light chain region from the IMGT and MOE software. Murine antibody CDRs 1 and 2 were transplanted into the corresponding humanized model and Petition 870240084342, dated 02 / 10 / 2024, pp. 55 / 104 34 / 59 matured in affinity to form a variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0109] The humanized light chain template of murine antibody 1 is IGKV4-1*01 and hjk4.1, the humanized heavy chain template is IGHV4-30-4*01 and hjh2, and the humanized variable region sequences are as follows:

[0110] 1 hVH-CDR Graft QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWN WIRQHPGKGLEWIGYIS YTGSTYYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDY WGRGTLVTVSS

[0111] SEQ ID NO: 24

[0112] 1 hVL CDR Graft DIVMTQSPDSLAVSLGERATINCKSSQSLFYRSNQKNSLA WYQQKPGQPPK LLIYGASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGYPYT FGGG TKVEIK

[0113] SEQ ID NO: 25

[0114] The humanized light chain model of murine antibody 2 is IGKV7-3*01 and hjk2.1, the humanized heavy chain model is IGHV1-46*01 and hjh6.1, and the humanized variable region sequence is as follows:

[0115] 2- hVH.1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMH WVRQAPGQGLEWMG RIHPNSGGTSYNEKFKN R VTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYD YFDY WGQGTTVTVSS

[0116] SEQ ID NO: 26

[0117] 2-hVL.1 DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMH WYQQKPGQPPKLLIY AASNLES GVPARFSGSGSGTDFTLTINPVEANDTANYYCQQSFEDPLT FGQGTKLE IK Petition 870240084342, dated 02 / 10 / 2024, pp. 56 / 104 35 / 59

[0118] SEQ ID NO: 27

[0119] Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the italicized part is the FR sequence and the underlined part is the CDR sequence. (5) Construction of a humanized clone

[0120] The primers were designed to construct the VH / VK gene fragment of each humanized antibody, and then recombined homologously with the pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC / CL)) to construct the complete antibody expression vector VH-CH1-FC-pHr / VK-CL-pHr.

[0121] 1. Primer design: The online software DNAWorks (v3.2.2) (http: / / helixweb.nih.gov / dnaworks / ) was used to design a plurality of primers to synthesize gene fragments containing VH / VK necessary for recombination: signal peptide 5'-30bp + VH / VK + 30bp CH1 / CL-3'. Principle of primer design: if there are different amino acids between target gene 2 and target gene 1, another primer comprising the mutation site was then designed, as shown in Fig. 1.

[0122] 2. Fragment splicing: according to the operating instructions of the STAR GXL DNA polymerase TaKaRa primer, the VH / VK-containing gene fragments required for recombination were obtained by two PCR amplification steps using a plurality of primers designed above.

[0123] 3. Construction and enzymatic digestion of the pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC / CL))

[0124] The pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC / CL)) was constructed using some special restriction endonucleases with a special design among which the recognition sequence was different from the restriction sites, such as BsmBI. The Petition 870240084342, dated 02 / 10 / 2024, page 57 / 104 The schematic diagram of the construction, shown in Figure 2, is shown. The vector was digested with BsmBI and the gel was extracted for use.

[0125] 4. Recombinant construction of the VH-CH1-FCpHr / VK-CL-pHr expression vector

[0126] VH / VK containing gene fragments necessary for recombination and the recovered and digested pHr expression vector with BsmBI (with signal peptide and constant region gene fragment (CH1-FC / CL)) were added to competent DH5H cells in a 3:1 molar ratio, maintained in an ice bath at 0°C for 30 minutes, subjected to heat shock at 42°C for 90 seconds, followed by the addition of 5 times the volume of LB medium, incubated at 37°C for 45 minutes and dispersed on the LB-Amp plate and incubated at 37°C overnight. The single clone was selected and sent for sequencing to obtain the target clone. (6) Affinity maturation of humanized anti-PD-L1 antibody 1. Construction of humanized PD-L1 antibody phagomid vectors 1 and 2

[0127] Humanized PD-L1 antibodies 1 and 2 were constructed in phagomid vectors in scFv mode (VH-(GGGGS)3-VL), respectively, as wild-type sequences (i.e., an original or initial sequence corresponding to mutant sequences screened for affinity maturation). The VH (GGGGS)3 and VL ligands were assembled by overlap PCR and ligated to the phagomid vector via NcoI and NotI restriction recognition sites. 2. Construction of the phage display library

[0128] The constructed wild-type scFv was used as a template and codon-based primers were used. In the primer synthesis process, each codon in the mutation region had 50% wild-type codon and 50% Petition 870240084342, dated 02 / 10 / 2024, pp. 58 / 104 37 / 59 of NNK (reverse primer was MNN), which introduced mutations in all CDR regions to construct a mutant library. The PCR fragment was digested with Ncol and Notl, ligated to a phagomid vector, and finally electrically transformed into E. coli TG1. An independent library was constructed for each codon-based primer, in which antibody 1 was split into 7 libraries and antibody 2 was split into 8 libraries. 3. Library screening

[0129] After the library was rescued and packaged into phage particles for screening, biotinylated human PD-L1 antigen (ECD) and streptavidin magnetic beads were used for liquid-phase screening, and the antigen concentration after each screening round decreased compared to the previous round. After three screening rounds, 250 clones were selected from antibody 1 and antibody 2 and subjected to phage-ELISA to detect binding activity, respectively, followed by sequencing of the positive clones. 4. Surface plasmon resonance (SPR) affinity detection

[0130] After sequencing the clones, redundant sequences were removed and non-redundant sequences were converted into full-length IG (γ1, κ) for mammalian cell expression. The complete IG after affinity purification was subjected to affinity detection using a BIAcore™ X-100 instrument (GE Life Sciences).

[0131] The variable region sequences of humanized antibody 2 after affinity maturation are shown below:

[0132] Variable heavy chain region: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMH WVRQAPGQGLEWMG RIGPNSGFTSYNEKFKN R VTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDY WGQGTTVTVSS Petition 870240084342, dated 02 / 10 / 2024, pp. 59 / 104 38 / 59

[0133] SEQ ID NO: 13

[0134] Where, X4 of CDR2 is G, X5 is F.

[0135] Variable light chain region: DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMH WYQQKPGQPPKLLIY AASNLES GVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLT FGQGTKLE IK

[0136] SEQ ID NO: 14

[0137] Note: The italicized part in the sequence is the FR sequence; the underlined part is the CDR sequence; and the site with double underlining is the site obtained after affinity maturation tracking.

[0138] The variable region sequences of humanized antibody 1 after affinity maturation are shown below:

[0139] Variable heavy chain region QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWT WIRQHPGKGLEYIGYIS YTGSTYYNPSLKS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDY WGRGTLVTVSS

[0140] SEQ ID NO: 28

[0141] Where, X4 of CDR1 is T.

[0142] Variable light chain region: DIVMTQSPDSLAVSLGERATINCKSSQSLFYHSNQKHSLA WYQQKPGQPPKLLIYG ASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGYPYT FGGGTKVEIK

[0143] SEQ ID NO: 29

[0144] Where, CDR1 X2 is H and X3 is H.

[0145] The clone obtained by affinity maturation was converted to the IgG4 type, and IgG4 whose central hinge region contains the S228P mutation was selected to obtain an antibody without ADCC and CDC, in which the antibody obtained from antibody 2 was named HRP00052. Petition 870240084342, dated 02 / 10 / 2024, pp. 60 / 104 39 / 59

[0146] The last three TGA nucleotides of the following genetic sequences SEQ ID NO: 16 and 18 are stop codons and do not code for any amino acid.

[0147] Heavy chain sequence of antibody HRP00052 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEW MGRIGPNSG FTSYN EKFKN RVTMTRDTSTSTVYME LSSLRSEDTAVYYCARG GSSYDYFDYWGQGTTVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0148] SEQ ID NO: 15

[0149] Heavy chain sequence encoding the genetic sequence of antibody HRP00052 CAGGTGCAACTGGTGCAGAGCGGTGCCGAGGTGAAGAAGCCTGGCG CAAGCGTGAAAGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTAC TGGATGCACTGGGTGAGGCAGGCCCCTGGACAGGGCCTGGAGTGGATGGG CAGGATCGGGCCCAACAGTGGTTTCACTAGCTACAATGAAAAGTTCAAGA ACAGGGTAACCATGACCAGGGACACCTCCACCAGCACAGTGTATATGGAG CTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGTGCCAGAGG CGGCAGCAGCTACGACTACTTCGACTATTGGGGCCAGGGCACCACCGTGA CCGTGAGCAGTGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCT GCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTC Petição 870240084342, de 02 / 10 / 2024, pág. 61 / 104 40 / 59 CCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCT ACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAG AGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGGC TGCTGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCT CATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC AGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTG CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGG AGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATTCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCT GCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA CGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGC AGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA

[0150] SEQ ID NO: 16

[0151] HRP00052 antibody light chain sequence DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLL IYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

[0152] SEQ ID NO: 17

[0153] Light chain sequence encoding the genetic sequence of antibody HRP00052 GACATCGTGCTGACCCAGAGTCCCGCCTCACTTGCCGTGAGCCCCGGT Petition 870240084342, dated 02 / 10 / 2024, pp. 62 / 104 41 / 59 CAGAGGGCCACATCACCTGTAGGGCCAGCGAGAGCGTGAGCATCCACGG CACCCACCTGATGCACTGGTATCAACAGAAACCCGGCCAGCCCCCCAAAC TGCTGATCTACGCCGCCAGCAACCTGGAGAGCGGCGTGCCCGCCAGGTTC AGCGGCTCCGGCAGCGGCACCGACTTCACCCTCACTATCAACCCCGTGGA GGCCGAGGACACCGCCAACTACTACTGCCAGCAGAGCTTCGAGGACCCCC TGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCA CCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGA CGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGCCTGAGCTCCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGTTGA

[0154] SEQ ID NO: 18

[0155] Note: The underlined part is the sequence of the variable region of the antibody heavy or light chain, or the nucleotide sequence that encodes it; the ununderlined part is the sequence of the constant region of the antibody and the nucleotide sequence that encodes it.

[0156] An expression plasmid was constructed to express the PD-L1 antibody HRP00052. The nucleotide sequences encoding the heavy and light chains, their corresponding promoters, and their polyadenylation signal sequences were confirmed by DNA sequence analysis. The expression vector was transfected into a CHO cell line. Antibody-expressing clones were selected based on growth stability and production, followed by the preparation of a master seed bank, which was used to prepare antibodies and subsequently generate a master cell bank. Petition 870240084342, dated 02 / 10 / 2024, pp. 63 / 104 42 / 59

[0157] Cells from the master cell bank were propagated in shake flasks, culture bags, and seed bioreactors, and the resulting seed cells were used to produce antibody products via a bioreactor. The obtained antibody underwent further purification by protein A affinity chromatography, cation exchange chromatography, and anion exchange chromatography, as well as low-pH virus inactivation and filtration steps to remove the virus. The specific purification steps were as follows. The cell expression supernatant sample was loaded onto a protein A column equilibrated with PBS buffer (Merck, 175118824). The column was washed with PBS until the A280 reading was reduced to baseline and then washed with PB buffer to remove impure protein. The target protein was eluted with 50mM sodium citrate at pH 3.5, and the eluate peak was collected.The purified protein was neutralized with 1M Tris and loaded onto an anion chromatography column (GE, 17-5316-10) equilibrated with PB buffer, and the flow peak was collected and adjusted to pH 5.0 with 1M citric acid. After anion chromatography, the protein was further purified on a cation chromatography column (Merck, 1.168882) equilibrated with citrate buffer (pH 5.0). The target protein was eluted with a citrate buffer (pH 5.0) containing 0.18 M sodium chloride, and the eluted peak was collected and aliquoted for subsequent use. MODE 2:

[0158] The experiments were designed based on the buffer system, buffer concentration, pH value, saccharide type, and saccharide concentration of the PD-L1 preparation (1 mg / mL). The sample value was determined by the DSC technique to initially screen the preparation formulation.

[0159] The buffer system, the buffer concentration, the pH value, the Petition 870240084342, dated 02 / 10 / 2024, pp. 64 / 104 43 / 59 The type of saccharide and the saccharide concentration were used as factors, and the Tm value was used as the response value to design the test and generate a drawing table. The Tm value was determined according to the experimental groups in the drawing table.

[0160] Table 2 DSC results with response to Tm value Buffer System Buffer Concentration (mM) pH Saccharide Type Saccharide Concentration (%) Tm 1 Sodium succinate-succinic acid 10 5 Trehalose 3 83.35 2 Sodium succinate-succinic acid 10 5.3 Sucrose 6 84.13 3 Sodium succinate-succinic acid 20 5.6 Sucrose 6 83.89 4 Sodium succinate-succinic acid 30 5 Sucrose 6 83.06 5 Sodium succinate-succinic acid 30 5.6 Trehalose 3 83.49 6 Sodium acetate-acetic acid 10 5 Sucrose 9 84.57 Petition 870240084342, dated 02 / 10 / 2024, pages 65 / 104 44 / 59 7 Sodium acetate-acetic acid 10 5.6 Sucrose 3 84.15 8 Sodium acetate-acetic acid 30 5 Trehalose 6 84.03 9 Sodium acetate-acetic acid 30 5.6 Sucrose 9 84.51 10 Histidine hydrochloride 10 5 Sucrose 6 82.41 11 Histidine hydrochloride 10 5.6 Trehalose 9 83.96 12 Histidine hydrochloride 20 5.3 Trehalose 3 81.92 13 Histidine hydrochloride 30 5 Trehalose 9 80.85 14 Histidine hydrochloride 30 5.6 Sucrose 6 83.03 15 Disodium phosphate citric acid 10 5 Trehalose 6 83.41 Petition 870240084342, dated 02 / 10 / 2024, pp. 66 / 104 45 / 59 16 Disodium phosphate citric acid 10 5.6 Sucrose 3 83.53 17 Disodium phosphate citric acid 30 5 Sucrose 3 82.61 18 Disodium phosphate citric acid 30 5.6 Trehalose 6 83.87

[0161] The Tm value was used as the response value and the model was fitted according to the experimental results. R2 was 0.99987, the adjusted R2 was 0.9979113 and P=0.0348<0.05 in the analysis of variance, indicating that the model was effective and that the results were reliable.

[0162] Note: the unit of concentration of saccharides is in g / 100 mL.

[0163] According to the principle of maximizing the Tm value, the formulation was preliminarily selected by the main effect diagrams of the Tm factors (Figure 3). For the buffer system, acetate (sodium) is best, followed by succinate (sodium); when the buffer concentration is 20 to 30 mM, the Tm value is higher; pH 5 to 5.6 had no significant effect on the Tm value; the best saccharide concentration is 6%. MODE 3:

[0164] The anti-PD-L1 antibody (HRP00052) was formulated in a preparation containing 10 mM succinate (sodium), acetate (sodium), 60 mg / mL sucrose, and 0.2 mg / mL polysorbate 20 at pH 5.0 to 5.5, respectively, and the protein concentration was 50 mg / mL. Each preparation was filtered and filled into a neutral borosilicate glass injection tube sealed with a bromobutyl rubber stopper for long-term stability observation. Petition 870240084342, dated 02 / 10 / 2024, pp. 67 / 104 46 / 59 time frame at 2 to 8°C. The stability of the sample was illustrated by the various characteristics shown in Table 3.

[0165] The color, appearance, and clarity of the sample were determined by visual inspection of the sample under white fluorescent light at room temperature with a black background. The purity of the samples was further evaluated by high-performance size exclusion chromatography (HP-SEC), in which the percentage of monomer and the percentage of high molecular weight substance (possibly aggregates) and late elution peaks (possibly degradation products) were determined. Purity was evaluated by revealing the presence of acidic or basic variants using high-performance ion-exchange chromatography (HP-IEX), and the results were expressed as the percentage of the total substance observed. The samples were analyzed by the CE-SDS technique, in which proteins were denatured with sodium dodecyl sulfate (SDS) under reducing and non-reducing conditions and separated by capillary electrophoresis (CE). The proteins were separated based on their apparent molecular weight.Under non-reducing conditions, all substances except the main IgG peak were classified as impurities. Under reducing conditions, IgG was split into heavy and light chains, and all other substances were classified as impurities.

[0166] The results showed that the stability of the anti-PDL1 antibody in the succinate buffer system was significantly better than that in the acetate buffer system; and the anti-PD-L1 antibody was very stable at pH 5.0 to 5.5. Table 3. Effect of pH and buffer system on the long-term stability of PD-L1 antibody at 2 to 8°C. pH Buffer Time (months) Appearance SEC (% of area) IEC (% of area) CE-SDS (%CPA) Mon Poly- Peak Peak Peak Reduction No Petition 870240084342, dated 02 / 10 / 2024, pages 68 / 104 47 / 59 omer mer principal acid basic -cotor reducing acetate (sodium) 5.0 0 Clear and transparent 98.30 1.0 21.0 63.7 15.3 97.40 96.00 3 Clear and transparent 98.30 1.0 24.1 62.2 13.7 98.76 95.30 6 With fine particles in suspension smoky N / AN / AN / AN / AN / AN / AN / AN acetate (sodium) 5.2 0 Clear and transparent 98.3 1.0 21.1 63.7 15.2 97.20 96.10 3 Clear and transparent 98.2 1.1 24.1 61.8 14.0 98.74 95.45 6 With fine particles in suspension, smoky N / AN / AN / AN / AN / AN / AN / AN Petition 870240084342, dated 02 / 10 / 2024, pp. 69 / 104 48 / 59 Sodium acetate 5.5 0 Clear and transparent 98.3 1.0 21.0 64.1 15.0 97.20 96.10 3 Clear and transparent 98.2 1.1 24.0 62.4 13.6 98.76 95.41 6 With fine particles in suspension, smoky N / AN / AN / AN / AN / AN / AN / AN Sodium succinate 5.0 0 Clear and transparent 98.4 1.0 21.4 63.6 14.9 97.80 96.10 3 Clear and transparent 98.3 1.0 24.4 61.5 14.1 98.65 95.85 6 Clear and transparent 97.2 0.7 25.0 60.6 14.4 98.43 96.11 succinate (sodium) 5.2 0 Clear and transparent 98.2 1.0 21.9 62.5 15.6 97.40 95.80 3 Clear and transparent 98.2 1.0 24.3 61.1 14.6 98.64 95.20 6 Clear and transparent 97.2 0.7 24.9 60.7 14.4 98.44 96.15 Petition 870240084342, dated 02 / 10 / 2024, pp. 70 / 104 49 / 59 Present succinate (sodium) 5, 5 0 Clear and transparent 98.2 1.1 23.0 62.4 14.7 97.50 95.90 3 Clear and transparent 98.2 1.1 24.1 61.6 14.3 98.61 95.45 6 Clear and transparent 97.2 0.7 24.9 60.6 14.5 97.66 96.19 MODE 4

[0167] The anti-PD-L1 antibody was formulated in a preparation containing 60 mg / mL of sucrose and 0.2 mg / mL of polysorbate 20 in 10 mM and 20 mM succinate (sodium) at pH 5.2, respectively, and the protein concentration was 50 mg / mL. Each preparation was filtered and filled into a neutral borosilicate glass injection tube sealed with a bromobutyl rubber stopper, followed by acceleration at 40°C and observation of long-term stability at 2 to 8°C. The results showed that the anti-PD-L1 antibody was quite stable in the 10 to 20 mM succinate buffer system. Table 4. Stability results of preparations with different concentrations of the buffer system at 40°C. Concentration Time (days) Appearance SEC (% of area) IEC (% of area) Non-reducing CE-SDS (%CPA) Monomer Polymer Acid peak Main peak Basic peak 10 mM 0 Clear and transparent 98.2 1.2 23.4 60.4 16.2 95.28 Petition 870240084342, dated 02 / 10 / 2024, pp. 71 / 104 50 / 59 28 Clear and transparent 96.9 2.0 35.0 51.8 13.1 93.66 20 mM 0 Clear and transparent 98.1 1.2 23.5 60.9 15.6 95.30 28 Clear and transparent 96.8 2.1 33.5 52.2 12.3 93.74 Table 5. Stability results of preparations with different concentrations of the buffer system at 40°C. Concentration Time (months) Appearance SEC (% of area) IEC (% of area) Non-reducing CE-SDS (%CPA) Monomer Polymer Acid peak Main peak Basic peak 10 mM 0 Clear and transparent 98.2 1.2 23.4 60.4 16.2 95.28 1 Clear and transparent 98.1 1.1 24.2 60.4 15.4 96.31 Petition 870240084342, dated 02 / 10 / 2024, pp. 72 / 104 51 / 59 3 Clear and transparent 96.9 1.0 24.8 58.7 16.5 96.10 6 Clear and transparent 98.2 1.1 23.6 60.9 15.5 94.86 20 mM 0 Clear and transparent 98.1 1.2 23.5 60.9 15.6 95.30 1 Clear and transparent 98.0 1.1 24.2 60.2 15.6 95.78 3 Clear and transparent 96.9 1.0 24.9 58.7 16.4 96.05 6 Clear and transparent 98.2 1.1 23.3 61.5 15.2 95.70 Petition 870240084342, dated 02 / 10 / 2024, pp. 73 / 104 52 / 59 relative MODE 5

[0168] Anti-PD-L1 antibody preparations containing 50 mg / mL of PD-LI antibody (HRP00052), 20 mM acetate (sodium) at pH 5.2 and 60 mg / mL of sucrose, and different types and concentrations of surfactant were prepared. Each preparation was filtered and filled into a neutral borosilicate glass vial, sealed with a bromobutyl rubber stopper and placed in a constant temperature shaker at 25°C, and shaken at 200 rpm.

[0169] DLS (Dynamic Light Scattering) was used to measure the average scattering coefficient, which was used to characterize the particle size and particle size distribution of nanometric particles in solution. The PDI (particle scattering index) distribution coefficient reflects the uniformity of particle size. The lower the PDI value, the narrower the particle size distribution and the more uniform the particle size.

[0170] Stability results indicated that 0.1 to 0.3 mg / mL of polysorbate 20 or polysorbate 80 effectively prevented the aggregation of anti-PD-L1 antibodies and the formation of large agglomerated particles. Table 6. Results of the agitation test of different types and concentrations of polysorbate. Type Concentration (mg / mL) Time (days) Appearance DLS (nm) SEC (% of area) Average Z-Ave PDI Monomer Polymer No polymer 0 0 Clear and transparent 17.27 0.29 98.8 1.2 Petition 870240084342, dated 02 / 10 / 2024, pp. 74 / 104 53 / 59 Close to the surface 3 Cloudy 16.23 0.25 98.2 1.3 7 Cloudy 15.41 0.25 97.9 1.4 Polysorbate 20 0.1 0 Clear and transparent 13.98 0.13 98.7 1.3 3 With suspended particles occasionally 14.08 0.13 98.2 1.3 7 With suspended particles occasionally 13.80 0.10 97.8 1.4 Polysorbate 20 0.2 0 Clear and transparent 14.08 0.14 98.7 1.3 3 With suspended particles occasionally 14.13 0.14 98.0 1.4 Petition 870240084342, dated 02 / 10 / 2024, pp. 75 / 104 54 / 59 7 With occasionally suspended particles 13.82 0.09 97.8 1.5 Polysorbate 20 0.3 0 Clear and transparent 14.13 0.14 98.7 1.3 3 With occasionally suspended particles 13.90 0.13 98.1 1.3 7 With occasionally suspended particles 14.13 0.15 97.8 1.5 Polysorbate 80 0.1 0 Clear and transparent 14.22 0.16 98.7 1.3 3 With suspended particles 14.07 0.12 98.1 1.4 Petition 870240084342, dated 02 / 10 / 2024, pp. 76 / 104 55 / 59 Occasionally with suspended particles 7 Occasionally with suspended particles 15.08 0.17 98.0 1.3 Polysorbate 80 0.2 0 Clear and transparent 14.51 0.18 98.7 1.3 3 Occasionally with suspended particles 13.73 0.10 98.2 1.2 7 Occasionally with suspended particles 14.15 0.13 97.4 1.9 Polysorbate 80 0.3 0 Clear and transparent 13.74 0.12 98.8 1.2 Petition 870240084342, dated 02 / 10 / 2024, pp. 77 / 104 56 / 59 3 With occasionally suspended particles 14.10 0.14 98.3 1.2 7 With occasionally suspended particles 14.15 0.13 97.9 1.4 MODE 6

[0171] Anti-PD-L1 antibody preparations containing 50 mg / mL PD-L1 antibody (HRP00052), 20 mM succinate (sodium) at pH 5.2, and 60 mg / mL sucrose, and different types and concentrations of surfactant were prepared. Each preparation was filtered and filled into a neutral borosilicate glass vial, sealed with a bromobutyl rubber stopper, and placed at 2 to 8°C for stability testing. The results showed that polysorbate 80 was significantly better than polysorbate 20, and there was no significant difference between each concentration. Table 7. Stability results of preparations with different types and concentrations of polysorbate at 2 to 8°C. Type Concentration mg / mL Time (months) Appearance SEC (% of area) IEC (% of area) CE- Non-reducing SDS Monomer Polymer Acid peak Primitive peak Basic peak Petition 870240084342, dated 02 / 10 / 2024, pp. 78 / 104 57 / 59 Principal Co. Tort. (% CPA) Polysorbate 20 0.2 0 Clear and transparent 98.1 1.20 24.3 60.5 15.2 95.9 3 With fine particles in suspension N / AN / A 24.5 59.6 15.9 96.6 6 With visible particles in suspension 98.3 1.15 23.8 60.3 15.9 96.2 0.4 0 Clear and transparent 98.2 1.20 24.4 59.4 16.2 96.0 3 With fine particles in suspension N / AN / A 24.7 59.5 15.8 95.9 Petition 870240084342, dated 02 / 10 / 2024, pp. 79 / 104 58 / 59 6 With suspended particles visible 98.2 1.17 23.6 58.7 17.7 95.9 0.6 0 Clear and transparent and 98.1 1.20 24.3 60.2 15.5 95.3 3 Clear and transparent and N / A / N 24.6 59.3 16.1 95.9 6 With suspended particles visible 98.3 1.16 24.1 60.0 15.9 95.9 Polysorbate 80 0.4 0 Clear and transparent and 98.1 1.20 23.7 60.5 15.8 96.0 3 Clear and transparent and N / A / N 24.7 59.5 15.8 95.3 6 Clear and transparent 98.3 1.17 23.3 58.7 18.0 95.8 Petition 870240084342, dated 02 / 10 / 2024, pp. 80 / 104 59 / 59 and 0.6 0 Clear and transparent and 98.2 1.10 24.7 59.8 15.5 94.8 3 Clear and transparent and N / A / N 25.0 58.7 16.3 96.6 6 Clear and transparent and 98.3 1.15 23.6 58.5 17.9 95.7 0.8 0 Clear and transparent and 98.1 1.20 24.2 59.8 16.0 95.7 3 Clear and transparent and N / A / N 24.8 59.4 15.9 95.7 6 Clear and transparent and 98.3 1.14 23.9 59.9 16.2 95.4 Petition 870240084342, dated 02 / 10 / 2024, pages 81 / 104

Claims

1 / 5 CLAIMS 1. Pharmaceutical composition, characterized in that it comprises: (a) 30 mg / mL to 80 mg / mL of an anti-PD-L1 antibody or antigen-binding fragment thereof; (b) 5 mM to 50 mM of a succinate buffer at a pH of 4.5 to 6.0; (c) 30 mg / mL to 90 mg / mL of saccharide, wherein the saccharide is trehalose or sucrose; and (d) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80 or polysorbate 20; The anti-PD-L1 antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2, and LCDR3 sequences, as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively, and HCDR1, HCDR2, and HCDR3 sequences, as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; wherein X4 is selected from H or G, and X5 is selected from G or F.

2. Pharmaceutical composition according to claim 1, characterized in that the pH of the pharmaceutical composition is from 5.0 to 6.

0.

3. Pharmaceutical composition according to claim 2, characterized in that the pH of the pharmaceutical composition is from 5.0 to 5.

5.

4. Pharmaceutical composition according to claim 2, characterized in that the pH of the pharmaceutical composition is 5.

2.

5. Pharmaceutical composition according to claim 1, characterized in that the concentration of the succinate buffer is from 10 mM to 30 mM.

6. Pharmaceutical composition according to claim 5, characterized in that the concentration of the succinate buffer is 10 mM to 20 mM.

7. Pharmaceutical composition according to claim 5, Petition 870260065318, dated 02 / 07 / 2026, page 18 / 22 2 / 5 characterized in that the concentration of the succinate buffer is 20 mM.

8. Pharmaceutical composition according to claim 1, characterized in that the concentration of the anti-PD-L1 antibody or antigen-binding fragment thereof is from 40 mg / mL to 60 mg / mL.

9. Pharmaceutical composition according to claim 8, characterized in that the concentration of the anti-PD-L1 antibody or antigen-binding fragment thereof is from 45 mg / mL to 55 mg / mL.

10. Pharmaceutical composition according to claim 8, characterized in that the concentration of the anti-PD-L1 antibody or antigen-binding fragment thereof is 50 mg / mL.

11. Pharmaceutical composition according to claim 1, characterized in that the saccharide is sucrose.

12. Pharmaceutical composition according to claim 1, characterized in that the concentration of the saccharide is from 40 mg / mL to 80 mg / mL.

13. Pharmaceutical composition according to claim 12, characterized in that the concentration of the saccharide is from 55 mg / mL to 65 mg / mL.

14. Pharmaceutical composition according to claim 12, characterized in that the concentration of the saccharide is 60 mg / mL.

15. Pharmaceutical composition according to claim 1, characterized in that the concentration of polysorbate 80 or polysorbate 20 is from 0.4 mg / mL to 0.8 mg / mL.

16. Pharmaceutical composition according to claim 15, characterized in that the concentration of polysorbate 80 or polysorbate 20 is from 0.5 mg / mL to 0.7 mg / mL.

17. Pharmaceutical composition according to claim 15, Petition 870260065318, dated 02 / 07 / 2026, page 19 / 22 3 / 5 characterized in that the concentration of polysorbate 80 or polysorbate 20 is 0.6 mg / mL.

18. Pharmaceutical composition according to claim 1, characterized in that it comprises: (a) 30 mg / mL to 80 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof; (b) 5 mM to 50 mM of succinate buffer at pH 5.0 to 6.0; (c) 30 mg / mL to 90 mg / mL of trehalose or sucrose; and (d) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80.

19. Pharmaceutical composition according to claim 18, characterized in that it comprises: 40 mg / mL to 60 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 10 mM to 30 mM of succinate buffer at pH 5.0 to 5.5, 40 mg / mL to 80 mg / mL of sucrose and 0.4 mg / mL to 0.8 mg / mL of polysorbate 80.

20. Pharmaceutical composition according to claim 18, characterized in that it comprises: 45 mg / mL to 55 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 10 mM to 20 mM of succinate buffer at pH 5.0 to 5.5, 55 mg / mL to 65 mg / mL of sucrose and 0.5 mg / mL to 0.7 mg / mL of polysorbate 80.

21. Pharmaceutical composition according to claim 18, characterized in that it is selected from one of the following compositions: pharmaceutical composition A comprising: 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.2, 60 mg / mL of sucrose and 0.4 mg / mL of polysorbate 80; Petition 870260065318, dated 02 / 07 / 2026, p.Pharmaceutical composition B comprising: 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.2, 60 mg / mL sucrose and 0.6 mg / mL polysorbate 80; Pharmaceutical composition C comprising: 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.2, 60 mg / mL sucrose and 0.8 mg / mL polysorbate 80; Pharmaceutical composition D comprising: 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.5, 60 mg / mL sucrose and 0.6 mg / mL polysorbate 80; Pharmaceutical composition E comprising: 50 mg / mL of anti-PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer at pH 5.8, 60 mg / mL of sucrose and 0.6 mg / mL of polysorbate 80.

22. Pharmaceutical composition according to claim 1, characterized in that the anti-PD-L1 antibody or the antigen-binding fragment thereof is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody and a human antibody.

23. Pharmaceutical composition according to claim 22, characterized in that the anti-PD-L1 antibody or the antigen-binding fragment thereof is a humanized antibody.

24. Pharmaceutical composition according to claim 1, characterized in that the variable region sequence of the heavy chain of the anti-PD-L1 antibody or of the antigen-binding fragment thereof is shown in SEQ ID NO: 13, and the variable region sequence of the light chain of the anti-PD-L1 antibody or of the antigen-binding fragment thereof is shown in SEQ ID NO:

14.

25. Pharmaceutical composition according to claim 1, Petition 870260065318, dated 02 / 07 / 2026, page 21 / 22 5 / 5 characterized in that the heavy chain sequence of the anti-PD-L1 antibody is shown in SEQ ID NO: 15, and the light chain sequence of the anti-PD-L1 antibody is shown in SEQ ID NO:

17.

26. Method for preparing the pharmaceutical composition defined in any one of claims 1 to 25, characterized in that it comprises mixing an anti-PD-L1 antibody or antigen-binding fragment thereof with a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient comprises succinate buffer, trehalose or sucrose and polysorbate 80 or polysorbate 20.

27. Use of the pharmaceutical composition defined in any one of claims 1 to 25, characterized in that it is for the manufacture of a medicament for the treatment of a disease or condition mediated by PDL1, wherein the disease or condition is cancer.

28. Use according to claim 27, characterized in that the disease or condition is a cancer that expresses PD-L1.

29. Use according to claim 27, characterized in that the disease or condition is breast cancer, lung cancer, stomach cancer, bowel cancer, kidney cancer, melanoma.

30. Use according to claim 27, characterized in that the disease or condition is non-small cell lung cancer, melanoma, bladder cancer, or kidney cancer. Petition 870260065318, dated 02 / 07 / 2026, page 22 / 22

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