ANTI-PD-L1 ANTIBODY, AND PREPARATION METHOD THEREFOR AND APPLICATION THEREOF
20230143294 · 2023-05-11
Inventors
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
Abstract
Provided are an anti-PD-L1 antibody, and a preparation method therefor and an application thereof. The antibody is obtained by a computer-aided design, and the anti-PD-L1 antibody comprises an antibody I and/or an antibody II; an antibody sequence is subjected to simulation analysis and design by bioinformatics modeling without complicated and cumbersome immune reactions; the antibody is designed and transferred into a host cell by molecular biological construction, and expressed in the host cell and purified to obtain the antibody with a PD-L1 antigen binding activity.
Claims
1. An anti-PD-L1 antibody, comprising an antibody I and/or an antibody II; wherein the antibody I comprises a light chain and a heavy chain, and a variable domain of the light chain of the antibody I comprises complementarity determining regions (CDRs) I-CDR-L1, I-CDR-L2 and I-CDR-L3 which are selected from the following amino acid sequences, respectively: I-CDR-L1: QX2X3X4X5T; I-CDR-L2: X7X8S; I-CDR-L3: LX12X13X14SX16X17FT; wherein X2=D, E, N, Q, X3=G, A, V, I, L, X4=G, A, V, I, L, X5=Q, N; X7=G, A, V, I, L, X8=S, T; X12=Q, Y, N, X13=Y, F, X14=G, A, V, I, L, X16=S, T, Y, X17=F, P; a variable domain of the heavy chain of the antibody I comprises CDRs I-CDR-H1, I-CDR-H2 and I-CDR-H3 which are selected from the following amino acid sequences, respectively: I-CDR-H1: GYX22X23X24X25YW; I-CDR-H2: IYX30X31X32SX34T; I-CDR-H3: TX37WX39X40X41X42X43HX45MX47H; wherein X22=S, T, X23=F, Y, X24=N, S, T, Y, Q, X25=S, T, F; X30=P, H, W, X31=G, A, V, I, L, X32=N, Q, H, X34=D, E; X37=R, H, X39=G, A, V, I, L, X40=D, E, X41=G, A, V, I, L, X42=Y, F, X43=Y, F, X45=G, A, V, I, L, X47=Q, N, D, E; wherein the antibody II comprises a light chain and a heavy chain, and a variable domain of the light chain of the antibody II comprises CDRs II-CDR-L1, II-CDR-L2 and II-CDR-L3 which are selected from the following amino acid sequences, respectively: II-CDR-L1: SX52X53X54Y; II-CDR-L2: X56X57S; II-CDR-L3: QX60RX62SX64X65YT; wherein X52=S, T, X53=G, A, V, I, L, X54=Y, T, S; X56=Y, T, S, X57=Y, T, S; X60=N, Q, Y, X62=F, T, S, X64=Y, F, X65=P, H, W; a variable domain of the heavy chain of the antibody II comprises CDRs II-CDR-H1, II-CDR-H2 and II-CDR-H3 which are selected from the following amino acid sequences, respectively: II-CDR-H1: GYX70X71X72SX74V; II-CDR-H2: INX.sub.77X78X79DX.sub.81T; II-CDR-H3: AX.sub.84SX86X87X88X89X90Y; wherein X70=S, T, Y, F, X71=T, Y, F, X72=T, S, X74=Y, F, P; X77=Y, H, P, X78=Y, F, X79=Q, N, Y, X81=G, A, V, I, L; X84=W, R, K, X86=G, A, V, I, L, X87=G, A, V, I, L, X88=G, A, V, I, L, X89=S, T, F, X90=D, E.
2. The antibody according to claim 1, wherein the CDR I-CDR-L1 in the variable domain of the light chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24; preferably, the CDR I-CDR-L1 in the variable domain of the light chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 18, 19, 21, 23; preferably, the CDR I-CDR-L2 in the variable domain of the light chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 25, 26, 27, 28, 29, 30; preferably, the CDR I-CDR-L2 in the variable domain of the light chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 25, 26, 29, 30; preferably, the CDR I-CDR-L3 in the variable domain of the light chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 31, 32, 33, 34, 35, 36, 37, 38; preferably, the CDR I-CDR-L3 in the variable domain of the light chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 33, 34, 36, 38; preferably, the CDR I-CDR-H1 in the variable domain of the heavy chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 39, 40, 41, 42, 43, 44, 45; preferably, the CDR I-CDR-H1 in the variable domain of the heavy chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 40, 42, 43, 44, 45; preferably, the CDR I-CDR-H1 in the variable domain of the heavy chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 40, 42, 44, 45; preferably, the CDR I-CDR-H2 in the variable domain of the heavy chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 46, 47, 48, 49, 50, 51, 52; preferably, the CDR I-CDR-H2 in the variable domain of the heavy chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 46, 48, 49, 51; preferably, the CDR I-CDR-H3 in the variable domain of the heavy chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 53, 54, 55, 56, 57, 58, 59, 60, 61, 62; preferably, the CDR I-CDR-H3 in the variable domain of the heavy chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 53, 54, 57, 59, 62; preferably, the CDR I-CDR-H3 in the variable domain of the heavy chain of the antibody I comprises one of amino acid sequences SEQ ID NO. 54, 57, 59, 62; preferably, the CDRs I-CDR-L1, I-CDR-L2 and I-CDR-L3 in the variable domain of the light chain of the antibody I comprise one of the following combinations of amino acid sequences, respectively: SEQ ID NO. 17, 26 and 38; SEQ ID NO. 17, 24 and 35; SEQ ID NO. 19, 26 and 31; SEQ ID NO. 19, 29 and 34; SEQ ID NO. 20, 30 and 32; SEQ ID NO. 21, 30 and 32; SEQ ID NO. 23, 25 and 33; SEQ ID NO. 22, 29 and 38; SEQ ID NO. 22, 30 and 34; SEQ ID NO. 21, 28 and 33; SEQ ID NO. 24, 27 and 36; SEQ ID NO. 18, 29 and 37; SEQ ID NO. 21, 25 and 38; SEQ ID NO. 18, 25 and 34; SEQ ID NO. 18, 28 and 33; SEQ ID NO. 23, 30 and 35; SEQ ID NO. 23, 29 and 38; preferably, the CDRs I-CDR-H1, I-CDR-H2 and I-CDR-H3 in the variable domain of the heavy chain of the antibody I comprise one of the following combinations of amino acid sequences, respectively: SEQ ID NO. 40, 52 and 53; SEQ ID NO. 42, 47 and 54; SEQ ID NO. 45, 49 and 55; SEQ ID NO. 45, 48 and 59; SEQ ID NO. 39, 51 and 55; SEQ ID NO. 43, 47 and 58; SEQ ID NO. 42, 49 and 56; SEQ ID NO. 41, 50 and 60; SEQ ID NO. 44, 48 and 62; SEQ ID NO. 40, 52 and 61; SEQ ID NO. 43, 51 and 57; SEQ ID NO. 45, 52 and 56; SEQ ID NO. 44, 46 and 57; SEQ ID NO. 42, 46 and 57; SEQ ID NO. 43, 48 and 62; SEQ ID NO. 42, 50 and 54; SEQ ID NO. 45, 46 and 61; preferably, amino acid sequences of the CDRs of the light chain and/or the heavy chain of the antibody I comprise those having 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequences of the CDRs of the light chain and/or the heavy chain of the antibody I and those functionally identical or similar to the amino acid sequences shown for the antibody I; preferably, an amino acid sequence of the variable domain of the light chain of the antibody I comprises one of SEQ ID NO. 107 to SEQ ID NO. 114; preferably, an amino acid sequence of the variable domain of the heavy chain of the antibody I comprises one of SEQ ID NO. 115 to SEQ ID NO. 122; preferably, the amino acid sequences of the CDRs of the light chain and/or the heavy chain of the antibody I comprise those obtained through substitutions, deletions or additions of one amino acid or a plurality of amino acids and those functionally identical or similar to the amino acid sequences of the CDRs of the light chain and/or the heavy chain shown for the antibody I; preferably, the plurality of amino acids are two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acids.
3. The antibody according to claim 1, wherein the CDR II-CDR-L1 of the light chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 63, 64, 65, 66, 67, 68; preferably, the CDR II-CDR-L1 of the light chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 63, 64, 65, 67; preferably, the CDR II-CDR-L2 of the light chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 69, 70, 71, 72; preferably, the CDR II-CDR-L2 of the light chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 69, 70, 72; preferably, the CDR II-CDR-L3 of the light chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 73, 74, 75, 76, 77, 78, 79, 80, 81; preferably, the CDR II-CDR-L3 of the light chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 76, 77, 78, 79, 80, 81; preferably, the CDR II-CDR-H1 of the heavy chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 82, 83, 84, 85, 86, 87, 88; preferably, the CDR II-CDR-H1 of the heavy chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 82, 83, 86, 88; preferably, the CDR II-CDR-H2 of the heavy chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 89, 90, 91, 92, 93, 94, 95; preferably, the CDR II-CDR-H2 of the heavy chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 89, 90, 91, 93; preferably, the CDR II-CDR-H3 of the heavy chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 96, 97, 98, 99, 100, 101, 102, 103, 104; preferably, the CDR II-CDR-H3 of the heavy chain of the antibody II comprises one of amino acid sequences SEQ ID NO. 96, 98, 99, 102; preferably, the CDRs II-CDR-L1, II-CDR-L2 and II-CDR-L3 of the light chain of the antibody II comprise one of the following combinations of amino acid sequences, respectively: SEQ ID NO. 63, 69 and 76; SEQ ID NO. 64, 70 and 81; SEQ ID NO. 65, 71 and 77; SEQ ID NO. 68, 70 and 73; SEQ ID NO. 67, 72 and 76; SEQ ID NO. 66, 72 and 80; SEQ ID NO. 64, 72 and 77; SEQ ID NO. 67, 72 and 81; SEQ ID NO. 63, 70 and 79; SEQ ID NO. 63, 70 and 74; SEQ ID NO. 64, 69 and 75; SEQ ID NO. 67, 72 and 78; SEQ ID NO. 63, 70 and 80; SEQ ID NO. 66, 69 and 78; SEQ ID NO. 65, 71 and 79; preferably, the CDRs II-CDR-H1, II-CDR-H2 and II-CDR-H3 of the heavy chain of the antibody II comprise one of the following combinations of amino acid sequences, respectively: SEQ ID NO. 82, 89 and 96; SEQ ID NO. 83, 90 and 98; SEQ ID NO. 84, 95 and 103; SEQ ID NO. 85, 91 and 97; SEQ ID NO. 88, 92 and 102; SEQ ID NO. 86, 91 and 99; SEQ ID NO. 87, 94 and 100; SEQ ID NO. 88, 93 and 101; SEQ ID NO. 88, 90 and 99; SEQ ID NO. 83, 91 and 99; SEQ ID NO. 82, 93 and 102; SEQ ID NO. 86, 95 and 98; SEQ ID NO. 86, 91 and 104; SEQ ID NO. 82, 89 and 102; SEQ ID NO. 84, 93 and 96; preferably, amino acid sequences of the CDRs of the light chain and/or the heavy chain of the antibody II comprise those having 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequences of the CDRs of the light chain and/or the heavy chain of the antibody II and those functionally identical or similar to the amino acid sequences shown for the antibody II; preferably, an amino acid sequence of the variable domain of the light chain of the antibody II comprises one of SEQ ID NO. 123 to SEQ ID NO. 132; preferably, an amino acid sequence of the variable domain of the heavy chain of the antibody II comprises one of SEQ ID NO. 133 to SEQ ID NO. 142; preferably, the amino acid sequences of the CDRs of the light chain and/or the heavy chain of the antibody II comprise those obtained through substitutions, deletions or additions of one amino acid or a plurality of amino acids and those functionally identical or similar to the amino acid sequences shown for the antibody II; preferably, the plurality of amino acids are two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acids.
4. The antibody according to claim 1, wherein a framework region (FR) in the variable domain of the light chain of the antibody I is selected from the following sequences: TABLE-US-00014 I-FR-L1: SEQ ID NO. 1: DIQMTQSPSSLSASVGDRVTITCRAS; I-FR-L2: SEQ ID NO. 2: LNWLQQKPGKAIKRLIY; I-FR-L3: SEQ ID NO. 3: SLDSGVPKRFSGSRSGSDYSLTISSLQPEDFATYYC; I-FR-L4: SEQ ID NO. 4: FGQGTKLEIK; preferably, a framework region in the variable domain of the heavy chain of the antibody I is selected from the following sequences: TABLE-US-00015 I-FR-H1: SEQ ID NO. 5: VQLVQSGAEVKKPGASVKVSCKAS; I-FR-H2: SEQ ID NO. 6: MHWVRQAPGQGLEWMGA; I-FR-H3: SEQ ID NO. 7: SYNQKFKGRVTITAVTSASTAYMELSSLRSEDTAVYYC; I-FR-H4: SEQ ID NO. 8: WGQGTLVTVS; preferably, a framework region in the variable domain of the light chain of the antibody II is selected from the following sequences: TABLE-US-00016 II-FR-L1: SEQ ID NO. 9: TQSPSFLSASVGDRVTITCSAS; II-FR-L2: SEQ ID NO. 10: MHWFQQKPGKSPKLWIY; II-FR-L3: SEQ ID NO. 11: NLASGVPARFSGSGSGTSYSLTISSLQPEDFATYYC; II-FR-L4: SEQ ID NO. 12: FGQGTKLEIK; preferably, a framework region in the variable domain of the heavy chain of the antibody II is selected from the following sequences: TABLE-US-00017 II-FR-H1: SEQ ID NO. 13: VQLVQSGAEVKKPGASVKVSCKAS; II-FR-H2: SEQ ID NO. 14: MHWVRQAPGQGLEWMGY; II-FR-H3: SEQ ID NO. 15: KYNEKFKGRVTITSDKSASTAYMELSSLRSEDTAVYYC; II-FR-H4: SEQ ID NO. 16: WGQGTLVTVS; preferably, the antibody further comprises a human constant region; preferably, the human constant region comprises any one or a combination of at least two of IgG1, IgG2, IgG3 or IgG4, preferably IgG1 and IgG4; preferably, the antibody further comprises a murine constant region; preferably, the murine constant region comprises any one or a combination of at least two of IgG1, IgG2A, IgG2B or IgG3, preferably IgG2A; preferably, the antibody is modified by glycosylation; preferably, the antibody comprises any one or a combination of at least two of a monoclonal antibody, a polyclonal antibody, an antibody fragment, a bispecific antibody, a trispecific antibody, a tetraspecific antibody or an antibody variant.
5. A nucleic acid for encoding the antibody according to claim 1.
6. A vector, comprising the nucleic acid according to claim 5.
7. A host cell, expressing the antibody according to claim 1; preferably, the host cell is a eukaryotic cell and/or a prokaryotic cell; preferably, the eukaryotic cell is a mammalian cell, further preferably any one or a combination of at least two of HEK 293 cells, simian COS cells, myeloma cells or Chinese hamster ovary cells; preferably, the prokaryotic cell comprises any one or a combination of at least two of E. coli, B. subtilis, P. aeruginosa or Salmonella typhimurium.
8. A method for preparing the antibody according to claim 1, comprising the following steps: (1) designing a sequence of the antibody; (2) selecting a host cell and constructing a vector containing the antibody sequence obtained in step (1); (3) transforming or transfecting the vector obtained in step (2) into the host cell for expression; and (4) isolating and purifying an antibody expressed in step (3); preferably, a method for designing the antibody sequence in step (1) comprises the following steps: (1′) antigen structure modeling: determining a homologous region of a protein sequence through multiple sequence alignment between a target antigen protein chain and a protein chain in a protein data bank; (2′) antibody structure modeling: generating an optimized homologous model of the antibody by a homology modeling method through alignment of a sequence having an unknown structure with the antibody having a known three-dimensional structure, performing loop region modeling and structure-based alignment, and constructing a structural model of the antibody; (3′) antigen and antibody structures optimizing: endowing a simulation system with a force field parameter, checking initial structure and preprocessing, and minimizing initial structural energy; and (4′) antigen and antibody docking: searching for and optimizing conformation in combination with a genetic algorithm, finding a best antibody binding pattern, calculating the binding pattern and affinity, and obtaining a best antibody sequence through a scoring function.
9. A composition, comprising the antibody according to claim 1; preferably, the composition further comprises a pharmaceutically acceptable carrier, buffer, excipient, stabilizer, preservative and other bioactive substances; preferably, the other bioactive substances comprise any one or a combination of at least two of a cytotoxic agent, a chemotherapeutic agent, a growth inhibitor, a radiation therapy agent, an anti-angiogenic agent, an apoptotic agent or an anti-tubulin agent.
10. A use of the composition according to claim 9 for preparing a drug and/or a kit for targeted treatment, preferably for preparing a drug and/or a kit for improving a condition of T cell dysfunction by antagonizing PD-L1 signaling; preferably, the condition of T cell dysfunction comprises cancer and a benign tumor; preferably, the cancer comprises any one or a combination of at least two of non-small cell lung cancer, small cell lung cancer, renal cell cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell cancer, liver cancer, renal cancer, salivary cancer, lung cancer, pancreatic cancer, colon cancer, ovarian cancer, bladder cancer, glioma, epithelioma or brain cancer.
Description
BRIEF DESCRIPTION OF DRAWINGS
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[0156]
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[0158]
[0159]
DETAILED DESCRIPTION
[0160] To further elaborate on the technical means adopted and the effects achieved in the present application, solutions of the present application are further described below through specific examples in conjunction with drawings, but the present application is not limited to the scope of the examples.
Example 1 Antibody Design
[0161] An antibody was analyzed and designed through bioinformatics with steps described below.
1. Antigen structure construction: Multiple sequence alignment was performed between a target antigen protein chain and a protein chain in a protein data bank (PDB): data and index files for the multiple sequence alignment were generated and a sequence alignment program was called so that the multiple sequence alignment was performed on the amino acid sequence of a target protein chain with protein chains in the data bank and a homologous region of a protein sequence was determined.
2. Antibody structure construction: An initial model of the antibody was constructed using MODELLER software by a homology modeling method through alignment of a sequence having an unknown structure with the antibody having a known three-dimensional structure to generate an optimized homologous model of the antibody; the model was optimized using ROSETTA software for loop region modeling and structure-based alignment, and a structural model of the antibody was constructed. To verify a model result, a Z-score distribution map and a Ramachandran plot were calculated to ensure that most amino acids of a protein fall into an allowable region of the Ramachandran plot.
3. Optimization of antigen and antibody structures
(1) Endowment of a simulation system with a force field parameter;
(2) Initial structure inspection and pretreatment; and
(3) Initial structure energy minimization.
[0162] Through gromacs4.5.7 software, the simulation system was endowed with the force field parameter, and the initial structure inspection and pretreatment and the initial structure energy minimization were performed. A specific method was that a protonation state of the protein close to a physiological environment (pH=7.4) was set through a PROPKA program, and DPPC, a TIP3P water model, neutralized ions and KCl of 0.15 mol/L were added for energy minimization so that the antibody structure optimized for an aqueous phase was obtained. Further, the rationality of the antibody structure may be determined through Rama plot evaluation and the like by the method in step 2.
4. Antigen and antibody docking
[0163] Conformation was searched for and optimized in combination with a genetic algorithm. An antigen epitope was determined firstly and the antibody and an antigen were docked so that a best antigen-antibody binding pattern was found. The binding pattern and affinity were calculated according to a principle of distance matching between the antigen epitope and antibody molecules, and calculation results were scored to evaluate the degree of binding of the antibody to the antigen so that a best target antibody sequence was obtained.
[0164] Rigid molecules were docked using zdock software, the affinity of the antigen to the antibody was evaluated using a scoring function consisting of ACE statistical potential energy, structure complementarity and static electricity, and the best antibody sequence was obtained in combination with information about the antigen epitope.
[0165] A series of antibody sequences were designed. The variable region (Fv) of the antibody consists of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Two types of antibody, an antibody I and an antibody II, were finally designed, as shown in Table 1 and Table 2.
[0166] Through a computer-aided design and bioinformatics modeling, an antibody structure was designed, and a series of antibody sequences were designed. The variable region of the antibody is composed of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Two types of antibody were designed: the antibody I and the antibody II. Some amino acid sequences of the antibody I, the antibody II and positive control respectively include amino acid sequences in Table 1, Table 2 and Table 3. Amino acid sequences of constant regions of the antibody I, the antibody II and a positive control antibody are shown in Table 4. Sequences of FRs of the antibody I and the antibody II are shown in Table 5.
TABLE-US-00005 TABLE 1 Sequence No. Amino Acid Sequence Light chain of the antibody I I-CDR-L1 SEQ ID NO. 17 QDLGNT I-CDR-L1 SEQ ID NO. 18 QNIGQT I-CDR-L1 SEQ ID NO. 19 QNIANT I-CDR-L1 SEQ ID NO. 20 QDVGQT I-CDR-L1 SEQ ID NO. 21 QEIGQT I-CDR-L1 SEQ ID NO. 22 QNGINT I-CDR-L1 SEQ ID NO. 23 QDIGNT I-CDR-L1 SEQ ID NO. 24 QEIGQT I-CDR-L2 SEQ ID NO. 25 ATS I-CDR-L2 SEQ ID NO. 26 ASS I-CDR-L2 SEQ ID NO. 27 GTS I-CDR-L2 SEQ ID NO. 28 STS I-CDR-L2 SEQ ID NO. 29 VSS I-CDR-L2 SEQ ID NO. 30 VTS I-CDR-L3 SEQ ID NO. 31 LQYASYPFT I-CDR-L3 SEQ ID NO. 32 LYFASSPFT I-CDR-L3 SEQ ID NO. 33 LQYASSPFT I-CDR-L3 SEQ ID NO. 34 LYYASYFFT I-CDR-L3 SEQ ID NO. 35 LQYASSFFT I-CDR-L3 SEQ ID NO. 36 LNYISSPFT I-CDR-L3 SEQ ID NO. 37 LQYASTFFT I-CDR-L3 SEQ ID NO. 38 LYYASTPFT Heavy chain of the antibody I I-CDR-H1 SEQ ID NO. 39 GYTFTSYW I-CDR-H1 SEQ ID NO. 40 GYSYYFYW I-CDR-H1 SEQ ID NO. 41 GYSFSSYW I-CDR-H1 SEQ ID NO. 42 GYTYYFYW I-CDR-H1 SEQ ID NO. 43 GYSFTTYW I-CDR-H1 SEQ ID NO. 44 GYSFTSYW I-CDR-H1 SEQ ID NO. 45 GYTYNFYW I-CDR-H2 SEQ ID NO. 46 IYPGNSDT I-CDR-H2 SEQ ID NO. 47 IYHGNSET I-CDR-H2 SEQ ID NO. 48 IYPVHSET I-CDR-H2 SEQ ID NO. 49 IYHVQSDT I-CDR-H2 SEQ ID NO. 50 IYPGQSET I-CDR-H2 SEQ ID NO. 51 IYWAHSET I-CDR-H2 SEQ ID NO. 52 IYHINSDT I-CDR-H3 SEQ ID NO. 53 TRWGEGYYHAMNH I-CDR-H3 SEQ ID NO. 54 THWADAYFHGMNH I-CDR-H3 SEQ ID NO. 55 THWLEVYFHGMDH I-CDR-H3 SEQ ID NO. 56 TRWAELFFHAMDH I-CDR-H3 SEQ ID NO. 57 TRWGDGYYHAMDH I-CDR-H3 SEQ ID NO. 58 TRWLDAFYHVMDH I-CDR-H3 SEQ ID NO. 59 THWVEVFYHAMEH I-CDR-H3 SEQ ID NO. 60 THWIDGYFHVMDH I-CDR-H3 SEQ ID NO. 61 THWAEGYYHAMEH I-CDR-H3 SEQ ID NO. 62 THWIEAFFHLMQH Variable SEQ ID NO. DIQMTQSPSSLSASVGDRVTITCRASQDLGNTLNWL domain of 107 QQKPGKAIKRLIYASSSLDSGVPKRFSGSRSGSDYS the light LTISSLQPEDFATYYCLYYASTPFTFGQGTKLEIK chain of the antibody I Variable SEQ ID NO. DIQMTQSPSSLSASVGDRVTITCRASQDIGNTLNWL domain of 108 QQKPGKAIKRLIYATSSLDSGVPKRFSGSRSGSDYS the light LTISSLQPEDFATYYCLQYASSPFTFGQGTKLEIK chain of the antibody I Variable SEQ ID NO. DIQMTQSPSSLSASVGDRVTITCRASQNIANTLNWL domain of 109 QQKPGKAIKRLIYVSSSLDSGVPKRFSGSRSGSDYS the light LTISSLQPEDFATYYCLYYASYFFTFGQGTKLEIK chain of the antibody I Variable SEQ ID NO. DIQMTQSPSSLSASVGDRVTITCRASQEIGQTLNWL domain of 110 QQKPGKAIKRLIYVTSSLDSGVPKRFSGSRSGSDYS the light LTISSLQPEDFATYYCLYFASSPFTFGQGTKLEIK chain of the antibody I Variable SEQ ID NO. DIQMTQSPSSLSASVGDRVTITCRASQNGINTLNWL domain of Ill QQKPGKAIKRLIYVTSSLDSGVPKRFSGSRSGSDYS the light LTISSLQPEDFATYYCLYYASYFFTFGQGTKLEIK chain of the antibody I Variable SEQ ID NO. DIQMTQSPSSLSASVGDRVTITCRASQEIGQTLNWL domain of 112 QQKPGKAIKRLIYGTSSLDSGVPKRFSGSRSGSDYS the light LTISSLQPEDFATYYCLNYISSPFTFGQGTKLEIK chain of the antibody I Variable SEQ ID NO. DIQMTQSPSSLSASVGDRVTITCRASQEIGQTLNWL domain of 113 QQKPGKAIKRLIYATSSLDSGVPKRFSGSRSGSDYS the light LTISSLQPEDFATYYCLYYASTPFTFGQGTKLEIK chain of the antibody I Variable SEQ ID NO. DIQMTQSPSSLSASVGDRVTITCRASQNIGQTLNWL domain of 114 QQKPGKAIKRLIYSTSSLDSGVPKRFSGSRSGSDYS the light LTISSLQPEDFATYYCLQYASSPFTFGQGTKLEIK chain of the antibody I Variable SEQ ID NO. VQLVQSGAEVKKPGASVKVSCKASGYSYYFYWMHWV domain of 115 RQAPGQGLEWMGAIYHINSDTSYNQKFKGRVTITAV the heavy TSASTAYMELSSLRSEDTAVYYCTRWGEGYYHAMNH chain of the WGQGTLVTVS antibody I Variable SEQ ID NO. VQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMHWV domain of 116 RQAPGQGLEWMGAIYPGNSDTSYNQKFKGRVTITAV the heavy TSASTAYMELSSLRSEDTAVYYCTRWGDGYYHAMDH chain of the WGQGTLVTVS antibody I Variable SEQ ID NO. VQLVQSGAEVKKPGASVKVSCKASGYTYYFYWMHWV domain of 117 RQAPGQGLEWMGAIYHGNSETSYNQKFKGRVTITAV the heavy TSASTAYMELSSLRSEDTAVYYCTHWADAYFHGMNH chain of the WGQGTLVTVS antibody I Variable SEQ ID NO. VQLVQSGAEVKKPGASVKVSCKASGYTYNFYWMHWV domain of 118 RQAPGQGLEWMGAIYHVQSDTSYNQKFKGRVTITAV the heavy TSASTAYMELSSLRSEDTAVYYCTHWLEVYFHGMDH chain of the WGQGTLVTVS antibody I Variable SEQ ID NO. VQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWV domain of 119 RQAPGQGLEWMGAIYWAHSETSYNQKFKGRVTITAV the heavy TSASTAYMELSSLRSEDTAVYYCTHWLEVYFHGMDH chain of the WGQGTLVTVS antibody I Variable SEQ ID NO. VQLVQSGAEVKKPGASVKVSCKASGYSFSSYWMHWV domain of 120 RQAPGQGLEWMGAIYPGQSETSYNQKFKGRVTITAV the heavy TSASTAYMELSSLRSEDTAVYYCTHWIDGYFHVMDH chain of the WGQGTLVTVS antibody I Variable SEQ ID NO. VQLVQSGAEVKKPGASVKVSCKASGYSFTTYWMHWV domain of 121 RQAPGQGLEWMGAIYWAHSETSYNQKFKGRVTITAV the heavy TSASTAYMELSSLRSEDTAVYYCTRWGDGYYHAMDH chain of the WGQGTLVTVS antibody I Variable SEQ ID NO. VQLVQSGAEVKKPGASVKVSCKASGYSFTTYWMHWV domain of 122 RQAPGQGLEWMGAIYPVHSETSYNQKFKGRVTITAV the heavy TSASTAYMELSSLRSEDTAVYYCTHWIEAFFHLMQH chain of the WGQGTLVTVS antibody I
TABLE-US-00006 TABLE 2 Sequence No. Amino Acid Sequence Light chain of the antibody II II-CDR-L1 SEQ ID NO. 63 SSVSY II-CDR-L1 SEQ ID NO. 64 STVYY II-CDR-L1 SEQ ID NO. 65 SSGYY II-CDR-L1 SEQ ID NO. 66 SSASY II-CDR-L1 SEQ ID NO. 67 STITY II-CDR-L1 SEQ ID NO. 68 SSISY II-CDR-L2 SEQ ID NO. 69 SYS II-CDR-L2 SEQ ID NO. 70 STS II-CDR-L2 SEQ ID NO. 71 TTS II-CDR-L2 SEQ ID NO. 72 YTS II-CDR-L3 SEQ ID NO. 73 QQRSSFPYT II-CDR-L3 SEQ ID NO. 74 QNRYSYPYT II-CDR-L3 SEQ ID NO. 75 QQRSSYHYT II-CDR-L3 SEQ ID NO. 76 QNRTSFHYT II-CDR-L3 SEQ ID NO. 77 QYRFSYWYT II-CDR-L3 SEQ ID NO. 78 QYRTSYPYT II-CDR-L3 SEQ ID NO. 79 QQRSSYPYT II-CDR-L3 SEQ ID NO. 80 QQRFSFHYT II-CDR-L3 SEQ ID NO. 81 QYRSSYWYT Heavy chain of the antibody II II-CDR-H1 SEQ ID NO. 82 GYSTSSFV II-CDR-H1 SEQ ID NO. 83 GYYTSSPV II-CDR-H1 SEQ ID NO. 84 GYYTTSYV II-CDR-H1 SEQ ID NO. 85 GYSFTSYV II-CDR-H1 SEQ ID NO. 86 GYTFTSYV II-CDR-H1 SEQ ID NO. 87 GYFYTSYV II-CDR-H1 SEQ ID NO. 88 GYFFSSFV II-CDR-H2 SEQ ID NO. 89 INYYNDAT II-CDR-H2 SEQ ID NO. 90 INYFYDGT II-CDR-H2 SEQ ID NO. 91 INPYNDGT II-CDR-H2 SEQ ID NO. 92 INYYNDAT II-CDR-H2 SEQ ID NO. 93 INHFQDAT II-CDR-H2 SEQ ID NO. 94 INPFQDIT II-CDR-H2 SEQ ID NO. 95 INPYYDGT II-CDR-H3 SEQ ID NO. 96 AWSGAIFEY II-CDR-H3 SEQ ID NO. 97 ARSIGITDY II-CDR-H3 SEQ ID NO. 98 AKSAIAFEY II-CDR-H3 SEQ ID NO. 99 AKSGGISDY II-CDR-H3 SEQ ID NO. 100 AWSAALSEY II-CDR-H3 SEQ ID NO. 101 ARSVGITDY II-CDR-H3 SEQ ID NO. 102 ARSAALSEY II-CDR-H3 SEQ ID NO. 103 ARSGGIFDY II-CDR-H3 SEQ ID NO. 104 AKSLVGSEY Variable SEQ ID NO. 123 TQSPSFLSASVGDRVTITCSASSSVSYMHWFQQKPG domain of KSPKLWIYSYSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQNRTSFHYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 124 TQSPSFLSASVGDRVTITCSASSTVYYMHWFQQKPG domain of KSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQYRSSYWYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 125 TQSPSFLSASVGDRVTITCSASSSGYYMHWFQQKPG domain of KSPKLWIYTTSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQYRFSYWYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 126 TQSPSFLSASVGDRVTITCSASSSISYMHWFQQKPG domain of KSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQQRSSFPYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 127 TQSPSFLSASVGDRVTITCSASSTITYMHWFQQKPG domain of KSPKLWIYYTSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQNRTSFHYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 128 TQSPSFLSASVGDRVTITCSASSSVSYMHWFQQKPG domain of KSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQQRSSYPYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 129 TQSPSFLSASVGDRVTITCSASSSVSYMHWFQQKPG domain of KSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQNRYSYPYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 130 TQSPSFLSASVGDRVTITCSASSTVYYMHWFQQKPG domain of KSPKLWIYSYSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQQRSSYHYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 131 TQSPSFLSASVGDRVTITCSASSSASYMHWFQQKPG domain of KSPKLWIYSYSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQYRTSYPYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 132 TQSPSFLSASVGDRVTITCSASSSGYYMHWFQQKPG domain of KSPKLWIYTTSNLASGVPARFSGSGSGTSYSLTISS the light LQPEDFATYYCQQRSSYPYTFGQGTKLEIK chain of the antibody II Variable SEQ ID NO. 133 VQLVQSGAEVKKPGASVKVSCKASGYSTSSFVMHWV domain of RQAPGQGLEWMGYINYYNDATKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCAWSGAIFEYWGQG chain of the TLVTVS antibody II Variable SEQ ID NO. 134 VQLVQSGAEVKKPGASVKVSCKASGYYTSSPVMHWV domain of RQAPGQGLEWMGYINYFYDGTKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCAKSAIAFEYWGQG chain of the TLVTVS antibody II Variable SEQ ID NO. 135 VQLVQSGAEVKKPGASVKVSCKASGYYTTSYVMHWV domain of RQAPGQGLEWMGYINPYYDGTKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCARSGGIFDYWGQG chain of the TLVTVS antibody II Variable SEQ ID NO. 136 VQLVQSGAEVKKPGASVKVSCKASGYSFTSYVMHWV domain of RQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCARSIGITDYWGQG chain of the TLVTVS antibody II Variable SEQ ID NO. 137 VQLVQSGAEVKKPGASVKVSCKASGYFFSSFVMHWV domain of RQAPGQGLEWMGYINYYNDATKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCARSAALSEYWGQG chain of the TLVTVS antibody II Variable SEQ ID NO. 138 VQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWV domain of RQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCAWSGAIFEYWGQG chain of the TLVTVS antibody II Variable SEQ ID NO. 139 VQLVQSGAEVKKPGASVKVSCKASGYFYTSYVMHWV domain of RQAPGQGLEWMGYINPFQDITKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCAWSAALSEYWGQG chain of the TLVTVS antibody II Variable SEQ ID NO. 140 VQLVQSGAEVKKPGASVKVSCKASGYFFSSFVMHWV domain of RQAPGQGLEWMGYINHFQDATKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCARSVGITDYWGQG chain of the TLVTVS antibody II Variable SEQ ID NO. 141 VQLVQSGAEVKKPGASVKVSCKASGYYTSSPVMHWV domain of RQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCAKSGGISDYWGQG chain of the TLVTVS antibody II Variable SEQ ID NO. 142 VQLVQSGAEVKKPGASVKVSCKASGYYTTSYVMHWV domain of RQAPGQGLEWMGYINHFQDATKYNEKFKGRVTITSD the heavy KSASTAYMELSSLRSEDTAVYYCAWSGAIFEYWGQG chain of the TLVTVS antibody II
TABLE-US-00007 TABLE 3 Positive Sequence Control No. Amino Acid Sequence Variable SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGK domain of a NO. 105 APKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFA light chain TYYCQQYLYHPATFGQGTKVEIK Variable SEQ ID EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPG domain of a NO. 106 KGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMN heavy chain SLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS
TABLE-US-00008 TABLE 4 Constant Sequence Region No. Amino Acid Sequence Constant SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK region of NO. 143 VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV a light YACEVTHQGLSSPVTKSFNRGEC chain Constant SEQ ID SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW region of NO. 144 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC a heavy NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP chain KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
TABLE-US-00009 TABLE 5 FR Sequence No. Amino Acid Sequence FR I-FR-L1 in the variable SEQ ID NO. 1 DIQMTQSPSSLSASVGDRV domain of the light chain TITCRAS of the antibody I FR I-FR-L2 in the variable SEQ ID NO. 2 LNWLQQKPGKAIKRLIY domain of the light chain of the antibody I FR I-FR-L3 in the variable SEQ ID NO. 3 SLDSGVPKRFSGSRSGSDY domain of the light chain SLTISSLQPEDFATYYC of the antibody I FR I-FR-L4 in the variable SEQ ID NO. 4 FGQGTKLEIK domain of the light chain of the antibody I FR I-FR-H1 in the variable SEQ ID NO. 5 VQLVQSGAEVKKPGASVKV domain of the heavy chain SCKAS of the antibody I FR I-FR-H2 in the variable SEQ ID NO. 6 MHWVRQAPGQGLEWMGA domain of the heavy chain of the antibody I FR I-FR-H3 in the variable SEQ ID NO. 7 SYNQKFKGRVTITAVTSAS domain of the heavy chain TAYMELSSLRSEDTAVYYC of the antibody I FR I-FR-H4 in the variable SEQ ID NO. 8 WGQGTLVTVS domain of the heavy chain of the antibody I FR II-FR-L1 in the variable SEQ ID NO. 9 TQSPSFLSASVGDRVTITC domain of the light chain SAS of the antibody II FR II-FR-L2 in the variable SEQ ID NO. 10 MHWFQQKPGKSPKLWIY domain of the light chain of the antibody II FR II-FR-L3 in the variable SEQ ID NO. 11 NLASGVPARFSGSGSGTSY domain of the light chain SLTISSLQPEDFATYYC of the antibody II FR II-FR-L4 in the variable SEQ ID NO. 12 FGQGTKLEIK domain of the light chain of the antibody II FR II-FR-H1 in the variable SEQ ID NO. 13 VQLVQSGAEVKKPGASVKV domain of the heavy chain SCKAS of the antibody II FR II-FR-H2 in the variable SEQ ID NO. 14 MHWVRQAPGQGLEWMGY domain of the heavy chain of the antibody II FR II-FR-H3 in the variable SEQ ID NO. 15 KYNEKFKGRVTITSDKSAS domain of the heavy chain TAYMELSSLRSEDTAVYYC of the antibody II FR II-FR-H4 in the variable SEQ ID NO. 16 WGQGTLVTVS domain of the heavy chain of the antibody II
[0167] Two types of antibody, the antibody I and the antibody II, were designed by this method.
[0168] The “positive control” antibody herein refers to an anti-PD-L1 antibody disclosed by F.Hoffmann-La Roche Ltd. A sequence of a variable domain of a light chain and a sequence of a variable domain of a heavy chain of the positive control antibody are respectively as shown in SEQ ID NO. 105 and SEQ ID NO. 106 in Table 3, and sequences of constant regions of the light chain and the heavy chain are respectively as shown in SEQ ID NO. 143 and SEQ ID NO. 144 in Table 4.
Example 2 Expression of Candidate Antibodies in Mammals
[0169] DNAs corresponding to amino acid sequences of a series of designed antibodies (an antibody I and an antibody II) and a positive control antibody were synthesized by conventional artificial DNA synthesis methods. With HEK 293 (or CHO cell) as a host cell and an ampicillin-resistant pTT5 plasmid as a vector, the DNAs of a series of designed antibodies (the antibody I and the antibody II) were inserted into vectors, and DNAs corresponding to the positive control antibody were inserted into a vector so that expression vectors were constructed. The constructed vectors were subjected to gene sequencing to determine correctly constructed vectors. The correctly constructed vectors (including positive control) were transiently or stably transfected into HEK 293 cells (or CHO cells), separately and incubated for 7 days. Candidate antibodies expressed in supernatants were preliminarily isolated, collected and filtered. A series of designed antibodies (the antibody I and the antibody II) were expressed so that a series of candidate antibodies (the antibody I and the antibody II) and the positive control antibody were obtained. Processes and steps used for the expression of the candidate antibodies were conventional methods and processes for the expression of antibodies in mammalian cells.
Example 3 Isolation and Purification of Candidate Antibodies
[0170] Candidate antibodies (an antibody I and an antibody II) and positive control were expressed in host cells and antibodies expressed in supernatants were preliminarily isolated and collected. The collected supernatants were isolated and purified by Protein A magnetic beads and then detected through SDS-PAGE. After each candidate antibody was isolated through SDS-PAGE, two clear electrophoretic bands were obtained at about 70 kDa and 30 kDa, respectively, and the expression of a heavy chain and a light chain of the antibody can be preliminarily determined. The SDS-PAGE electrophoresis diagrams of some antibodies (the antibody I and the antibody II) and a positive control antibody are shown in
Example 4 Determination of Affinity of Candidate Antibodies
[0171] To determine the binding affinity of a candidate antibody to an antigen, Bio-layer Interferometry (ForteBIO Octet system) was used for determining the affinity of the antibody to the antigen. The association constant, dissociation constant and affinity constant of the antibody with the antigen were determined through qualitative analyses. A positive control antibody was the positive control antibody in Example 1. The antigen was a human PD-L1 antigen (purchased from Beijing Sino Biological Inc, Catalog No. #10084-H08H-100). The isolated and purified antibody I and antibody II samples were taken to test the affinity of the antibody I, the antibody II and positive control to human PD-L1.
(1) Test of Binding Kinetics Based on Protein A
[0172] Protein A was used to bind to a candidate antibody I sample, a candidate antibody II sample and the positive control antibody to form Protein A-candidate antibody I sample, Protein A-candidate antibody II sample and Protein A-positive control antibody complexes. The human PD-L1 antigen was added, and binding kinetic curves were determined by a ForteBIO Octet platform. As can be known from test results, with continuous binding of Protein A-candidate antibodies to the antigen, an interference spectrum curve moves toward the direction of wavelength increase and finally tends to be stable. The candidate antibody I sample and the candidate antibody II sample both have strong binding activity to the human PD-L1 antigen. The test results of the candidate antibody I sample are shown in
(2) Test of Binding Kinetics Based on Anti-Human Fc
[0173] Anti-human Fc fragments were used to bind to the candidate antibody I sample, the candidate antibody II sample and the positive control antibody to form anti-human Fc fragment-candidate antibody I sample, anti-human Fc fragment-candidate antibody II sample and anti-human Fc fragment-positive control antibody complexes. The human PD-L1 antigen was added, and binding kinetic curves were determined by the ForteBIO Octet platform. As can be known from test results, with continuous binding of anti-human Fc fragment-candidate antibody I sample, anti-human Fc fragment-candidate antibody II sample and anti-human Fc fragment-positive control antibody to the human PD-L1 antigen, the detected interference spectrum curve moves toward the direction of wavelength increase. The test results of the candidate antibody I sample, the candidate antibody II sample and the positive control antibody are shown in
[0174] Kinetic constants were obtained from the preceding determined kinetic curves. The kinetic constants of the antibody I binding to the human PD-L1 antigen are shown in Table 6 and the kinetic constants of the antibody II binding to the human PD-L1 antigen are shown in Table 7.
TABLE-US-00010 TABLE 6 Disassociation Equilibrium Detection Sample Association Rate Rate Constant Disassociation of Antibody I Constant kon (1/Ms) kdis (1/s) Constant KD (M) Full R{circumflex over ( )}2 Antibody I-HX-1 8.92E+03 4.10E−07 4.60E−11 0.995131 Antibody I-HX-2 9.28E+03 2.70E−07 2.91E−11 0.992429 Antibody I-HX-3 7.08E+03 1.92E−06 2.71E−10 0.994982 Antibody I-HX-4 8.10E+03 8.95E−07 1.10E−10 0.991726 Antibody I-HX-5 7.12E+03 3.27E−06 4.59E−10 0.992477 Antibody I-HX-6 8.93E+03 4.29E−07 4.81E−11 0.993229 Antibody I-HX-7 9.09E+03 4.27E−07 4.70E−11 0.996294 Antibody I-HX-8 6.99E+03 1.81E−06 2.59E−10 0.996221 Positive control 7.29E+04 8.90E−07 1.22E−11 0.996965 antibody
[0175] As can be seen from Table 6, the KD of the antibody I obtained through affinity tests is between 2.91×10.sup.−11 to 4.59×10.sup.−10, which indicates that the antibody I has strong affinity to the human PD-L1 antigen. The antibody I-HX-2 has the strongest affinity to the human PD-L1 antigen and its equilibrium dissociation constant is 2.91×10.sup.−11, which is very close to that of the positive control.
TABLE-US-00011 TABLE 7 Association Disassociation Equilibrium Detection Rate Rate Disassociation Sample Constant Constant Constant of Antibody II kon (1/Ms) kdis (1/s) KD (M) Full R{circumflex over ( )}2 Antibody 2.51E+03 9.20E−06 3.67E−09 0.996271 II-HX-10 Antibody 2.75E+03 7.71E−06 2.80E−09 0.995129 II-HX-11 Antibody 3.47E+03 7.93E−07 2.28E−10 0.996629 II-HX-12 Antibody 3.01E+03 2.13E−06 7.07E−10 0.994516 II-HX-13 Antibody 2.99E+03 9.55E−06 3.19E−09 0.993947 II-HX-14 Antibody 3.76E+03 5.60E−07 1.49E−10 0.995068 II-HX-15 Antibody 2.50E+03 9.19E−06 3.71E−09 0.995161 II-HX-16 Antibody 2.71E+03 4.92E−06 1.82E−09 0.993928 II-HX-17 Antibody 3.08E+03 1.92E−06 6.24E−10 0.994701 II-HX-18 Antibody 3.04E+03 3.75E−06 1.23E−09 0.996247 II-HX-19 Positive control 7.29E+04 8.90E−07 1.22E−11 0.996965 antibody
[0176] As can be seen from Table 7, the equilibrium dissociation constant of the antibody II obtained through affinity tests is between 1.49×10.sup.−10 to 3.71×10.sup.−9, which indicates that the antibody II sample has relatively strong affinity to the human PD-L1 antigen. The antibody II-HX-15 has an equilibrium dissociation constant of 1.49×10.sup.−10 and has relatively strong affinity to human PD-L1 among test samples.
[0177] Amino acid sequences corresponding to test samples of the antibody I and the antibody II in Table 6 and Table 7 are shown in Table 8 and Table 9, respectively.
TABLE-US-00012 TABLE 8 Antibody Light Chain Variable Heavy Chain Light Chain Heavy Chain I Region Variable Region Constant Region Constant Region Antibody SEQ ID NO.107 SEQ ID NO.115 SEQ ID NO.143 SEQ ID NO.144 I-HX-1 Antibody SEQ ID NO.108 SEQ ID NO.116 SEQ ID NO.143 SEQ ID NO.144 I-HX-2 Antibody SEQ ID NO.109 SEQ ID NO.117 SEQ ID NO.143 SEQ ID NO.144 I-HX-3 Antibody SEQ ID NO.110 SEQ ID NO.118 SEQ ID NO.143 SEQ ID NO.144 I-HX-4 Antibody SEQ ID NO.111 SEQ ID NO.119 SEQ ID NO.143 SEQ ID NO.144 I-HX-5 Antibody SEQ ID NO.112 SEQ ID NO.120 SEQ ID NO.143 SEQ ID NO.144 I-HX-6 Antibody SEQ ID NO.113 SEQ ID NO.121 SEQ ID NO.143 SEQ ID NO.144 I-HX-7 Antibody SEQ ID NO.114 SEQ ID NO.122 SEQ ID NO.143 SEQ ID NO.144 I-HX-8 Positive SEQ ID NO.105 SEQ ID NO.106 SEQ ID NO.143 SEQ ID NO.144 Control Antibody
TABLE-US-00013 TABLE 9 Antibody Light Chain Variable Heavy Chain Light Chain Heavy Chain II Region Variable Region Constant Region Constant Region Antibody SEQ ID NO123 SEQ ID NO133 SEQ ID NO.143 SEQ ID NO.144 II-HX-10 Antibody SEQ ID NO124 SEQ ID NO134 SEQ ID NO.143 SEQ ID NO.144 II-HX-11 Antibody SEQ ID NO125 SEQ ID NO135 SEQ ID NO.143 SEQ ID NO.144 II-HX-12 Antibody SEQ ID NO126 SEQ ID NO136 SEQ ID NO.143 SEQ ID NO.144 II-HX-13 Antibody SEQ ID NO127 SEQ ID NO137 SEQ ID NO.143 SEQ ID NO.144 II-HX-14 Antibody SEQ ID NO128 SEQ ID NO138 SEQ ID NO.143 SEQ ID NO.144 II-HX-15 Antibody SEQ ID NO129 SEQ ID NO139 SEQ ID NO.143 SEQ ID NO.144 II-HX-16 Antibody SEQ ID NO130 SEQ ID NO140 SEQ ID NO.143 SEQ ID NO.144 II-HX-17 Antibody SEQ ID NO131 SEQ ID NO141 SEQ ID NO.143 SEQ ID NO.144 II-HX-18 Antibody SEQ ID NO132 SEQ ID NO142 SEQ ID NO.143 SEQ ID NO.144 II-HX-19 Positive SEQ ID NO.105 SEQ ID NO.106 SEQ ID NO.143 SEQ ID NO.144 Control Antibody
Example 5 PD-L1 Antigen Epitope Binding Competition Experiment
[0178] To analyze and determine the binding epitopes of an antibody I and an antibody II to an antigen, Bio-layer Interferometry (ForteBIO Octet system) was used for antigen epitope analysis experiments. Amino acid sequences of a positive control antibody are the sequences of the positive control antibody in Table 7 or Table 8. The antigen is a human PD-L1 antigen (purchased from Beijing Sino Biological Inc, catalog No. #10084-H08H-100). Antibody I-HX-2 is selected as an antibody I sample, and antibody II-HX-15 is selected as an antibody II sample.
[0179] The Bio-layer Interferometry (ForteBIO Octet system) was used for antigen epitope binding competition experiments of a candidate antibody I, a candidate antibody II and the positive control antibody. The test results are shown in
[0180] An ability of the human PD-L1 antigen to bind to the Anti-HIS and NTA biosensor of the ForteBIO Octet platform was determined to exclude the interference of the binding of the biolayer to the antigen. The results are shown in
[0181] As can be seen from
[0182] To conclude, the present application provides the anti-PD-L1 antibody, the method for preparing the same and the use thereof. An antibody sequence is simulated, analyzed and designed through bioinformatics modeling without complicated and cumbersome immune reactions, and the antibody is designed, transferred into a host cell through molecular biology construction, expressed in the host cell, and purified so that the antibody I and the antibody II can be obtained. The method is efficient and convenient. The antibody II has different binding epitopes from the positive control antibody and the antibody I and the antibody II have great market value and application prospect.
[0183] The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosed scope of the present application.