CN112759647B - anti-PD-L1 antibody and pharmaceutical application thereof - Google Patents

anti-PD-L1 antibody and pharmaceutical application thereof Download PDF

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CN112759647B
CN112759647B CN201910999788.4A CN201910999788A CN112759647B CN 112759647 B CN112759647 B CN 112759647B CN 201910999788 A CN201910999788 A CN 201910999788A CN 112759647 B CN112759647 B CN 112759647B
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宁金鹰
彭浩
郝锋
贺锋
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Shanghai Hongcheng Pharmaceutical Co ltd
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Abstract

The present invention provides an antibody molecule or antigen-binding fragment thereof that binds human PD-L1. The invention also provides the use of the antibody molecule or antigen binding fragment thereof in the manufacture of a medicament for the treatment of a tumour or cancer. Compared with the existing anti-PD-L1 antibody, the antibody provided by the invention has better affinity and dissociation rate for PD-L1, lower immunogenicity and better tumor inhibiting effect.

Description

anti-PD-L1 antibody and pharmaceutical application thereof
Technical Field
The invention belongs to the field of biological medicine, and relates to a novel anti-PD-L1 antibody or a functional fragment thereof. The invention also relates to the use of said antibodies or functional fragments thereof.
Background
PD-L1, which encodes a gene also known as CD274, is a ligand molecule for the programmed death factor PD1, often expressed on hematopoietic and non-hematopoietic cells such as T cells and B cells, as well as on various types of tumor cells. PD-L1 belongs to a type 1 transmembrane protein with immunoglobulin V-like and C-like domains. When PD-L1 interacts as a ligand with its receptor molecule PD-1, it inhibits T cell activation and cytokine production. This interaction is important to prevent autoimmunity by maintaining homeostasis of the immune response during normal tissue infection or inflammation. At the same time, this interaction plays a critical role in inducing and maintaining immune tolerance to itself, which in the tumor microenvironment often results in inactivation of toxic T cells, thereby providing immune escape to tumor cells. Expression of this gene in tumor cells is considered one of the criteria for whether a variety of human malignancies, including melanoma and non-small cell lung cancer, can be treated with PD-1 or PD-L1 antibody drugs.
Several PD-L1 mab drugs have been marketed, for example, the first PD-L1 mab drug, atezolizumab (trade name tecantrioq), developed and available by GENETECH under rocarvensis, for the treatment of advanced bladder cancer that failed or progressed to platinum-based chemotherapy. In 2017, the U.S. Food and Drug Administration (FDA) has accelerated approval of the new drug baventio for the treatment of Merck Cell Carcinoma (MCC) in adults and 12 year old children. The medicine contains Avelumab as main ingredient, and has sustained relieving effect on metastatic merck cell carcinoma (mMCC). In europe, the application of a license to PD-L1 antibody biologicals for the treatment of metastatic urothelial cancer is also under FDA priority. The 3 rd month of 2018, aslicon, also published its immunotherapy drug Imfinzi (Durvalumab) as formally approved by the U.S. Food and Drug Administration (FDA) for the treatment of non-small cell lung cancer stage III (NSCLC) patients who cannot be surgically resected.
Then, currently, there is room for improvement in affinity and dissociation rate of the monoclonal antibody against PDL1, and the immunogenicity of the Atezolizumab drug is relatively high according to clinical test data, which results in more anti-antibody production and greatly influences the therapeutic effect of the drug.
Disclosure of Invention
The invention aims to solve the technical problems that antibodies which specifically bind to human PD-L1, particularly have high affinity to human PD-L1, are obtained through hybridoma screening and humanization technologies.
In view of the above-mentioned problems, it is an object of the present invention to provide an antibody or a fragment thereof against human PD-L1, and to provide uses thereof based on the antibody or the fragment thereof. The "fragments" of the antibody molecules of the invention encompass various functional fragments of antibodies, for example, antigen-binding portions thereof, such as Fab, F (ab') 2 Or an scFv fragment.
The invention provides the following technical scheme.
In one aspect, the invention provides an antibody or fragment thereof capable of specifically binding to PD-L1, in particular human PD-L1, the amino acid sequence of which is set forth in SEQ ID NO. 3. According to a specific embodiment of the present invention, the antibody of the present invention is a murine antibody obtained using the extracellular region of human PD-L1 shown in SEQ ID NO. 3 as an immunogen, and a chimeric antibody and a humanized antibody obtained based on the murine antibody.
Specifically, the invention provides an antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a CDR combination (H-CDR 1, H-CDR2, H-CDR3; L-CDR1, L-CDR2, L-CDR 3) selected from the group consisting of:
(1) H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO:35 (EYTFTNNWIA), SEQ ID NO:36 (DIHPGGGFTNYNEKFKV), SEQ ID NO:37 (SKTRDYDAWFAY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:38 (KSSQSLLYTGNQKNYLA), SEQ ID NO:39 (WASTRES), SEQ ID NO:40 (QQYYTYRT);
(2) H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO:41 (GYTFTNYVVH), SEQ ID NO:42 (YVNPNNDGTIFNEKFKD), SEQ ID NO:43 (SPFAH); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:44 (SASESVDFYGTSLMQ), SEQ ID NO:45 (TASNVDS), SEQ ID NO:46 (HQTRKVPYT);
(3) H-CDR1, H-CDR2, H-CDR3 shown in sequence SEQ ID NO. 47 (GYIFTEYIIH), SEQ ID NO. 48 (WFYPGSDNIKYNEKFKD), SEQ ID NO. 49 (HETGYFFDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:50 (SASSSVSKMN), SEQ ID NO:51 (DTSKLAS), SEQ ID NO:52 (QQWSSNPLT);
(4) H-CDR1, H-CDR2, H-CDR3 shown in sequence SEQ ID NO:53 (GYSFTGYNMN), SEQ ID NO:54 (NIDPYYGVTHYNQKFKG), SEQ ID NO:55 (GIPFYGLDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:56 (GASSSVSFMH), SEQ ID NO:51 (DTSKLAS), SEQ ID NO:57 (QQWNTNPFT);
(5) H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO 58 (AFNIDDTYIH), SEQ ID NO 59 (RIDPANGNTDYDPECQG), SEQ ID NO 60 (GLRLPGLVY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:61 (RASQDISNYLN), SEQ ID NO:62 (YTILYS), SEQ ID NO:63 (QQGNTLPWT);
(6) H-CDR1, H-CDR2, H-CDR3 shown in sequence SEQ ID NO:64 (GFNIEDTYLH), SEQ ID NO:65 (RIDPANGNTYYDPKFQG), SEQ ID NO:66 (GLRLPGFPY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:67 (RASQDISNYLS), SEQ ID NO:68 (YTILS), SEQ ID NO:63 (QQGNTLPWT);
(7) H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO 69 (GDSIISGYWN), SEQ ID NO 70 (YISYTGSTYYNPSLKS), SEQ ID NO 71 (RGEWLLHFDV); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:72 (KSSQSLLYSSNQKNSLA), SEQ ID NO:39 (WASTRES), SEQ ID NO:73 (QQYYSYPLT);
(8) H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO:74 (GFSLTGYGVN), SEQ ID NO:75 (KIWGDGITDYNSALKS), SEQ ID NO:76 (DVMDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:77 (SASSSISYMH), SEQ ID NO:51 (DTSKLAS), SEQ ID NO:78 (HHHRSPYPT);
(9) H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO:74 (GFSLTGYGVN), SEQ ID NO:79 (KIWGDGSTDYTSALKS), SEQ ID NO:76 (DVMDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:77 (SASSSISYMH), SEQ ID NO:51 (DTSKLAS), SEQ ID NO:80 (HQRSPYPT);
(10) H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO:74 (GFSLTGYGVN), SEQ ID NO:75 (KIWGDGITDYNSALKS), SEQ ID NO:76 (DVMDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:81 (RSSQSIEQSNGNTYLE), SEQ ID NO:82 (KVSNSRFS), SEQ ID NO:83 (FQGSHVPYT);
(11) H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO:74 (GFSLTGYGVN), SEQ ID NO:75 (KIWGDGITDYNSALKS), SEQ ID NO:76 (DVMDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:84 (SASSSINYMH), SEQ ID NO:51 (DTSKLAS), SEQ ID NO:78 (HHHRSPYPT);
(12) H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO:35 (EYTFTNNWIA), SEQ ID NO:85 (DIHPGGGYTNYNEKFKG), SEQ ID NO:86 (SKTRDYDSWFAY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:50 (SASSSVSKMN), SEQ ID NO:51 (DTSKLAS), SEQ ID NO:52 (QQWSSNPLT).
The light and heavy chain CDRs provided by the present invention are combined from an antibody or fragment thereof of the present invention, and the CDRs contained therein can be routinely determined by one of skill in the art based on the variable region amino acid sequence contained in a given antibody or fragment thereof. For example, according to a specific embodiment of the present invention, the CDRs in the variable region amino acid sequence are defined using the AbM definition method.
In the antibodies or fragments thereof provided herein, preferably, the heavy chain variable region comprises a sequence selected from the group consisting of:
an amino acid sequence shown in SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 or SEQ ID NO. 33 or an amino acid sequence having at least 75% identity to said amino acid sequence; and/or
In the antibodies or fragments thereof provided herein, preferably, the light chain variable region comprises a sequence selected from the group consisting of:
the amino acid sequence shown in SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32 or SEQ ID NO. 34 or an amino acid sequence having at least 75% identity to said amino acid sequence.
More preferably, the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(1) An amino acid sequence as set forth in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 9; and, an amino acid sequence as set forth in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 10;
(2) An amino acid sequence as set forth in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 11; and, an amino acid sequence as set forth in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 12;
(3) An amino acid sequence as set forth in SEQ ID NO. 13 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 13; and, an amino acid sequence as set forth in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 14;
(4) An amino acid sequence as set forth in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 15; and, an amino acid sequence as set forth in SEQ ID NO. 16 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 16;
(5) An amino acid sequence as set forth in SEQ ID NO. 17 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 17; and, an amino acid sequence as set forth in SEQ ID NO. 18 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 18;
(6) An amino acid sequence as set forth in SEQ ID NO. 19 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 19; and, an amino acid sequence as set forth in SEQ ID NO. 20 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 20;
(7) An amino acid sequence as set forth in SEQ ID NO. 21 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 21; and, an amino acid sequence as set forth in SEQ ID NO. 22 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 22;
(8) An amino acid sequence as set forth in SEQ ID NO. 23 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 23; and, an amino acid sequence as set forth in SEQ ID NO. 24 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 24;
(9) An amino acid sequence as set forth in SEQ ID NO. 25 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 25; and, an amino acid sequence as set forth in SEQ ID NO. 26 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 26;
(10) An amino acid sequence as set forth in SEQ ID NO. 27 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 27; and, an amino acid sequence as set forth in SEQ ID NO. 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 28;
(11) An amino acid sequence as set forth in SEQ ID NO. 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 29; and, an amino acid sequence as set forth in SEQ ID NO. 30 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 30;
(12) An amino acid sequence as set forth in SEQ ID NO. 31 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 31; and, an amino acid sequence as set forth in SEQ ID NO. 32 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 32;
(13) The amino acid sequence shown in SEQ ID NO. 33 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 33; and, the amino acid sequence shown in SEQ ID NO. 34 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 34.
In the context of the present invention, "at least 75% identity" is any percent numerical identity between 75% and 100%, such as 75%, 80%, 85%, 90%, even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
In particular, the antibodies or fragments thereof of the invention comprise at least a heavy chain variable region and a light chain variable region, both comprising CDRs as described above and a framework region (frame work) at intervals, the arrangement of the individual domains being: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further alternatively, up to 25% difference in amino acid sequence resulting from said "at least 75% identity" may be present in any framework region in the heavy chain variable region or the light chain variable region, or in any domain or sequence outside the heavy chain variable region and the light chain variable region in an antibody or fragment thereof of the invention. The differences may result from amino acid deletions, additions or substitutions at any position.
As for the antigen, the antibody or fragment thereof of the present invention is an anti-PD-L1 antibody or fragment thereof; preferably, the antibody is a monoclonal antibody, a single chain antibody, a bifunctional antibody, a single domain antibody, a nanobody, a fully or partially humanized antibodyOr chimeric antibodies, or the antigen-binding fragments are half antibodies or antigen-binding fragments of antibodies or half antibodies, e.g., scFv, bsFv, dsFv, (dsFv) 2 、Fab、Fab'、F(ab') 2 Or Fv; more preferably, the antibody is IgG.
In addition to the variable region, the antibody or fragment thereof comprises a constant region of human or murine origin, preferably comprises a heavy chain constant region (CH) and/or a light chain constant region (CL) of human or murine origin; preferably, the antibody or fragment thereof comprises a heavy chain and a light chain; more preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, igA, igM, igD or IgE and/or a kappa or lambda type light chain constant region.
According to a specific embodiment of the invention, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type;
preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO. 1 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO. 1;
preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 2.
In another aspect, the invention also provides a nucleic acid molecule encoding an antibody or fragment thereof of the invention or encoding a heavy chain CDR, a light chain variable region, a heavy chain or a light chain comprised in said antibody or fragment thereof.
The nucleic acid molecules of the invention may be cloned into vectors to transform or transfect host cells. Thus in a further aspect, the invention also provides a vector comprising a nucleic acid molecule of the invention. The vector can be eukaryotic expression vector, prokaryotic expression vector, artificial chromosome, phage vector and the like. The vectors or nucleic acid molecules of the invention may be used to transform or transfect host cells for purposes such as preservation or antibody expression. Thus, in a further aspect, the invention provides a host cell comprising or transformed or transfected with a nucleic acid molecule and/or vector of the invention. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
The antibodies or fragments thereof provided herein may be obtained using any method known in the art. For example, the heavy and/or light chain variable regions of the antibody may be obtained from the nucleic acid molecules provided herein, or the heavy and/or light chain of the antibody may be obtained, and then assembled with the optional other domains of the antibody to form an antibody; alternatively, the host cell provided by the invention is cultured under conditions that allow the host cell to express the heavy and/or light chain variable regions of the antibody or the heavy and/or light chain of the antibody to assemble into the antibody. Optionally, the method further comprises the step of recovering the produced antibodies.
The antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells provided herein may be included in pharmaceutical compositions, more particularly in pharmaceutical formulations, for use for a variety of purposes as desired. Thus, in a further aspect, the invention also provides a pharmaceutical composition comprising an antibody or fragment thereof, a nucleic acid molecule, a vector and/or a host cell according to the invention, and optionally a pharmaceutically acceptable adjuvant.
In yet another aspect, the invention also provides the use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition in the manufacture of a medicament for the prevention or treatment of a disease, including a tumor or cancer, such as non-small cell lung cancer, melanoma, bladder cancer, merck lymphoma, cutaneous squamous cell carcinoma, lung cancer, hodgkin lymphoma, renal cancer, liver cancer, esophageal cancer, non-hodgkin lymphoma, breast cancer, thyroid cancer, gastric cancer, intestinal cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, leukemia, laryngeal cancer, oral cancer, ear eye tumors, biliary tract cancer, gall bladder cancer, adrenal gland cancer, reproductive system tumors, multiple myeloma, nervous system tumors, urothelial cell carcinoma.
Accordingly, the invention also provides a method of preventing or treating a disease comprising administering to a subject in need thereof an antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell and/or a pharmaceutical composition of the invention, the disease comprising a tumor or cancer, e.g., non-small cell lung cancer, melanoma, bladder cancer, merck lymphoma, cutaneous squamous cell carcinoma, lung cancer, hodgkin lymphoma, kidney cancer, liver cancer, esophageal cancer, non-hodgkin lymphoma, breast cancer, thyroid cancer, gastric cancer, intestinal cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, leukemia, laryngeal cancer, oral cancer, ear-eye tumors, biliary tract cancer, gall bladder cancer, adrenal cancer, reproductive system tumors, multiple myeloma, nervous system tumors, urothelial cell carcinoma. Preferably, the subject is a mammal; more preferably, the subject is a human.
In yet another aspect, the invention provides a kit comprising an antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell and/or a pharmaceutical composition of the invention. The kit may be for therapeutic or diagnostic use.
Compared with the prior art, the invention provides a novel antibody capable of binding PD-L1, especially human PD-L1 with high affinity. Compared with the existing anti-PD-L1 antibody, the antibody provided by the invention has the following characteristics: compared with the existing antibody (Atezolizumab (Tecentriq) of Genetech is taken as an example), the affinity and dissociation rate of the antibodies screened by the invention are improved; meanwhile, based on the murine antibody obtained by initial screening, the immunogenicity of the antibody is reduced by performing humanized transformation to the maximum extent; in addition, pharmacodynamic experiments prove that the antibody provided by the invention has better tumor inhibiting effect.
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Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the results of FACS detection of binding of the culture supernatant of the hybridoma cell lines of the invention to the antigen PD-L1.
FIG. 2 shows the results of FACS detection of the culture supernatant of hybridoma cells of the invention blocking the binding of PD-L1 to cells.
FIG. 3 shows the results of FACS detection of binding of the antibodies of the invention to the antigen PD-L1.
FIG. 4 shows the results of FACS detection of the antibodies of the invention blocking the binding of PD-L1 to cells.
FIG. 5 shows the results of a ForteBio Octet assay for binding of an antibody of the invention to the antigen PD-L1, wherein 5A:14A7 (hz); 5B: reference(s); 5C:14A7 (chi); 5D:14A7.
FIG. 6 shows the results of an in vivo efficacy test of an antibody of the invention in animals.
Detailed Description
The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials and reagent materials used in the examples below are all commercially available products unless otherwise specified.
Atezolizumab (Reference): genetech under the trade name Tecentriq, disclosed in US2016/0319022A1, see SEQ ID NO. 6 and SEQ ID NO. 7 for the light and heavy chains, respectively.
Example 1Preparation of hybridoma cells of the invention
Mice were immunized with a fusion protein comprising the extracellular region (position 19 to 238 of SEQ ID NO: 3) of the human PD-L1 protein (from Genbank accession number NP-054862.1,SEQ ID NO:3) with mouse IgG2a-FC (from Genbank accession number AAH 31470.1) as an immunogen, 5 mice were immunized subcutaneously, 5 mice were immunized intramuscularly, the adjuvant was quick anti 5W water-soluble adjuvant, titers were measured 2 weeks after boosting, two mice with high titers were selected for immunization impact, and cell fusion as described below was performed after 3 days.
Taking 2 mice to be fused, taking serum, dissecting, taking spleen, separating spleen cells, fusing with cultured myeloma cells, paving a 96-well plate, adding a selective culture medium for screening, changing liquid after 7 days, performing ELISA detection after 10 days, and selecting a flow cytometer with an OD value more than 10 times that of a negative control for detection.
Double positive cells were selected, subcloned plated by cell limiting dilution, and monoclonal cells were selected. And (3) taking culture supernatant of the selected monoclonal cells, performing ELISA detection and flow cytometry detection, and selecting double-positive cells for expansion culture.
Example 2ELISA detection of the binding of culture supernatants of hybridoma cells of the invention to PD-L1
Fusion proteins comprising the extracellular region (positions 19 to 238 of SEQ ID NO: 3) of human PD-L1 protein (from Genbank accession number NP-054862.1,SEQ ID NO:3) and human IgG1-FC (from Genbank accession number CAC 20454.1) were diluted to 1-2. Mu.g/ml with coating solution, then added to the wells of the enzyme-labeled plate at 50-100. Mu.l/well and allowed to adsorb at 4℃overnight or 37℃for 2 hours. The liquid in the wells was discarded and simultaneously washed 3 times with wash liquid for 3-5 minutes each time, and the wells were dried by patting. 200 μl of blocking solution was added to each well and blocked for 2 hours at 4deg.C or 37deg.C. Washing with washing liquid for 3 times, and storing the coated plate at-20deg.C or 4deg.C.
To each well 50-100. Mu.l of culture supernatant of hybridoma cells to be examined was added, while positive control (addition of serum of fusion mice), negative control (addition of serum of normal mice) and blank control (addition of medium) were established. Incubate at 37℃for 1-2 hours, wash, and pat dry. Then, enzyme-labeled secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG) (SIGMA, cat. No. A9044-2 ml) diluted 1:10000 was added to each well, and incubated at 37℃for 1-2 hours at 50-100. Mu.l per well, washed, and then dried. To each well, 50-100. Mu.l of freshly prepared substrate chromogenic solution TMB was added and incubated at 37℃for 10-30 minutes.
By adding 2mol/L H 2 SO 4 The reaction was terminated and OD values were read on an enzyme-linked immunosorbent assay reader.
And (3) result judgment: positive with P/N >2:1 (P represents positive value, N represents normal mouse serum value). If the negative control Kong Mose is or is near colorless, the positive control wells are clearly colored, and the results can be directly observed with the naked eye.
Example 3FACS detection of the binding of culture supernatants of hybridoma cells of the invention to PD-L1
Synthesis of the extracellular region (19 th to 238 th positions of SEQ ID NO: 3) gene fragment of human PD-L1 protein (from Genbank accession No. NP-054862.1,SEQ ID NO:3), construction into PLVX virus packaging vector (Clontech, virus package mix, cat. No. 631275), transfection of 293T cell packaging virus, infection of HEK293 cells with the virus, selection of drug-resistant cell lines by puromycin (puromycin) to obtain HEK293 stably expressing PD-L1, designated 293T-h-PDL1, and preparation of 1X 10 cell concentration in PBS containing 2% FBS 7 Cell suspensions of individual cells/ml.
Into each flow tube (sample tube) 50. Mu.l of the cell suspension was placed, and then 50. Mu.l of culture supernatant of hybridoma cells to be examined or positive control antibody Atezolizumab (Reference) was added and incubated at 4℃for 60 minutes. To each flow tube was added 1ml of flow buffer, centrifuged at 1200rpm for 5 minutes, the supernatant was discarded, and washing was repeated three times. Control tube 1 (no culture supernatant and secondary antibody below, only cell suspension) and control tube 2 (no culture supernatant, only cell suspension and secondary antibody below) were simultaneously set.
Then, 100. Mu.l of flow buffer was added to each flow tube for resuspension, 5. Mu.l of PE-labeled anti-murine Fc labeled secondary antibody (Biolegend, cat. No. 409304) was added according to the experimental requirements, incubated at 4℃for 30 minutes in the absence of light, then 1ml of flow buffer was added, centrifuged at 1200rpm for 5 minutes at room temperature, the supernatant was discarded, and washing was repeated three times.
250 μl of flow buffer was added to each flow tube again, resuspended, and checked on the machine.
The results of FACS binding assays are shown in table 1 and figure 1.
TABLE 1 FACS detection results of binding of culture supernatants of hybridoma cells to PD-L1
EC50(μg/mL)
13C9-1 0.4401
4G7-3 0.543
8A2-2 0.336
15G10-3 1.45
14A7-4 0.5383
28B1-2 0.3904
22B10-1 0.5172
18B4-2 0.1249
Reference 1.33
The left column of the table indicates the hybridoma cell line number and subclone number, e.g., "13C9-1" is the first clone of subclone number hybridoma cell line 13C 9.
Example 4FACS detection of culture supernatant of hybridoma cells of the invention blocking binding of PD-L1 to cells
The extracellular region (24 th to 170 th positions of SEQ ID NO: 8) gene fragment of human PD-1 protein (SEQ ID NO: 8) was synthesized, and was constructed to PLVX-IRES-PURO (Clontech), DH 5. Alpha. Competent cells were transformed, plated, and cultured overnight at 37 ℃. Selecting monoclonal colony, shaking, enzyme cutting, identifying, extracting plasmid, sequencing, selecting correct clone, extracting plasmid, and storing.
293T cells were resuscitated and passaged 2 times in 2X 10 cells 6 Plating individual cells/10 ml/dish, when the cell density grows to 70% -80% after 16-24 hours, transfecting the cells with the eukaryotic expression plasmid, placing at 37 ℃ and 5% CO 2 Culturing in an incubator. After culturing for 12-16 h, the culture medium is discarded, the liquid is changed, and after 48-56 h, the corresponding amount of Puromycin (final concentration is 10 mug/ml) is added. The cells were grown well when cultured continuously with DMEM+10. Mu.g/ml Puromycin containing 10% FBS for 10 days. And (5) primarily judging that the polyclonal cells are taken out, and performing flow detection.
Positive polyclonal cells were plated at 0.5 cells per well, and after 7 days, monoclonal cells were selected for expanded culture. FACS detection is carried out on the selected monoclonal, and positive cells, namely 293T cells stably expressing h-PD1, are selected and named as 293T-h-PD1 cells.
The cells were washed once with FACS buffer at 2-5X 10 5 Each cell/well was plated onto a 96-well plate, and 50. Mu.L of a premix of hybridoma supernatant or positive control antibody Atezolizumab (Reference) and 500ng of fusion protein PD-L1 (hFC tag) of example 2 was added to each well and incubated for 2 hours. Then 400 μl FACS buffer was added to wash twice per well, PE anti-human IgG Fc secondary antibody was added, and incubated for 1 hour, after which 400 μl FACS buffer was added to wash twice per well. And (5) detecting on the machine.
The detection results of FACS blocking are shown in table 2 and fig. 2.
TABLE 2 FACS detection of culture supernatants of hybridoma cells blocking binding of PD-L1 to cells
IC50(μg/mL)
13C9-1 1.95
4G7-3 3.42
8A2-2 5.17
15G10-3 4.00
14A7-4 2.31
28B1-2 2.31
22B10-1 4.31
18B4-2 2.89
Reference 2.97
Example 5Affinity detection of culture supernatants of hybridoma cells of the invention for binding to PD-L1
The experimental method comprises the following steps:
1. capture Surface Dip of the same genus is selected.
2. A96-well plate was prepared, 200. Mu.l SD buffer (1 XPBS+0.02% Tween20+0.1% BSA) was added to each well, and the mixture was placed in a ForteBio Octet for pre-cycling.
3. Antibody was immobilized and the concentration of the antibody after supernatant purification was 10. Mu.g/ml.
4. The extracellular region of the human PD-L1 protein (SEQ ID NO:3, 19 th to 238 th) was diluted by 6 gradients of 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, respectively, and added to the corresponding wells.
5. And (5) detecting on the machine.
The results are shown in Table 3.
TABLE 3 affinity assay results of culture supernatants of hybridoma cells for binding to PD-L1
Based on the experiment, corresponding hybridoma cell strains are selected, RNA is extracted from monoclonal cells, and is reversely transcribed into cDNA, and the cDNA is connected into a sequencing vector for sequencing analysis.
The sequences of exemplary murine antibodies are as follows, with the CDRs (obtained according to the AbM definition method) underlined. The murine antibody was named according to the hybridoma cell line from which it was derived.
A1 (murine antibody 14A 7)
>14A7-VH
QVQLKQSGAELVRPGTSVKMSCKATEYTFTNNWIAWVKQRPGHGLEWVGDIHPGGGFTNYNEKFKVKATLTADTSSSTAYMQLRSLTSEDSAIYYCAGSKTRDYDAWFAYWGQGTLVTVSA
>14A7-VLDIVMTQAPSSLAVSVGEKVTLNCKSSQSLLYTGNQKNYLAWYQQKPGQSPKLLIYWASTR ESGVPDRFTGSGSGTDFTLTISNVKAEDLAVYFCQQYYTYRTFGGGTKLEIK
A2 (murine antibody 15G 10)
>15G10-VH
QVQLKQSGPELVKPGASVKTSCKASGYTFTNYVVHWVKQNPGQGLEWIGYVNPNNDGTIFNEKFKDKAILTSDKSSSTAYMELSSLTSEDSAVYYCARSPFAHWGQGTLVTVSA
>15G10-VL
QIVLTQSPASLTVSLGQRATISCSASESVDFYGTSLMQWFQQKPGQPPKLLVYTASNVDSEVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCHQTRKVPYTFGGGTKLEIK
A3 (murine antibody 13C 9)
>13C9-VH
QVQLKQSGAGLVKPGASVKLSCKASGYIFTEYIIHWVKQRSGQGLEWIGWFYPGSDNIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARHETGYFFDYWGQGTTLTVSS
>13C9-VL
DIVITQSPAIMSASPGEKVTMTCSASSSVSKMNWYQQKSGTSPKRWIYDTSKLASGVPSRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLELKRA
A4 (murine antibody 18B 4)
>18B4-VH
QVQLKQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIGNIDPYYGVTHYNQKFKGKATLTVDESSSTASMQLKSLTSEDSAIYYCARGIPFYGLDYWGQGTSVTVSS
>18B4-VL
QIVLTQSPAIMSASPGEKVTMTCGASSSVSFMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNTNPFTFGSGTKLEIKRA
A5 (murine antibody 28B 1)
>28B1-VH
QVQLKQSGAELVKPGASVMLSCTASAFNIDDTYIHWVKQRPEQGLEWIGRIDPANGNTDYDPECQGKATITTDMSSNTAYLQLSSLTSEDTAVYFCARGLRLPGLVYWGRGTLVTVSA
>28B1-VL
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSILYSGVPSRFSGSGSGTDYSLTINTLEEEDIATYFCQQGNTLPWTFGGGTKLEIIRA
A6 (murine antibody 22B 10)
>22B10-VH
QVQLKQSGAELVKPGASVELSCTASGFNIEDTYLHWVNQRPEQGLEWIGRIDPANGNTYYDPKFQGKATITTDTSSNTAYLQLSRLTSEDTAVYYCARGLRLPGFPYWGQGTLVTVSA
>22B10-VL
DIQMTQTPSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLAISNLDQEDIATYFCQQGNTLPWTFGGGTKLEIKRA
A7 (murine antibody 4G 7)
>4G7-VH
QVQLKESGPSLVKPSQTLSLTCSVTGDSIISGYWNWIRKFPGNELEYMGYISYTGSTYYNPSLKSRVSIIRDTFKNQYYLQLNSVTTEDTATYYCARRGEWLLHFDVWGAGTTVTVSS
>4G7-VL
DIVMTQAPSSLAVSVGEKVTVSCKSSQSLLYSSNQKNSLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK
A8 (murine antibody 8A 2)
>8A2-VH
QVQLKQSGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGKIWGDGITDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARDVMDYWGQGTSVTVSS
>8A2-VL
QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSESGTSYSLTISSMEAEDAATYYCHHRSPYPTFGAGTKLELK
A9 (murine antibody 7E 5)
>7E5-VH
QVQLKQSGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGKIWGDGSTDYTSALKSRLSISKDNSKSQVFLKVNSLQTDDTARYYCARDVMDYWGQGTSVTVSS
>7E5-VL
QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWFQQKPGTSPKRWIYDTSKLASGVPARFSGSESGTSYSLTISSMEAEDAATYYCHQRSPYPTFGAGTKLELK
A10 (murine antibody 2E 5)
>2E5-VH
QVQLKQSGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGKIWGDGITDYNSALKSRLSISKDNSKSQVFLKMNSLQTEDTARYYCARDVMDYWGQGTSVTVSS
>2E5-VL
DVLMTQIPLSLPVSLGDQASISCRSSQSIEQSNGNTYLEWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVESEDLGVYYCFQGSHVPYTFGGGTKLEIK
A11 (murine antibody 10A 3)
>10A3-VH
QVQLKQSGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGKIWGDGITDYNSALKSRLSISKDNSKSQVFLKMNSLQTEDTARYYCARDVMDYWGQGTSVTVSS
>10A3-VL
QIVLTQSPAIMSASPGEKVTMTCSASSSINYMHWFQQKPGTSPKRWIYDTSKLASGVPARFSGSESGTSYSLTISSMEAEDAATYYCHHRSPYPTFGAGTKLELKRA
A12 (murine antibody 6G 5)
>6G5-VH
QVQLKQSGAELVRPGTSVKMSCKATEYTFTNNWIAWVKQRPGHGLEWIGDIHPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAIYYCAGSKTRDYDSWFAYWGQGTLVTVSA
>6G5-VL
DIVITQSPAIMSASPGEKVTMTCSASSSVSKMNWYQQKSGTSPKRWIYDTSKLASGVPSRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLELKRA
Example 6Humanized engineering of antibodies of the invention
The A1 antibody (murine antibody 14A 7) was selected for humanization of the antibody. Comprehensively considers the sequence similarity, the expression quantity and the light and heavy chain combination of the antibody and different humanized templates, and the like, whether the antibody is used by the antibody prepared by the patent medicine. According to factors such as that the light and heavy chain similarity value is greater than 200, the expression quantity is greater than 50mg/ml, the light and heavy chain combination is used as an antibody of the patent medicine, and finally, the human sequences are respectively selected as templates of the heavy chain and the light chain: IGHV1-46 x 01 (SEQ ID NO: 4) and IGKV1-5 x 01 (SEQ ID NO: 5). And carrying out homologous modeling on the monoclonal antibody of the A1 murine origin, and carrying out structural simulation on the Fab region. And finally obtaining the predicted Fab structure of the A1 antibody through homologous modeling calculation.
The CDR regions are completely replaced by templates of human origin by aligning and analyzing the predicted Fab structure and heavy chain with IGHV1-46 x 01 sequences, and then structural simulation and kinetic calculation are carried out. Through structural simulation and kinetic analysis, a plurality of amino acids affecting the CDR structure are kept to be of a murine origin, and finally, other murine amino acids except the CDR region, 68A, 70L and the like are all replaced by corresponding amino acids of IGHV1-46 x 01 template human.
And (3) replacing all CDR regions with templates of human sources through comparison and analysis of the predicted Fab structure and the light chain and IGKV1-5 x 01 sequences, and then carrying out structural simulation and kinetic calculation. Through structural simulation and kinetic analysis, several amino acids affecting the CDR structure are kept in murine form, and the final light chain version has all the other murine amino acids replaced with corresponding IGKV1-5 x 05 template human amino acids except the original murine amino acids in the CDR region.
The sequence of the humanized antibody of the A1 antibody is as follows:
VH humanized sequence 14A7-VH-hz
QVQLVQSGAEVKKPGASVKVSCKASEYTFTNNWIAWVRQAPGQGLEWVGDIHPGGGFTNYNEKFKVRATLTADTSTSTAYMELSSLRSEDTAVYYCAGSKTRDYDAWFAYWGQGTTVTVSS
VL humanized sequence 14A7-VL-hz
DIQMTQSPSTLSASVGDRVTITCKSSQSLLYTGNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYTYRTFGQGTKVEIK
The sequence shown in SEQ ID NO. 1 is used as a heavy chain constant region, the sequence shown in SEQ ID NO. 2 is used as a light chain constant region, primers are redesigned for the antibody DNA sequence aiming at the PD-L1 target point obtained by sequencing, corresponding chimeric antibody and humanized antibody genes are synthesized, the genes are connected into eukaryotic expression vectors, DH5alpha competent cells are transformed, the culture is carried out in a constant temperature incubator at 37 ℃ for overnight, and monoclonal strains are selected for sequencing identification. The strain with correct sequence is selected, shaken, and the plasmid is extracted, transfected into mammal expression cell 293F, placed in an incubator with 5% CO2 at 37 ℃ for expression culture for 7 days.
Collecting the expression supernatant, centrifuging, filtering, selecting protein G affinity chromatography column, purifying, detecting purity of the purified antibody by SDS-PAGE electrophoresis, detecting antibody concentration by BCA protein detection kit, packaging, and storing in-80deg.C refrigerator for use. Among them, the chimeric antibody was named "mouse antibody abbreviation (chi)", and the humanized antibody was named "mouse antibody abbreviation (hz)".
Example 7FACS detection of binding of antibodies of the invention to PD-L1
Experimental procedure referring to example 3, except that after 50. Mu.l of cell suspension was placed in each flow tube, the antibodies to be tested, positive control antibody (Reference) or negative Reference antibody (hIgG 1) were added at the concentrations shown in FIG. 3. Control tube 1 (no antibody and secondary antibody added, only cell suspension added) and control tube 2 (no antibody added, only cell suspension and secondary antibody added) were set simultaneously.
The results of FACS binding assays are shown in table 4 and fig. 3.
TABLE 4 FACS detection of antibody binding to PD-L1
EC50(μg/mL)
14A7-4(hz) 0.77
14A7-4(chi) 1.32
Reference 1.32
14A7-4 0.80
13C9-1(chi) 1.42
13C9-1 0.49
Example 8FACS detection of blocking binding of PD-L1 to cells by antibodies of the invention
Experimental procedure referring to example 4, except that the antibodies to be tested, the positive control antibody Atezolizumab (Reference) or the negative reference antibody (hIgG 1) were added per well, the concentrations are shown in fig. 4.
The detection results of FACS blocking are shown in table 5 and fig. 4.
TABLE 5 FACS detection results of antibody blocking binding of PD-L1 to cells
Example 9Affinity detection of binding of antibodies of the invention to PD-L1
Extracellular domains (19 th to 238 th positions of SEQ ID NO: 3) of human PD-L1 protein were prepared as a solution of 30nM maximum concentration in PBS buffer, and diluted 3-fold to 6 concentrations and 0-concentration control was set. Murine, chimeric and humanized antibodies of the present invention were prepared as a 20nM solution in PBS.
The affinity of the antibody antigen was determined using a ForteBio Octet as an affinity detection instrument. Specific methods of operation refer to example 5.
The experimental results are shown in table 6 and fig. 5.
TABLE 6 affinity assay results for binding of antibodies to the antigen PD-L1
Antibodies to Antigens KD(M) kdis(1/s)
Reference Human PD-L1-his 2.23E-09 7.96E-04
14A7(hz) Human PD-L1-his 8.84E-10 3.58E-04
14A7(chi) Human PD-L1-his 4.56E-09 1.73E-03
14A7 Human PD-L1-his 5.45E-10 3.59E-04
Example 10Efficacy test of the humanized PD-L1 antibody of the invention in animals
Referring to example 4, a fragment of the extracellular region (19 th to 238 th positions of SEQ ID NO: 3) gene of human PD-L1 protein (SEQ ID NO: 3) was synthesized to construct PLVX-IRES-PURO
(Clontech), a eukaryotic expression vector carrying h-PDL1 was obtained, and the plasmid was extracted.
Also referring to example 4, the 293T cells were replaced with MC38 cells, and MC38 cells stably expressing h-PDL1, designated MC38-h-PDL1 cells, were obtained.
Recovering MC38-h-PDL1 cells, and culturing in vitro to obtain 0.3X10E8 cells. 30 male BALB/C mice were subjected to 1 week of adaptive breeding and weighed. Cells were seeded according to the seeding conditions shown in table 7.
TABLE 7 mouse vaccination information
Animal number (only) Inoculation site Cell inoculum size/seed Inoculation of cell suspension volume/volume (mL)
30 Subcutaneous tissue 1 X 10 6 0.1mL
Tumor volume and body weight were measured 2 times per week after inoculation, when the average tumor volume reached about 80-120mm 3 The tumor volumes and body weights were randomly divided into 3 groups of 10 animals each. Administration is started immediately after grouping. The start date of administration was considered day 0. Drug administration and grouping information are shown in Table 8.
TABLE 8 grouping and administration information
After the start of the administration, the body weight and tumor volume of the mice were measured. The results are shown in FIG. 6.
The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, and shall fall within the scope of the appended claims.
Sequence listing
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<120> an anti-PD-L1 antibody and pharmaceutical use thereof
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Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr
245 250 255
Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys
260 265 270
Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu
275 280 285
Glu Thr
290
<210> 4
<211> 98
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 1-46.01 template
<400> 4
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg
<210> 5
<211> 95
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 1-5.01 template
<400> 5
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser
85 90 95
<210> 6
<211> 448
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> Atezolizumab heavy chain
<400> 6
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 7
<211> 214
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> Atezolizumab light chain
<400> 7
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 8
<211> 288
<212> PRT
<213> Homo sapiens
<400> 8
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 9
<211> 121
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 14A7-VH
<400> 9
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Thr Glu Tyr Thr Phe Thr Asn Asn
20 25 30
Trp Ile Ala Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile His Pro Gly Gly Gly Phe Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Val Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Gly Ser Lys Thr Arg Asp Tyr Asp Ala Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 10
<211> 112
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 14A7-VL
<400> 10
Asp Ile Val Met Thr Gln Ala Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Leu Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Asn Val Lys Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 11
<211> 114
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 15G10-VH
<400> 11
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Thr Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Val Val His Trp Val Lys Gln Asn Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Val Asn Pro Asn Asn Asp Gly Thr Ile Phe Asn Glu Lys Phe
50 55 60
Lys Asp Lys Ala Ile Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Pro Phe Ala His Trp Gly Gln Gly Thr Leu Val Thr Val
100 105 110
Ser Ala
<210> 12
<211> 111
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 15G10-VL
<400> 12
Gln Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Thr Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Ser Ala Ser Glu Ser Val Asp Phe Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Val Tyr Thr Ala Ser Asn Val Asp Ser Glu Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys His Gln Thr Arg
85 90 95
Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 13
<211> 118
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 13C9-VH
<400> 13
Gln Val Gln Leu Lys Gln Ser Gly Ala Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Glu Tyr
20 25 30
Ile Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg His Glu Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 14
<211> 108
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 13C9-VL
<400> 14
Asp Ile Val Ile Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met
20 25 30
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys Arg Ala
100 105
<210> 15
<211> 118
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 18B4-VH
<400> 15
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Val Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Ser
65 70 75 80
Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Gly Ile Pro Phe Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 16
<211> 108
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 18B4-VL
<400> 16
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Gly Ala Ser Ser Ser Val Ser Phe Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala
100 105
<210> 17
<211> 118
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 28B1-VH
<400> 17
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Met Leu Ser Cys Thr Ala Ser Ala Phe Asn Ile Asp Asp Thr
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Asp Tyr Asp Pro Glu Cys
50 55 60
Gln Gly Lys Ala Thr Ile Thr Thr Asp Met Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Leu Arg Leu Pro Gly Leu Val Tyr Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 18
<211> 109
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 28B1-VL
<400> 18
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Ile Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Asn Thr Leu Glu Glu
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ile Arg Ala
100 105
<210> 19
<211> 118
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 22B10-VH
<400> 19
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Glu Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr
20 25 30
Tyr Leu His Trp Val Asn Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Tyr Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Arg Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Leu Arg Leu Pro Gly Phe Pro Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 20
<211> 109
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 22B10-VL
<400> 20
Asp Ile Gln Met Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Ala Ile Ser Asn Leu Asp Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala
100 105
<210> 21
<211> 118
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 4G7-VH
<400> 21
Gln Val Gln Leu Lys Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Ile Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Glu Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Ser Ile Ile Arg Asp Thr Phe Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Arg Gly Glu Trp Leu Leu His Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 22
<211> 113
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 4G7-VL
<400> 22
Asp Ile Val Met Thr Gln Ala Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Val Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 23
<211> 113
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 8A2-VH
<400> 23
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
20 25 30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Lys Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Asp Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 24
<211> 105
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 8A2-VL
<400> 24
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Glu Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His His Arg Ser Pro Tyr Pro Thr Phe
85 90 95
Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 25
<211> 113
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 7E5-VH
<400> 25
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
20 25 30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Lys Ile Trp Gly Asp Gly Ser Thr Asp Tyr Thr Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Val Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Asp Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 26
<211> 105
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 7E5-VL
<400> 26
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Glu Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Pro Tyr Pro Thr Phe
85 90 95
Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 27
<211> 113
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 2E5-VH
<400> 27
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
20 25 30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Lys Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Asp Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 28
<211> 112
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 2E5-VL
<400> 28
Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Glu Gln Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ser Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 29
<211> 113
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 10A3-VH
<400> 29
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
20 25 30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Lys Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Asp Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 30
<211> 107
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 10A3-VL
<400> 30
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Asn Tyr Met
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Glu Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His His Arg Ser Pro Tyr Pro Thr Phe
85 90 95
Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala
100 105
<210> 31
<211> 121
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 6G5-VH
<400> 31
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Thr Glu Tyr Thr Phe Thr Asn Asn
20 25 30
Trp Ile Ala Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
Gly Asp Ile His Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Gly Ser Lys Thr Arg Asp Tyr Asp Ser Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 32
<211> 108
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 6G5-VL
<400> 32
Asp Ile Val Ile Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met
20 25 30
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys Arg Ala
100 105
<210> 33
<211> 121
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 14A7-VH-hz
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Glu Tyr Thr Phe Thr Asn Asn
20 25 30
Trp Ile Ala Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile His Pro Gly Gly Gly Phe Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Val Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Ser Lys Thr Arg Asp Tyr Asp Ala Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 34
<211> 112
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> 14A7-VL-hz
<400> 34
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 35
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 35
Glu Tyr Thr Phe Thr Asn Asn Trp Ile Ala
1 5 10
<210> 36
<211> 17
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 36
Asp Ile His Pro Gly Gly Gly Phe Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Val
<210> 37
<211> 12
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 37
Ser Lys Thr Arg Asp Tyr Asp Ala Trp Phe Ala Tyr
1 5 10
<210> 38
<211> 17
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 38
Lys Ser Ser Gln Ser Leu Leu Tyr Thr Gly Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210> 39
<211> 7
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 39
Trp Ala Ser Thr Arg Glu Ser
1 5
<210> 40
<211> 8
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 40
Gln Gln Tyr Tyr Thr Tyr Arg Thr
1 5
<210> 41
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 41
Gly Tyr Thr Phe Thr Asn Tyr Val Val His
1 5 10
<210> 42
<211> 17
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 42
Tyr Val Asn Pro Asn Asn Asp Gly Thr Ile Phe Asn Glu Lys Phe Lys
1 5 10 15
Asp
<210> 43
<211> 5
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 43
Ser Pro Phe Ala His
1 5
<210> 44
<211> 15
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 44
Ser Ala Ser Glu Ser Val Asp Phe Tyr Gly Thr Ser Leu Met Gln
1 5 10 15
<210> 45
<211> 7
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 45
Thr Ala Ser Asn Val Asp Ser
1 5
<210> 46
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 46
His Gln Thr Arg Lys Val Pro Tyr Thr
1 5
<210> 47
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 47
Gly Tyr Ile Phe Thr Glu Tyr Ile Ile His
1 5 10
<210> 48
<211> 17
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 48
Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Asp
<210> 49
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 49
His Glu Thr Gly Tyr Phe Phe Asp Tyr
1 5
<210> 50
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 50
Ser Ala Ser Ser Ser Val Ser Lys Met Asn
1 5 10
<210> 51
<211> 7
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 51
Asp Thr Ser Lys Leu Ala Ser
1 5
<210> 52
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 52
Gln Gln Trp Ser Ser Asn Pro Leu Thr
1 5
<210> 53
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 53
Gly Tyr Ser Phe Thr Gly Tyr Asn Met Asn
1 5 10
<210> 54
<211> 17
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 54
Asn Ile Asp Pro Tyr Tyr Gly Val Thr His Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 55
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 55
Gly Ile Pro Phe Tyr Gly Leu Asp Tyr
1 5
<210> 56
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 56
Gly Ala Ser Ser Ser Val Ser Phe Met His
1 5 10
<210> 57
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 57
Gln Gln Trp Asn Thr Asn Pro Phe Thr
1 5
<210> 58
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 58
Ala Phe Asn Ile Asp Asp Thr Tyr Ile His
1 5 10
<210> 59
<211> 17
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 59
Arg Ile Asp Pro Ala Asn Gly Asn Thr Asp Tyr Asp Pro Glu Cys Gln
1 5 10 15
Gly
<210> 60
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 60
Gly Leu Arg Leu Pro Gly Leu Val Tyr
1 5
<210> 61
<211> 11
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 61
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 10
<210> 62
<211> 7
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 62
Tyr Thr Ser Ile Leu Tyr Ser
1 5
<210> 63
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 63
Gln Gln Gly Asn Thr Leu Pro Trp Thr
1 5
<210> 64
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 64
Gly Phe Asn Ile Glu Asp Thr Tyr Leu His
1 5 10
<210> 65
<211> 17
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 65
Arg Ile Asp Pro Ala Asn Gly Asn Thr Tyr Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210> 66
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 66
Gly Leu Arg Leu Pro Gly Phe Pro Tyr
1 5
<210> 67
<211> 11
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 67
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ser
1 5 10
<210> 68
<211> 7
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 68
Tyr Thr Ser Ile Leu His Ser
1 5
<210> 69
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 69
Gly Asp Ser Ile Ile Ser Gly Tyr Trp Asn
1 5 10
<210> 70
<211> 16
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 70
Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 71
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 71
Arg Gly Glu Trp Leu Leu His Phe Asp Val
1 5 10
<210> 72
<211> 17
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 72
Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Ser Leu
1 5 10 15
Ala
<210> 73
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 73
Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr
1 5
<210> 74
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 74
Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn
1 5 10
<210> 75
<211> 16
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 75
Lys Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys Ser
1 5 10 15
<210> 76
<211> 5
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 76
Asp Val Met Asp Tyr
1 5
<210> 77
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 77
Ser Ala Ser Ser Ser Ile Ser Tyr Met His
1 5 10
<210> 78
<211> 8
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 78
His His Arg Ser Pro Tyr Pro Thr
1 5
<210> 79
<211> 16
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 79
Lys Ile Trp Gly Asp Gly Ser Thr Asp Tyr Thr Ser Ala Leu Lys Ser
1 5 10 15
<210> 80
<211> 8
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 80
His Gln Arg Ser Pro Tyr Pro Thr
1 5
<210> 81
<211> 16
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 81
Arg Ser Ser Gln Ser Ile Glu Gln Ser Asn Gly Asn Thr Tyr Leu Glu
1 5 10 15
<210> 82
<211> 7
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 82
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 83
<211> 9
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 83
Phe Gln Gly Ser His Val Pro Tyr Thr
1 5
<210> 84
<211> 10
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 84
Ser Ala Ser Ser Ser Ile Asn Tyr Met His
1 5 10
<210> 85
<211> 17
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 85
Asp Ile His Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 86
<211> 12
<212> PRT
<213> Artificial (Artifical)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 86
Ser Lys Thr Arg Asp Tyr Asp Ser Trp Phe Ala Tyr
1 5 10

Claims (18)

1. An anti-PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise CDRs of:
H-CDR1, H-CDR2, H-CDR3 shown in sequence as SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO: 37; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence as SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO: 40.
2. The anti-PD-L1 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(1) The amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 9; and, the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 10;
(2) The amino acid sequence shown in SEQ ID NO. 33 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 33; and, the amino acid sequence shown in SEQ ID NO. 34 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 34.
3. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is a monoclonal antibody, a single chain antibody, a bifunctional antibody, a single domain antibody, a nanobody, a fully or partially humanized antibody or a chimeric antibody; the antigen binding fragment is scFv, bsFv, dsFv, (dsFv) 2 、Fab、Fab'、F(ab') 2 Or Fv.
4. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is IgG.
5. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises a constant region of human or murine origin.
6. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 5, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) and/or a light chain constant region (CL) of human or murine origin.
7. The anti-PD-L1 antibody or antigen-binding fragment thereof of claim 5, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain.
8. The anti-PD-L1 antibody or antigen-binding fragment thereof of claim 5, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain constant region of IgG, igA, igM, igD or IgE and/or a kappa or lambda light chain constant region.
9. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-PD-L1 antibody is a murine, chimeric or humanized monoclonal antibody.
10. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 9, wherein the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type.
11. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 9, wherein the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID No. 1.
12. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 9, wherein the light chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID No. 2 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID No. 2.
13. A nucleic acid molecule encoding the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-12.
14. A vector comprising the nucleic acid molecule of claim 13.
15. A host cell comprising the nucleic acid molecule of claim 13 and/or the vector of claim 14, or transformed or transfected with the nucleic acid molecule of claim 13 and/or the vector of claim 14.
16. A pharmaceutical composition comprising the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the vector of claim 14 or the host cell of claim 15, and optionally a pharmaceutically acceptable adjuvant.
17. Use of the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the vector of claim 14, the host cell of claim 15, or the pharmaceutical composition of claim 16 in the manufacture of a medicament for preventing or treating a disease that is non-small cell lung cancer, melanoma, bladder cancer, merck lymphoma, cutaneous squamous cell carcinoma, lung cancer, hodgkin lymphoma, renal cancer, liver cancer, esophageal cancer, non-hodgkin lymphoma, breast cancer, thyroid cancer, gastric cancer, intestinal cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, laryngeal cancer, oral cancer, ear-eye tumors, biliary tract cancer, gall bladder cancer, adrenal cancer, tumors of the reproductive system, multiple myeloma, tumors of the nervous system, urothelial cell carcinoma.
18. A kit comprising the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the vector of claim 14, the host cell of claim 15, or the pharmaceutical composition of claim 16.
CN201910999788.4A 2019-10-21 2019-10-21 anti-PD-L1 antibody and pharmaceutical application thereof Active CN112759647B (en)

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CN108456251A (en) * 2017-02-21 2018-08-28 上海君实生物医药科技股份有限公司 Anti- PD-L1 antibody and its application
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CN108699146A (en) * 2016-01-04 2018-10-23 江苏怀瑜药业有限公司 Anti- PD-L1 antibody and application thereof
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