anti-PD-1 antibody and application thereof in preparation of medicine for treating cervical cancer patient
阅读说明：本技术 抗-pd-1抗体及其在制备治疗宫颈癌患者的药物中的用途 (anti-PD-1 antibody and application thereof in preparation of medicine for treating cervical cancer patient ) 是由 徐天 于 2021-10-16 设计创作，主要内容包括：本发明涉及抗PD-1抗体或其抗原结合片段在治疗宫颈癌中的用途。具体而言,本发明涉及抗PD-1抗体或其抗原结合片段在治疗一线或以上含铂标准化疗后的复发或转移、PD-L1表达阳性宫颈癌中的用途。(The present invention relates to the use of an anti-PD-1 antibody or antigen-binding fragment thereof in the treatment of cervical cancer. In particular, the invention relates to the use of an anti-PD-1 antibody or antigen-binding fragment thereof for the treatment of recurrence or metastasis following one or more platinum-containing standard chemotherapies, cervical cancer positive for PD-L1 expression.)
1. Use of an anti-PD-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of cervical cancer, wherein the antibody or antigen-binding fragment thereof comprises:
a) a heavy chain variable region comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 3 and CDR3 of SEQ ID NO. 5; and a light chain variable region comprising CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 9 and CDR3 of SEQ ID NO. 11;
b) a heavy chain variable region comprising CDR1 of SEQ ID NO 13, CDR2 of SEQ ID NO 15 and CDR3 of SEQ ID NO 5; and a light chain variable region comprising CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 17 and CDR3 of SEQ ID NO. 11;
c) a heavy chain variable region comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 15 and CDR3 of SEQ ID NO. 5; and a light chain variable region comprising CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 17 and CDR3 of SEQ ID NO. 19;
d) a heavy chain variable region comprising CDR1 of SEQ ID NO:21, CDR2 of SEQ ID NO:23 and CDR3 of SEQ ID NO: 25; and a light chain variable region comprising CDR1 shown in SEQ ID NO:27, CDR2 shown in SEQ ID NO:29 and CDR3 shown in SEQ ID NO:31
e) A heavy chain variable region comprising CDR1 of SEQ ID NO:33, CDR2 of SEQ ID NO:35 and CDR3 of SEQ ID NO: 37; and a light chain variable region comprising CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:41 and CDR3 of SEQ ID NO: 43; or
f) A heavy chain variable region comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 15 and CDR3 of SEQ ID NO. 5; and a light chain variable region comprising CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 17, and CDR3 shown in SEQ ID NO. 65.
2. The use of claim 1, the antibody or antigen-binding fragment thereof comprising:
a) a heavy chain variable region comprising SEQ ID NO 45; and a light chain variable region comprising SEQ ID NO 47;
b) a heavy chain variable region comprising SEQ ID NO 49; and a light chain variable region comprising SEQ ID NO 51;
c) a heavy chain variable region comprising SEQ ID NO 53; and a light chain variable region comprising SEQ ID NO: 55;
d) a heavy chain variable region comprising SEQ ID NO 57; and a light chain variable region comprising SEQ ID NO 59; or
e) A heavy chain variable region comprising SEQ ID NO 61; and a light chain variable region comprising SEQ ID NO 63;
f) a heavy chain variable region comprising SEQ ID NO 53; and a light chain variable region comprising SEQ ID NO 67.
3. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof being administered in an amount of no more than 10-8The Kd value of M, which is determined by plasmon resonance binding, specifically binds to human PD-1.
4. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof having an EC of no more than 100nM, or no more than 10nM50Binding to monkey PD-1, and/or no binding to mouse PD-1.
5. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof which blocks the binding of human or monkey PD-1 to its ligand with an IC50 of no more than 100 nM.
6. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof which does not bind to CD28 or CTLA 4.
7. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof that does not mediate ADCC or CDC or both.
8. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof, which is a fully human monoclonal antibody.
9. The use of claim 8, the antibody or antigen-binding fragment thereof, wherein the fully human monoclonal antibody is produced by a transgenic rat.
10. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof, which blocks binding of human PD-1 to its ligand and thus provides at least one of the following activities:
a) in CD4+Inducing IL-2 production in T cells;
b) in CD4+Inducing production of IFN γ in T cells;
c) induction of CD4+Proliferation of T cells; and
d) reversing the T reg inhibitory function.
11. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof, which is a bifunctional antibody (diabody), scFv dimer, dsFv, (dsFv)2, dsFv-dsFv ', Fv fragment, Fab ', or F (ab ') 2.
12. The use of claim 11, the antibody or antigen-binding fragment thereof, the diabody is BsFv or ds diabody (ds diabody).
13. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof, further comprising an immunoglobulin constant region.
14. The use of claim 1 or 2, the antibody or antigen-binding fragment thereof, further comprising a conjugate.
15. The use according to claim 1 or 2, wherein the occurrence of cervical cancer is associated with immunosuppression by PD-1.
16. The use of claim 1 or 2, wherein the cervical cancer is recurrent or metastatic cervical cancer.
17. The use of claim 1 or 2, wherein the cervical cancer is a recurrent or metastatic and/or PD-L1 expression positive cervical cancer following first-line or above platinum-containing standard chemotherapy.
18. The use according to claim 1 or 2, wherein the cervical cancer is advanced cervical cancer.
Technical Field
The invention relates to the field of antibody medicines, in particular to an anti-PD-1 antibody related to cervical cancer.
Background
Cervical Cancer (CC) is the second most common malignancy that severely affects female health, with new cases of Cervical Cancer accounting for approximately 1/4 of the total number of worldwide occurrences each year in our country. Evidence of increasing preclinical and clinical outcomes suggests that targeting immune checkpoints is becoming the most promising approach to treating cancer patients. Programmed cell death molecule 1 is one of the immune checkpoint proteins that plays a major role in limiting T cell activity, which provides the major immune resistance mechanism by which tumor cells can evade immune surveillance. The interaction of PD-1 expressed on activated T cells with PD-L1 expressed on tumor cells down-regulates the immune response and attenuates anti-tumor immunity. The expression of PD-L1 on tumors is associated with a decreased survival rate in esophageal, pancreatic and other types of cancer, highlighting the potential of this pathway as a new promising target for tumor immunotherapy. Pharmaceutical companies have developed a variety of drugs for the PD-1 pathway, such as BMSs, merck, roche, and Glatiramer Smith (GSK). Data from clinical trials show early evidence of persistent clinical activity and good safety in patients with various tumor types. Nivolumab is a PD-1 drug developed by BMS that is being put into the central phase of the next generation field. Currently in 6 late stage studies, treatment promoted tumor shrinkage in 3 of the 5 cancer groups studied, including 18% in 72 patients with lung cancer, nearly one-third in 98 patients with melanoma, and 27% in 33 patients with renal cancer. Lambrolizumab, developed by merck corporation, is a fully human monoclonal IgG4 antibody, and results from phase IB studies showed an objective anti-tumor response in 51% of 85 cancer patients and a complete response in 9% of patients. The experimental drug MPDL3280A from roche demonstrated a reduction in tumor size in 29 (21%) of 140 advanced cancer patients with tumors of various sizes.
The prognosis of advanced cervical cancer is poor, the first-line standard treatment is combined chemotherapy mainly based on platinum, and the overall survival time of the cisplatin single-drug treatment is about 6 to 9 months; the overall survival of cisplatin-based combination chemotherapy is about 9 to 18 months, with progression-free survival of about 4 months. There is a need to provide more treatment options for patients with recurrent or metastatic cervical cancer who have failed first-line standard chemotherapy. Pembrolizumab is approved by the FDA in the United states for treatment of patients with solid tumors with high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) with or without satisfactory replacement therapy for disease progression after treatment and for treatment of patients with recurrent or metastatic cervical cancer with PD-L1 positive (CPS ≧ 1) in or after chemotherapy for disease progression. The NCCN guideline in 2018 recommends Pembrolizumab for second-line treatment of PD-L1 positive (CPS ≧ 1) or MSI-H/dMMR relapse or metastatic cervical cancer (class 2A evidence). Most of patients with recurrent and metastatic cervical cancer are treated by multi-line platinum-containing regimen chemotherapy, after platinum drug resistance occurs, the selectable regimens are very limited, the treatment effect is poor, the development of clinical tests of immunotherapy for cervical cancer at home and abroad is not enough, and the field of cervical cancer in China has more unmet medical requirements, so that the treatment of the patients at the stage still has great requirements, and the treatment regimens of the patients at the stage need to be continuously supplemented and replaced.
Disclosure of Invention
The invention aims at the problem of poor clinical cervical cancer treatment effect, and provides the application of the anti-PD-1 antibody or the antigen binding fragment thereof in the treatment of cervical cancer, in particular the application in the treatment of recurrence or metastasis after one or more platinum-containing standard chemotherapies and positive cervical cancer expressed by PD-L1.
In a first aspect of the invention, there is provided a use of an anti-PD-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of cervical cancer, wherein the antibody or antigen-binding fragment thereof comprises:
a) a heavy chain variable region comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 3 and CDR3 of SEQ ID NO. 5; and a light chain variable region comprising CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 9 and CDR3 of SEQ ID NO. 11;
b) a heavy chain variable region comprising CDR1 of SEQ ID NO 13, CDR2 of SEQ ID NO 15 and CDR3 of SEQ ID NO 5; and a light chain variable region comprising CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 17 and CDR3 of SEQ ID NO. 11;
c) a heavy chain variable region comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 15 and CDR3 of SEQ ID NO. 5; and a light chain variable region comprising CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 17 and CDR3 of SEQ ID NO. 19;
d) a heavy chain variable region comprising CDR1 of SEQ ID NO:21, CDR2 of SEQ ID NO:23 and CDR3 of SEQ ID NO: 25; and a light chain variable region comprising CDR1 of SEQ ID NO:27, CDR2 of SEQ ID NO:29 and CDR3 of SEQ ID NO: 31;
e) a heavy chain variable region comprising CDR1 of SEQ ID NO:33, CDR2 of SEQ ID NO:35 and CDR3 of SEQ ID NO: 37; and a light chain variable region comprising CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:41 and CDR3 of SEQ ID NO: 43; or
f) A heavy chain variable region comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 15 and CDR3 of SEQ ID NO. 5; and a light chain variable region comprising CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 17, and CDR3 shown in SEQ ID NO. 65.
Further, the antibody or antigen binding fragment thereof comprises:
a) a heavy chain variable region comprising SEQ ID NO 45; and a light chain variable region comprising SEQ ID NO 47;
b) a heavy chain variable region comprising SEQ ID NO 49; and a light chain variable region comprising SEQ ID NO 51;
c) a heavy chain variable region comprising SEQ ID NO 53; and a light chain variable region comprising SEQ ID NO: 55;
d) a heavy chain variable region comprising SEQ ID NO 57; and a light chain variable region comprising SEQ ID NO 59; or
e) A heavy chain variable region comprising SEQ ID NO 61; and a light chain variable region comprising SEQ ID NO 63;
f) a heavy chain variable region comprising SEQ ID NO 53; and a light chain variable region comprising SEQ ID NO 67.
Further, the antibody or antigen binding fragment thereof is present in an amount of no more than 10-8The Kd value of M, which is determined by plasmon resonance binding, specifically binds to human PD-1.
Further, the antibody or antigen binding fragment thereof binds monkey PD-1 with an EC50 of no more than 100nM, or no more than 10nM, and/or does not bind mouse PD-1.
Further, the antibody or antigen binding fragment thereof blocks binding of human or monkey PD-1 to its ligand with an IC50 of no more than 100 nM.
Further, the antibody or antigen binding fragment thereof, which does not bind to CD28 or CTLA 4.
Further, the antibody, or antigen-binding fragment thereof, does not mediate ADCC or CDC or both.
Further, the antibody or antigen binding fragment thereof is a fully human monoclonal antibody.
Further, the antibody or antigen-binding fragment thereof, wherein the fully human monoclonal antibody is produced by a transgenic rat.
Further, the antibody or antigen binding fragment thereof, which blocks binding of human PD-1 to its ligand, and thus provides at least one of the following activities:
a) inducing IL-2 production in CD4+ T cells;
b) inducing production of IFN γ in CD4+ T cells;
c) inducing proliferation of CD4+ T cells; and
d) reversing the T reg inhibitory function.
Further, the antibody or antigen binding fragment thereof is a bifunctional antibody (diabody), scFv dimer, dsFv, (dsFv)2, dsFv-dsFv ', Fv fragment, Fab ', or F (ab ') 2.
Further, the antibody or antigen binding fragment thereof, wherein the diabody is BsFv or ds diabody (ds diabody).
Further, the antibody or antigen binding fragment thereof, further comprises an immunoglobulin constant region.
Further, the antibody or antigen binding fragment thereof, further comprising a conjugate.
Further, the cervical cancer is associated with immunosuppression by PD-1.
Further wherein the cervical cancer is recurrent or metastatic cervical cancer.
Further wherein the cervical cancer is a recurrent or metastatic and/or PD-L1 expression positive cervical cancer following first line or above platinum-containing standard chemotherapy.
Further wherein the cervical cancer is an advanced cervical cancer patient.
Drawings
FIG. 1 flow chart of research and design of cervical cancer of stage II
FIG. 2 investigator-assessed progression-free survival Kaplan-Meier survival curves (FAS) (survival curves for first and second stages, respectively)
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1: generation of antibody hybridomas
1.1 Generation of immunogen: the full length DNA encoding the ECD of PD-1 and PD-L1 or both was synthesized and inserted into the expression vector pcDNA3.3. Plasmid DNA was prepared in large quantities and the inserted DNA sequence was verified by sequencing. Fusion proteins of PD-1ECD and PD-L1ECD, which contain a variety of tags, including human Fc, mouse Fc and His tags, were prepared by transfection of the human PD-1ECD gene into CHO-S or HEK293 cells. After 5 days, the supernatant harvested from the transiently transfected cell culture was used for protein purification. The fusion protein was purified and quantified for immunization and screening.
1.2 establishment of stable cell lines. To obtain tools for antibody screening and validation, PD-1 and PD-L1 transfected cell lines were established. Briefly, the pCND3.3 expression vectors containing full-length PD-1 or PD-L1 were transfected into CHO-K1, 293F or Ba/F3 cells using the Lipofectamine 2000 transfection kit according to the manufacturer's instructions. 48-72 hours after transfection, the transfected cells were cultured in medium containing Blasticidin (Blasticidin) or G418 for selection. After a period of time, cells stably incorporating the PD-1 or PD-L1 gene in the genomic DNA will be selected. Meanwhile, whether the cells have the expression of the target genes PD-1 and PD-L1 is verified. Once the expression is verified, individual clones of interest are picked by limited dilution and scaled up to large capacity. The established monoclonal cell lines were then maintained in medium containing low doses of the antibiotics Blasticidin (Blasticidin) or G418.
1.3 establishment of antibody hybridomas.
1.3.1 immunization and cell fusion: primary challenge immunizations were performed using 8-10 week old OMT-rats (available from Open Monoclonal Technology, inc., Palo Alto, US) on the footpad with 10 μ g of human PD-1ECD protein in TiterMax, repeated every 3 days with PD-1ECD formulated with aluminum. Sera were collected from rats every 2 weeks and antibody titers were determined by ELISA or FACS assays. When the antibody titer reached a sufficiently high value, rats were administered the final freeAdjuvant challenge (100. mu.l 1XPBS addition instead) cell fusion was performed as follows: b lymphocytes isolated from lymph nodes of immunized OMT-rats were cell fused with myeloma cells (in a 1:1 ratio). The cell mixture was washed and suspended with 5-10ml of ECF solution. The ECF solution was added to adjust the concentration to 2x106Cells/ml. Immediately after electrofusion of the cells, the cell suspension in the fusion chamber was transferred into a sterile tube containing a larger volume of medium. After incubation at 37 ℃ for more than 24 hours, the cell suspensions were mixed and pipetted into 96-well plates (0.5 × 10)6Cells/plate). At 37 deg.C, 5% CO2The cells are cultured under conditions. When the clones were large enough, 100 μ Ι of supernatant was transferred from the 96-well plate for antibody screening test.
1.3.2 first round and confirmation screening of hybridoma supernatants: an ELISA test was used as a first round of screening to test hybridoma supernatants for binding to PD-1 protein. Briefly, plates were coated with 1. mu.g/ml of soluble protein of the extracellular domain of human PD-1 overnight at 4 ℃. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubated at room temperature for 1 hour. The plates were then washed and then incubated for 45 minutes with goat anti-rat IgG1HRP (Bethyl) and goat anti-rat IgG2b HRP (Bethyl) secondary antibody. After washing, TMB substrate was added and the reaction was stopped with 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device). To confirm the natural binding of the PD-1 antibody to the conformational PD-1 molecule expressed on the cell membrane, FACS analysis was performed on PD-1 transfected CHO-S cell lines. At 1x106Concentration of cells/ml CHO-S cells expressing PD-1 were transferred to a 96-well U-bottom plate (BD). The hybridoma supernatants were then transferred to the plates and incubated at 4 ℃ for 1 hour. After washing with 1 XPBS/1% BSA, goat anti-rat FITC (Jackson Immunoresearch Lab) secondary antibody was added and the cells were incubated at 4 ℃ for 1 hour in the dark. The cells were then washed and resuspended in 1 XPBS/1% BSA or fixed in 4% formalin and analyzed by flow cytometry (BD). Binding of the antibody to the parental CHO-S cell line was performed using the same method.
1.3.3 hybridoma subcloning: once passed the first wheelAnd confirmation screening to verify specific binding and blocking, the positive hybridoma cell line can be used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted in cloning media to 5 cells/well, 1 cell/well, and 0.5 cells/well. 200 μ l/well were plated into 96-well plates, one plate at 5 cells/well, one plate at 1 cell/well and four plates at 0.5 cells/well. All plates were incubated at 37 ℃ with 5% CO2. Incubate until all cell lines can be checked by ELISA assay.
Example 2: antibody hybridoma cell sequencing and fully human antibody characterization
2.1 antibody hybridoma cell sequencing: RNA was isolated from monoclonal hybridoma cells using Trizol reagent. VH and VL of PD-1 antibody were amplified using the following protocol: briefly, RNA is first reverse transcribed into cDNA using reverse transcriptase as described in the present application, reaction system (20 μ l):
mu.l of the PCR reaction product was taken for ligation with pMD18-T vector. Top10 competent cells were transformed with 10. mu.l of the ligation product and the mixture was transferred to 2-YT + Cab plates pre-warmed according to standard protocols and incubated overnight. Positive clones were checked by PCR using M13-48 and M13-47 primers and subsequently sequenced.
2.2 construction of fully human antibody molecules: VH and VL of PD-1 antibody were amplified as described above. The PCR reaction product was purified by PCR clean-up kit and VL and pCI vectors were digested with restriction enzymes Pme I and BssH II at 37 ℃ for 2 hours. The reaction products were electrophoresed in a 1% agarose gel and gel extracted according to the manufacturer's instructions. The digested VL and pCI vectors were ligated using the following steps:
the mixture was incubated at 16 ℃ for 30 minutes. Transformation and clonal propagation were performed with 10. mu.l of reaction product. Plasmid pCI-VL DNA extraction was performed using the confirmed clones. The pCI-VL vector and VH fragment were then digested with Xbal and Sal I and the purified digested VH and vector were ligated with T4DNA ligase at 16 ℃ for 30 min. Once the inserted VL and VH sequences were verified by sequencing, transient transfection and establishment of stable cell lines were performed using an expression vector containing whole IgG of fully human PD-1 antibody.
Example 3: characterization of fully human antibodies
3.1 full kinetic binding affinity of Surface Plasmon Resonance (SPR) assay: the affinity and binding kinetics of the antibody to PD-1 were characterized by SPR using ProteOn XPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized on a GLM sensor chip by amine coupling (Bio-Rad). The purified antibody was flowed over the sensor chip and captured by protein a. The chip was rotated 90 ℃ and washed with running buffer (1 XPBS/0.01% Tween20, Bio-Rad) until the baseline stabilized. 5 concentrations of human PD-1 and running buffer were flowed through the antibody flow cell at a flow rate of 100. mu.L/min, first for 240s for the bound phase and then 600s for the dissociated phase. After each run with H pH 1.73PO4Regenerating the chip. Binding and dissociation curves were fitted to a 1:1 Langmiur binding model using ProteOn software.
3.2 binding affinity of PD-1 antibodies to cell surface PD-1 molecules as determined by flow cytometry (FACS): the binding affinity of the antibodies to cell surface PD-1 was tested by FACS analysis. At 5x105Concentration of cells/ml CHO-S cells expressing PD-1 were transferred to a 96-well U-bottom plate (BD). The test antibody was diluted 1:2 serially with wash buffer (1 XPBS/1% BSA) and incubated at 4 ℃ for 1 hour. Secondary goat anti-human IgG Fc FITC (3.0 moles FITC per mole IgG, (Jackson Immunoresearch Lab)) was added and incubated at 4 ℃ for 1 hour protected from light. The cells were then washed once and resuspended in 1 XPBS/1% BSA and analyzed using flow cytometry (BD). Based on the quantified beads (Quantum. MESF Kit (Bangs Laboratories, Inc.), fluorescence intensity was converted to bound molecules per cell. K was calculated using Graphpad Prism5D。
3.3 Effect of human PD-1 antibody on T cell proliferation. The effect of PD-1 antibodies on T lymphocyte proliferation was tested using an allogeneic response. Primary Dendritic Cell (DC) -stimulated MLR was performed in 96-well U-bottomed tissue culture plates in 200. mu.l RPMI 1640 containing 10% FCS and antibiotics. Mixing DC with 1X105Allogeneic total CD4+T cells were mixed at a ratio of DC to T cells of 1:10 and 1: 100. Neutralizing mAb in the Presence or absence of conditionsThe cultivation was carried out as follows: the human PD-1 antibody and the reference antibodies A and B were used at a concentration of 10. mu.g/ml. Incubation for 5 days, 1 uCi/well of [ 2 ] was added during the last 16 hours3H]Thymidine. Measurement by scintillation counting3H]Incorporation of thymidine using a triple-well mean [ 2 ]3H]Thymidine incorporation (counts per minute) indicates a proliferative response. The DC-only counting is conventionally<1000 cpm. The results shown are representative examples of a minimum of 5 trials.
Human Dendritic Cells (DCs) and CD4 used in the above allogeneic MLR+T、CD8+T and total cells were generated from PBMCs as follows: human monocytes were purified from PBMCs by negative selection using a human monocyte concentration kit (human monocyte enrichment cocktail kit) according to the instructions of the manufacturer (StemCell Meylan). Briefly, PBMCs were isolated from healthy donor blood using a Ficoll-Paque gradient. Cells were washed twice with PBS, followed by 1X10 in isolation buffer8Cells/ml were resuspended and the Ab mix was concentrated with monocytes and incubated at 4 ℃ for 30 min. Unlabeled monocytes passed through the MACS column were collected. To generate iDCs, monocytes were cultured with GM-CSF (PeproTech, Rocky Hill, N.J.; 800U/ml) and IL-4 (PeproTech; 500U/ml) in RPMI 1640 medium containing 10% FCS and antibiotics at a cell concentration of 2X106Cells/ml. Half of the medium was replaced daily with medium containing GM-CSF and IL-4. iDCs were stimulated with LPS (026: B6; Sigma-Aldrich, St. Louis, MO; 1. mu.g/ml) for an additional 24 hours on day 5 to generate mature DCs. PBMC were prepared by contacting human CD4 with PBMC according to the manufacturer's instructions (Stemsep)+T、CD8+Purification of CD4 by negative selection of concentrated mixtures of T and Total T cells incubated with magnetic colloids+T、CD8+T and total T cells.
Stimulation of human CD4 with allogeneic DCs in the presence or absence of PD-1 antibodies 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb and 1.153.7hAb+T cells. Warp 23H]Evaluation of Thymidine incorporation CD4+Proliferation of T cells. 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb and 1.153.7hAb enhanced concentration dependent T cell proliferation.
3.5.2 in vitro humanEffect of the source PD-1 antibody on cytokine IFN γ secretion: to evaluate the blocking effect of human PD-1 antibody on the production of cytokine IFN γ, we performed an experiment of IFN γ production in allogeneic-MLR. Briefly, CD4 was used according to the manufacturer's instructions+T cell concentration kit (CD 4)+T cell enrichment cocktail kit) human CD4 was negative screened+T cells were purified from PBMC. Immature DCs were generated from monocytes cultured in GM-CSF and IL-4 for 5 days and differentiated into mature DCs by overnight stimulation with LPS at 1. mu.g/ml. Will CD4+T cells and iDC/mDC were mixed at T: DC ratios of 10:1 and 100: 1. The culture is performed in the presence or absence of a human PD-1 antibody and a standard antibody. After 5 days, supernatants from each culture were collected for determination of the cytokine IFN γ. IFN γ levels in the supernatant were determined by ELISA assay. Briefly, Maxisorp plates (0.75. mu.g/ml; i.e., 1/1360 diluted) were coated with anti-human IFN γ mAb diluted in coating buffer, 50. mu.l/well (i.e., 3.7. mu.l of antibody was added to a full 96-well plate in 5ml of coating buffer) and incubated overnight at 4 ℃. Add 200. mu.l/well of blocking buffer for 2 hours to block excess protein binding capacity. Dilutions of recombinant IFN γ were prepared as standards and were diluted two-fold with complete medium from 8000pg/ml to 125pg/ml, plus only complete medium. Plates were washed, standard and test supernatants (100. mu.l/well) were added and incubated for 2-4 hours. Biotinylated anti-IFN γ mAb (1/1333) in blocking buffer was added followed by additional avidin peroxidase. The reaction was performed by adding TMB substrate and quenched with 2M HCl. Absorbance was measured at 450 nm.
The results show that allogeneic DCs stimulate human CD4 in the presence or absence of the 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb, and 1.153.7hAb antibodies+T cells. IFN γ levels were determined by ELISA. The results show that fully human PD-1 antibody increases IFN γ secretion in a dose-dependent manner.
3.5.3 Effect of human PD-1 on Interleukin 2(IL-2) production in vitro: will CD4+T cells and iDC/mDC were mixed at T: DC ratios of 10:1 and 100: 1. In the presence or absence of a human PD-1 antibody and a reference antibodyThen, the culture was carried out. After 5 days, supernatants from each culture were collected for cytokine determination. IL-2 levels in the supernatants were determined by ELISA assays.
The results show stimulation of human CD4 with allogeneic DCs in the presence or absence of the antibodies of the present application or control antibodies+T cells. IL-2 levels were determined by ELISA. The results show that fully human PD-1 antibody increases IFN γ secretion in a dose-dependent manner. The results show that anti-PD-1 antibodies increase IL-2 secretion in a dose-dependent manner.
3.5.4 Effect of human PD-1 antibody on cell proliferation and cytokine production by autoantigen specific immune response: in this assay, T cells and DC cells are from the same donor. Briefly, CD4 was purified from PBMCs+T cells were cultured in CMV pp65 peptide and low dose IL2(20U/ml) while DCs were generated from monocytes cultured in PBMCs of the same donor in GM-CSF and IL-4. After 5 days, CD4 treated with CMV pp65 peptide+T cells were co-cultured with DCs to which pp65 peptide was pulsed in the presence or absence of human PD-1 antibody and a reference antibody (as a control).
On day 5, 100. mu.l of supernatant from each culture was used for the determination of the cytokines IFN. gamma. and IL-2. The level of production of IFN γ and IL-2 was detected by ELISA assay. A specific T cell proliferation targeting DC pulsed with CMV pp65 peptide3H]Thymidine incorporation assay.
The results show that the PD-1 antibody increased the concentration-dependent CMV stimulated by autologous DC loaded with CMV pp65 peptide+-CD4+Proliferation of T cells.
3.5.5 Effect of human PD-1 antibodies on regulatory T cell (Tregs) suppressive function: tregs are a subset of T cells, which are key immune regulators and play a key role in maintaining self-tolerance.
CD4+CD25+Regulatory T cells are associated with tumors because of the increased number of Tregs found in patients with a variety of cancers and are associated with a poorer prognosis. To directly assess the effect of human PD-1 antibodies on the immunosuppressive response, we performed TreAnd g, performing gs experiment. Separation of CD4 Using specific anti-CD 25 Microbeads (Miltenyi Biotec, Auburn, Calif.) and Positive or negative selection, respectively+CD25+And CD4+CD25-T cells. Initially, human CD4 was used according to the manufacturer's instructions (Stemsep)+T cell concentration mixture and magnetic colloid incubation of PBMC, negative selection purification of CD4+T cells. Then resuspend CD4 in MACS buffer+T cells, on ice with CD25+The beads were incubated for 30 minutes, washed and packed. Collecting non-column bound CD4 from the effluent solution+CD25-T cells, and washed prior to use. CD4 was subsequently recovered from the column+CD25+T cells and washed prior to use. Tregs were combined with CD4 in the presence or absence of human PD-1 antibody at a concentration of 10. mu.g/ml+CD25-T cells and DCs (Treg: Teff ratio 1:1) were cultured. No antibody or isotype antibody was used as a negative control. On day 5, the supernatant of the culture was taken for detection of cytokines by ELISA, by adding at a concentration of 1 uCi/well3H]Thymidine and further cultured for 18 hours, and cell proliferation was detected. [3H]Thymidine incorporation was counted by scintillation. The results show that the PD-1 antibody abolished Treg inhibitory function and restored responsive T cell proliferation and IFN γ secretion.
3.6ADCC/CDC assay: for healthy PD-1+The undesired cytotoxicity was minimized and the selected anti-PD-1 fully human antibodies were confirmed to be free of ADCC and CDC functions.
3.6.1 ADCC: activated T cells expressing high levels of cell surface PD-1 were used as target cells and pre-incubated with varying concentrations of fully human antibody in 96-well plates for 30 minutes, followed by the addition of IL-2 activated PBMC (used as a source of Natural Killer (NK) cells, i.e., effector cells) at an effector/target ratio of 50: 1. [ 5% CO at 37 ℃%2Incubate the plate in the incubator for 6 hours. Target cell lysis was determined by cytotoxicity detection kit (Roche). The optical density was determined by Molecular Devices SpectraMax M5e microplate reader. The results show that the fully human anti-PD-1 antibodies tested do not mediate ADCC.
3.6.2 CDC: target cells (activated T cells)) Diluted human serum complement (Quidel-A112) and various concentrations of fully human PD-1 antibody were mixed in 96-well plates. At 37 deg.C, 5% CO2Incubate the plate in the incubator for 4 hours. Target cell lysis was measured by CellTiter glo (Promega-G7573). Rituxan (Roche) and human B lymphocyte cell fine Raji (CD20 positive) were used as positive controls. The data show that the PD-1 antibody does not mediate CDC.
Example 4 clinical trial results of treating cervical cancer with the recombinant fully human anti-PD-1 monoclonal antibody
The recombinant human anti-PD-1 monoclonal antibody injection (such as GLS-010 injection) is used for researching the relapse or transfer of GLS-010 after first-line or above platinum-containing standard chemotherapy, the anti-tumor curative effect in the treatment of cervical cancer patients with positive PD-L1 expression (CPS is more than or equal to 1), the relapse or transfer of GLS-010 after first-line or above platinum-containing standard chemotherapy, the safety and tolerance of patients with positive PD-L1 expression cervical cancer, and the Pharmacokinetic (PK) characteristics and the exposure-reaction relationship of GLS-010; the immunogenicity of the recombinant fully human anti-PD-1 monoclonal antibody injection is observed, and the correlation between the microsatellite instability (MSI)/mismatch repair deficiency (dMMR) and/or tumor mutation coincidence (TMB) and the curative effect is preliminarily evaluated along with the evaluation of a diagnostic method (PD-L1).
1. The administration mode comprises the following steps:
all subjects were infused intravenously with GLS-010 at a dose of 240 mg/time, 1 time every two weeks, until either definitive disease progression or any of the following (whichever occurred first): 1) end of treatment period, 2) death, 3) development of intolerable toxic side effects, 4) pregnancy of the subject, 5) termination of the study as judged by the researcher from the subject's greatest benefit, 6) withdrawal of the subject or their guardian, 7) subject loss of visit, 8) poor subject compliance, failure to comply with the study protocol, etc.
The treatment period is 2 years. After 2 years of administration, if the investigator assessed that the subject could continue to benefit without intolerable adverse reactions, the subjects could enter the study for an extended treatment period (the extended treatment period is defined as the continued administration phase 2 years after administration) according to the wishes of the subjects and with approval of the applicant, and continue to receive study drug treatment.
The infusion time is not less than 45min each time, if the subject has mild anaphylaxis such as dizziness, shivering, and erythra during infusion, the infusion speed can be properly reduced (judged by the researcher), and the infusion time can be correspondingly prolonged (judged by the researcher).
2. And (3) experimental design:
the study included cervical cancer patients who had progressed to relapse or metastasis after receiving first-line or more standard platinum-containing chemotherapy and who had positive PD-L1 expression.
Phase I clinical studies show that anti-PD-1 monoclonal antibody GLS-010 has good safety and tolerance in patients with advanced tumors, and shows primary anti-tumor efficacy. The results of single-arm clinical study (KEYNOTE-158) of Pembrolizumab, a drug of the same class, on treatment of advanced cervical cancer showed a proportion of objective remission rates (ORR 14.3%), with 91% of patients responding over 6 months.
Pembrolizumab is approved by the FDA in the United states for treatment of patients with solid tumors with high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) with or without satisfactory replacement therapy for disease progression after treatment and for treatment of patients with recurrent or metastatic cervical cancer with PD-L1 positive (CPS ≧ 1) in or after chemotherapy for disease progression. The NCCN guideline in 2018 recommends Pembrolizumab for second-line treatment of PD-L1 positive (CPS ≧ 1) or MSI-H/dMMR relapse or metastatic cervical cancer (class 2A evidence). Considering that most of patients with recurrent and metastatic cervical cancer are treated by multi-line platinum-containing chemotherapy, the selectable scheme is very limited after platinum drug resistance occurs, and the treatment effect is poor, and the similar anti-PD-1 monoclonal antibody drug Pembrolizumab obtains a certain curative effect in clinical tests; clinical benefit was also observed in patients with advanced cervical cancer in the GLS-010 phase Ia study, where subjects were expected to benefit from the study, and ethical issues with placebo as a control may exist. The objective of the invention can be met by single-arm research by balancing scientific and ethical considerations.
3. The conditions of the subjects to be enrolled are as follows, and the conditions of the subjects are shown in Table 1:
subjects must meet all of the following inclusion criteria to be enrolled in the study:
1) voluntarily attending the clinical study; fully understand, inform this study and sign informed consent; willing to follow and able to complete all the test procedures;
2) women, age 18 to 75 years (including 18 and 75 years);
3) proved by histology, positive expression (CPS is more than or equal to 1) of PD-L1;
4) patients with recurrent or metastatic cervical cancer who have progressed after one or more standard chemotherapies with platinum as confirmed by imaging;
5) at least one measurable focus exists according to RECIST1.1 standard, namely the major diameter of a non-lymph node focus is more than or equal to 10mm or the minor diameter of a lymph node focus is more than or equal to 15mm according to CT cross section images or MRI;
6) eastern american tumor cooperative group (ECOG) physical status score 0 to 1;
7) the expected life span is not less than 12 weeks;
8) organ function and hematopoiesis must meet the following requirements:
hemoglobin (HGB) no less than 90 g/L;
white blood cell count (WBC) ≥ 3X 109/L；
Absolute Neutrophil Count (ANC) ≥ 1.5X 109/L；
Platelet count (PLT) ≥ 100X 109/L；
Total Bilirubin (TBIL) is less than or equal to 1.5 times the Upper Limit of Normal (ULN);
aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) of 2.5 × ULN; AST and ALT are less than or equal to 5 × ULN if the abnormal liver function is caused by tumor liver metastasis;
serum creatinine (Cr) is less than or equal to 1.5 × ULN; or creatinine clearance (CrCl) is more than or equal to 50mL/min
The International Normalized Ratio (INR) or the plasma Prothrombin Time (PT). ltoreq.1.5 × ULN.
9) Female subjects in the fertile age group must agree to effective contraceptive measures to be taken within 5 months after the informed consent was signed, during the study and after the last dose of GLS-010.
10) The subject must agree to provide sufficient tumor tissue samples for PD-L1 expression detection. Including archived tumor samples (paraffin blocks or unstained sections in quantities meeting the test requirements specified in the study); without the archived tumor tissue samples, the subjects agreed to receive a tumor lesion re-biopsy.
TABLE 1 distribution of subjects
N is the number of people to be studied in each stage.
Percentages are denominated in the number of study cohorts at each stage and are calculated only when the numerator is not 0.
4. The test results are shown in the following table and fig. 2.
The results show that all the subjects with positive PD-L1 expression are treated by other antitumor drugs, including cisplatin, paclitaxel, docetaxel, carboplatin, nedaplatin, albumin paclitaxel and bevacizumab, and relapse or metastasis occurs after treatment.
The recombinant fully human anti-PD-1 monoclonal antibody has good safety and anti-tumor curative effect on the whole data, has obvious anti-tumor curative effect in the treatment of cervical cancer patients with positive PD-L1 expression (CPS is more than or equal to 1) and relapse or transfer after first-line or above platinum-containing standard chemotherapy, has more excellent objective remission rate and total survival time evaluated by subject researchers compared with other similar medicines, has the total effective rate of more than 22 percent, is higher than the treatment effective rate of the similar medicines in the patients, can obviously prolong the median total survival time of the patients, obviously shortens the time from the first day of medication to the first evaluation of complete remission or partial remission, has good safety and tolerance, and has fewer adverse events of the subjects in the treatment period.
TABLE 1 expression of PD-L1 in subjects
N is the total analysis population number of each stage.
The percentages are denominated in the total analysis set number for each stage and are only calculated when the numerator is not 0.
BMI (body weight kg/height)2(m2)。
TABLE 2 previous tumor treatment history of subjects
N is the total analysis population number of each stage.
The percentages are denominated in the total analysis set number for each stage and are only calculated when the numerator is not 0.
Encoding is performed with WHODrug 201909 and MedDRA encoding dictionary (V24.0).
TABLE 3 Objective remission Rate (FAS) assessed by Subjects
Objective remission rate refers to the percentage of subjects whose overall efficacy achieved Complete Remission (CR) or Partial Remission (PR) according to RECIST1.1 criteria. The first stage 44 subjects can be evaluated, the second stage 98 subjects can be evaluated;
estimate 95% confidence intervals using the capper-Pearson exact method.
Two-term precision method assay.
N is the total analysis population number of each stage.
The percentages are denominated in the total analysis set number for each stage and are only calculated when the numerator is not 0.
TABLE 4 Total survival of Subjects (FAS)
Overall survival was defined as the time from the first dose to death due to any cause.
Median overall survival was calculated by Kaplan-Meier method with bilateral 95% confidence intervals calculated by brookmmeyer and Crowley method.
N is the total analysis population number of each stage.
The percentages are denominated in the total analysis set number for each stage and are only calculated when the numerator is not 0.
TABLE 5 median to remission time assessed by Subjects
Time to remission is defined as the time from the start of the first day of dosing to the first assessment of complete or partial remission, whichever occurs first. Only suitable for subjects who achieve complete or partial remission.
Median to remission times were calculated by the Kaplan-Meier method, with bilateral 95% confidence intervals calculated by the brookmmeyer and Crowley method.
N is the total analysis population number of each stage.
TABLE 6 evaluation of the safety and tolerability of the subjects (adverse events in the treatment of the subjects (TEAE) Profile (SS))
N is the safe population number of each stage.
The percentages are denominated in the safety pool number for each stage and are only calculated when the numerator is not 0.
The number of occurrences of an adverse event is exemplified.
Except for the AE analysis cases, the calculation was performed only once for the same subject in each row.
SAE: subject to evaluation by the investigator.
Encoding is performed using the MedDRA coding dictionary (V24.0).
Table 7 summary of immune-related adverse events during treatment for organ classification, first-choice terminology and greatest-severity classification
TABLE 7-1
TABLE 7-2
Tables 7 to 3
Tables 7 to 4
N is the safe population number of each stage.
The percentages are denominated in the safety pool number for each stage and are only calculated when the numerator is not 0.
The baseline severity is missing, and defaults to grade 1.
Severity of the same PT in the same subject in each row was calculated only once to the heaviest extent.
The same AE occurred multiple times in the same row of subjects in the test group, and if the severity was completely absent, the default was grade 3, and if the severity was absent at least once, only the remaining one with the highest severity was calculated.
Encoding is performed using the MedDRA coding dictionary (V24.0).
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
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cagcccccag ggaagggact ggagtggatt gggagtatct cttatagtgg gagcacctac 180
tacaatccgt ccctcaagag tcgagtcacc gtatccgtag acacgtccaa gaaccagttc 240
tccctgaagc tgaactctgt ggccgccaca gacacggctc tatattactg tgcgagacat 300
ctagggtata atgggaggta cctccccttt gactactggg gccagggaac cctggtcacc 360
gtctcctcc 369
<210> 55
<211> 110
<212> PRT
<213> Intelligent people
<400> 55
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Glu Leu
35 40 45
Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Asn Ile
85 90 95
Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 56
<211> 330
<212> DNA
<213> Intelligent people
<400> 56
cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60
tcctgcactg gaaccagcag tgacgttggt ttttataact atgtctcctg gtaccaacag 120
cacccaggca aagcccccga actcatgatt tatgatgtca gtaatcggcc ctcaggggtt 180
tctgatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240
caggctgagg acgaggctga ttattactgc agctcatata caaacatcag cacttgggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210> 57
<211> 123
<212> PRT
<213> Intelligent people
<400> 57
Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Thr
20 25 30
Thr Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly Ser Ile Ser Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Val Thr Ile Pro Val Asp Thr Ser Lys Asn Gln Ile
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ser Leu Tyr Tyr
85 90 95
Cys Ala Arg His Leu Gly Tyr Asn Ser Asn Trp Tyr Pro Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 58
<211> 369
<212> DNA
<213> Intelligent people
<400> 58
cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cctcggagac cctgtccctc 60
acctgcactg tctctggtgg ctccatcagc agtactactt actactgggg ctggatccgc 120
cagcccccag ggaaggggct ggagtggatt gggagtatct cttatagtgg gaccacctac 180
tacaacccgt ccctcaagag tcgagtcacc atccccgtag acacgtccaa gaaccagatc 240
tccctgaaac tgagctctgt gaccgccgca gacacgtctt tgtattattg tgcgagacat 300
ctcgggtata acagcaactg gtaccctttt gactactggg gccagggaac cctggtcacc 360
gtctcctca 369
<210> 59
<211> 110
<212> PRT
<213> Intelligent people
<400> 59
Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr
20 25 30
Asn Arg Val Ser Trp Tyr Gln Gln Pro Pro Gly Thr Ala Pro Glu Val
35 40 45
Ile Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
85 90 95
Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 60
<211> 330
<212> DNA
<213> Intelligent people
<400> 60
cagtcggccc tgactcagcc tccctccgtg tccgggtctc ctggacagtc agtcaccatc 60
tcctgcactg gaaccagcag tgacgttggt agttataacc gtgtctcctg gtaccagcag 120
cccccaggca cagcccccga agtcattatt tatgaggtca gtaatcggcc ctcaggggtc 180
cctgatcgct tctctgggtc caagtctggc aacacggcct ccctgaccat ctctgggctc 240
caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cacttgggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210> 61
<211> 119
<212> PRT
<213> Intelligent people
<400> 61
Glu Val Gln Leu Leu 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 Ser His
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Thr Gly Gly Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Lys Asn Arg Ala Gly Glu Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 62
<211> 357
<212> DNA
<213> Intelligent people
<400> 62
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactg 60
tcctgcgcag cctctggatt cacctttagc agccatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaact attactggtg gtggtggtag catatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attattgtgc gaaaaaccgc 300
gctggggagg gttactttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357
<210> 63
<211> 105
<212> PRT
<213> Intelligent people
<400> 63
Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Ala Arg Ile Thr Cys Gly Gly Asp Asn Ile Gly Asn Lys Asp Val
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Gly Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ile Trp Val Phe
85 90 95
Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 64
<211> 315
<212> DNA
<213> Intelligent people
<400> 64
tcctatgagc tgactcagcc actctcagtg tcagtggccc tgggacagac ggccaggatt 60
acctgtgggg gagacaacat tggaaataaa gatgtgcact ggtaccagca gaagccaggc 120
caggcccctg tgctggtcat ctatagggat agcaaccggc cctctgggat ccctgaggga 180
ttctctggct ccaactcggg gaacacggcc accctgacca tcagcagagc ccaagccggg 240
gatgaggctg actattactg tcaggtgtgg gacagcattt gggtgttcgg cggagggacc 300
aagctgaccg tccta 315
<210> 65
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 65
Ser Ser Tyr Thr Ser Ile Ser Thr Trp Val
1 5 10
<210> 66
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 66
agctcatata caagcatcag cacttgggtg 30
<210> 67
<211> 110
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 67
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Glu Leu
35 40 45
Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ile
85 90 95
Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 68
<211> 330
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 68
cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60
tcctgcactg gaaccagcag tgacgttggt ttttataact atgtctcctg gtaccaacag 120
cacccaggca aagcccccga actcatgatt tatgatgtca gtaatcggcc ctcaggggtt 180
tctgatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240
caggctgagg acgaggctga ttattactgc agctcatata caagcatcag cacttgggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
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