CN116510010A - anti-PD-1 antibodies and their use in the preparation of a medicament for treating non-small cell lung cancer patients - Google Patents
anti-PD-1 antibodies and their use in the preparation of a medicament for treating non-small cell lung cancer patients Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to the use of an anti-PD-1 antibody or antigen-binding fragment thereof in the treatment of non-small cell lung cancer. Provides a new way for treating non-small cell lung cancer.
Description
Technical Field
The invention belongs to the field of antibodies, and particularly relates to an anti-PD-1 antibody and application thereof in preparing a medicament for treating a non-small cell lung cancer patient.
Background
Non-small cell lung cancer is a type of lung malignancy that originates in the bronchial mucosa, bronchial glands and alveolar epithelium. In recent years, the occurrence rate and the death rate of lung cancer are continuously increased, and the data show that the global number of cases of lung cancer is about 220.6 ten thousand, and the number of patients dying from lung cancer is about 179.6 ten thousand. The number of new and dead lung cancer in China is 78.7 ten thousand and 63.1 ten thousand respectively. Undoubtedly, lung cancer is one of malignant tumors threatening human health, and the World Health Organization (WHO) classifies lung cancer into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC), with NSCLC accounting for 80% -85% and mortality also remaining high. Many researches show that NSCLC is cancer with the main characteristics of abnormal proliferation of lung adenocarcinoma cells and lung squamous epithelial cancer cells with the clinical survival rate of far less than 10%, the lung cancer cases are characterized by high recurrence rate and drug resistance and the survival rate of 15% -20% in 5 years, the incidence rate is increased year by year, advanced patients often have the phenomena of high malignancy degree, rapid cell proliferation speed, rapid disease development speed after incidence and the like, and therefore, the effective treatment of NSCLC is particularly important for improving and improving the life quality of the patients.
The clinical treatment means of NSCLC usually uses the comprehensive treatment of surgical excision, radiotherapy and auxiliary chemotherapy as the main treatment, and the side effects brought by the surgery, the chemotherapy and the radiotherapy have great influence on the prognosis of patients, so that the repeated condition of the patients can occur, and the life quality of the patients is reduced to a certain extent. For middle and late stage non-small cell lung cancer, comprehensive interventional therapy is an important way for improving the overall curative effect. The comprehensive intervention treatment is based on the comprehensive intervention treatment mainly comprising the bronchial artery approach and the comprehensive intervention treatment not comprising the bronchial artery approach, and has obvious clinical curative effects, can prolong the survival time of patients, improve the life quality of the patients and improve the survival rate of the patients. However, nearly one third of patients diagnosed with NSCLC are locally advanced non-small cell lung cancer, and only standard therapeutic chemotherapy can be adopted when the optimal surgical treatment machine is lost. Although research and development of early diagnosis, surgical treatment and chemotherapy drugs for lung cancer have achieved a certain research result in recent years, mortality rate and recurrence rate of lung cancer within 5 years remain high, and on the other hand, the necessity of developing new drug therapies and therapeutic strategies has been demonstrated.
Disclosure of Invention
In order to overcome the problems in the background art, the invention provides an anti-PD-1 antibody and application thereof in preparing medicaments for treating non-small cell lung cancer patients.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
use of an anti-PD-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for treating a patient with non-small cell lung cancer, wherein the antibody or antigen-binding fragment thereof comprises:
a heavy chain variable region comprising CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3; and a light chain variable region comprising CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6.
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) In CD4 + Induction of IL-2 production in T cells;
b) In CD4 + Induction of ifnγ production in T cells;
c) Induction of CD4 + Proliferation of T cells; and
d) Reversing Treg inhibition 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, the bifunctional antibody is BsFv or ds bifunctional antibody (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 comprises a conjugate.
The invention has the beneficial effects that: the invention provides an anti-PD-1 antibody, and provides a new way for treating non-small cell lung cancer.
Drawings
FIG. 1 is a graph of tumor growth curves for groups of mice in the HCC827 human non-small cell lung cancer Mixeno model of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding by the skilled person.
Example 1: antibody hybridoma production
1.1 production of immunogens
Full length DNA encoding the ECDs of PD-1 and PD-L1 or both was synthesized and inserted into 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-L1 ECD, prepared by transfection of the human PD-1ECD gene into CHO-S or HEK293 cells, contain a variety of tags, including human Fc, mouse Fc and His tags. After 5 days, the supernatant harvested from the transiently transfected cell culture was used for protein purification. The fusion proteins were 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, pCND3.3 expression vectors containing full length PD-1 or PD-L1 were transfected into CHO-K1, 293F or Ba/F3 cells using Lipofectamine 2000 transfection kit according to the manufacturer's instructions. 48-72 hours after transfection, the transfected cells are 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 genomic DNA are selected. At the same time, it was verified whether the cells had the expression of the genes of interest PD-1 and PD-L1. Once the expression was verified, individual clones of interest were picked by limiting dilution and amplified to large capacity. The established monoclonal cell line is then maintained in medium containing the low dose antibiotic Blasticidin (Blasticidin) or G418.
1.3 establishment of antibody hybridomas
1.3.1 immunization and cell fusion: primary challenge immunization was performed on footpads using 8-10 week old OMT-rats (available from Open Monoclonal Technology, inc., palo Alto, US) with 10 μg of human PD-1ECD protein in TiterMax, and the immunization was repeated every 3 days with PD-1ECD formulated with aluminum. Rats were bled every 2 weeks and serum was collected and antibody titers were determined by ELISA or FACS tests. When the antibody titer reached high enough, the final adjuvant-free challenge (100 μl 1XPBS substitution) was given to the rats and cell fusion was performed as follows: b lymphocytes isolated from lymph nodes of immunized OMT-rats were cell fused with myeloma cells (at 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 2X10 6 cells/mL. Immediately after cell electrofusion, the cell suspension in the fusion chamber was transferred into a sterile tube containing more volume of medium. After incubation at 37 ℃ for more than 24 hours, the cell suspension was mixed and pipetted into a 96-well plate (0.5x10) 6 Cell/plate). The cells were cultured at 37℃under 5% CO2. When the clones were large enough, 100 μl of supernatant was transferred from the 96-well plate for antibody screening test.
1.3.2 first round and confirmation screening of hybridoma supernatants: ELISA assays were used as the first round of screening to test binding of hybridoma supernatants to PD-1 proteins. Briefly, plates were coated overnight at 4℃with 1. Mu.g/mL of soluble protein of the extracellular domain of human PD-1. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubated for 1 hour at room temperature. The plates were then washed and then incubated with goat anti-rat IgG1 HRP (Bethyl) and goat anti-rat IgG2b HRP (Bethyl) secondary antibodies for 45 minutes. After washing, TMB substrate was added and the reaction was quenched with 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device). To confirm the natural binding of PD-1 antibodies to conformational PD-1 molecules expressed on the cell membrane, FACS analysis was performed on PD-1 transfected CHO-S cell lines. At 1x10 6 cell/mL concentration CHO-S cells expressing PD-1 were transferred to 96 well U-bottom plates (BD). SubsequentlyThe hybridoma supernatants were transferred to the plates and incubated for 1 hour at 4 ℃. After washing with 1XPBS/1% BSA, goat anti-rat FITC (Jackson Immunoresearch Lab) secondary antibody was added and incubated with cells at 4℃for 1 hour in the dark. The cells were then washed and resuspended in 1XPBS/1% BSA or fixed in 4% formalin and analyzed by flow cytometry (BD). Binding of antibodies to maternal CHO-S cell lines was performed using the same method.
1.3.3 subcloning of hybridomas: once specific binding and blocking is verified by the first round and confirmation of screening, subcloning can be performed using the positive hybridoma cell line. Briefly, for each hybridoma cell line, cells were counted and diluted to 5 cells/well, 1 cell/well, and 0.5 cells/well in cloning medium. 200. Mu.L/well was 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 placed at 37℃with 5% CO2. Incubate until all cell lines can be checked by ELISA test.
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 antibodies were amplified using the following protocol: briefly, first RNA was reverse transcribed into cDNA using reverse transcriptase as described herein, reaction system (20. Mu.L):
mu.L of the PCR reaction product was taken and subjected to ligation with the pMD18-T vector. Top10 competent cells were transformed with 10. Mu.L of 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, followed by sequencing.
2.2 construction of fully human antibody molecules: VH and VL of PD-1 antibodies were amplified as described above. The PCR reaction products were 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 manufacturer's instructions. The digested VL and pCI vectors were ligated using the following procedure:
the mixture was incubated at 16℃for 30 minutes. Transformation and clonal expansion were performed with 10. Mu.L of reaction product. Plasmid pCI-VL DNA was extracted using the confirmed clone. The pCI-VL vector and VH fragment were then digested with Xbal and Sal I and purified digested VH and vector were ligated using T4 DNA ligase at 16℃for 30 min. Once the sequence of the inserted VL and VH was verified by sequencing, transient transfection and establishment of stable cell lines were performed using expression vectors containing whole IgG of fully human PD-1 antibodies.
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 antibodies to PD-1 were characterized by the SPR method using Proteon XPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized on a GLM sensor chip (Bio-Rad) by amine coupling. Purified antibodies were flowed through 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 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, followed by a combined phase flow of 240s and then a dissociated phase of 600s. After each run with H at pH 1.7 3 PO 4 Regenerating 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 5x10 5 cell/mL concentration CHO-S cells expressing PD-1 were transferred to 96 well U-bottom plates (BD). The antibodies to be tested were serially diluted 1:2 (1 XPBS/1% BSA) with wash buffer and incubated for 1 hour at 4 ℃. Secondary anti-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 in the dark. Cells were then washed once and resuspended in 1XPBS/1% BSA and analyzed using flow cytometry (BD). Based on the quantified beads (QuantumTM MESF Kit (Bangs Laboratories, inc.)), the fluorescence intensity was converted into a bound molecule on each cell. KD was calculated using Graphpad Prism 5.
3.3 effects of human PD-1 antibodies on T cell proliferation. The effect of PD-1 antibodies on T lymphocyte proliferation was tested using an allogeneic response. Primary Dendritic Cells (DC) -stimulated MLR were performed in 200. Mu.L RPMI 1640 containing 10% FCS and antibiotics in 96-well U-bottom tissue culture plates. DC was combined with 1X10 5 Allogeneic total CD4 of (C) + T cells were mixed in a ratio of 1:10 and 1:100 DC to T cells. Culturing in the presence or absence of neutralizing mAb: the human PD-1 antibody and reference antibodies A and B were used at a concentration of 10. Mu.g/ml. Incubation test for 5 days, 1 uCi/well added during the last 16 hours [ 3 H]Thymidine. Determination by scintillation counting [ 3 H]Thymidine incorporation, with three-well averaging [ 3 H]Thymidine incorporation (counts per minute) indicates a proliferative response. DC only counts were conventionally<1000cpm. The results shown are representative examples of a minimum of 5 trials performed.
Human Dendritic Cells (DC) and CD4 for use 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 human monocyte concentration kit (human monocyte enrichment cocktail kit) according to the instructions of the manufacturer (StemCell menu). Briefly, PBMC were isolated from healthy donor blood using Ficoll-Paque gradient. Cells were washed twice with PBS, then 1X10 in separation buffer 8 cells/mL were resuspended and the Ab mixture was incubated with monocyte concentration for 30 min at 4 ℃. Unlabeled monocytes passed through MACS column were collected. To generate iDC, monocytes are cultured with GM-CSF (PeproTech, rocky Hill, NJ; 800U/ml) and IL-4 (PeproTech; 500U/ml) at a cell concentration of 2X10 in a medium of RPMI 1640 containing 10% FCS and antibiotics 6 cells/mL. Half of the medium was replaced daily with medium containing GM-CSF and IL-4. The iDC was 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. By combining PBMC with human CD4 according to the manufacturer's instructions (Stemsep) + T、CD8 + Negative selection of T and Total T cell concentrate mixtures and magnetic colloid incubation, purification of CD4 + 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 [ warp ] 3 H]Assessment of thymidine incorporation CD4 + Proliferation of T cells. 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb and 1.153.7hAb increased concentration-dependent T cell proliferation.
3.4 effects of in vitro human PD-1 antibodies on cytokine IFNγ secretion: to evaluate the blocking effect of human PD-1 antibodies on the production of the cytokine IFNγ, we performed experiments on the production of IFNγ in allogeneic-MLR. Briefly, CD4 was used according to the manufacturer's instructions + Negative selection of T cell concentrate kit (CD4+ T cell enrichment cocktail kit) for human CD4 + T cells were purified from PBMCs. Immature DCs were generated in monocytes cultured for 5 days in GM-CSF and IL-4 and differentiated to mature DCs by stimulation with LPS at 1 μg/mL overnight. CD4 + T cells and iDC/mDC were mixed in a T:1 and 100:1 ratio of T to DC. The culture is performed in the presence or absence of the human PD-1 antibody and the reference antibody. After 5 days, the supernatant of each culture was collected and assayed for cytokine ifnγ. Ifnγ levels in the supernatant were determined by ELISA test. Briefly, maxisorp plates (0.75. Mu.g/mL; i.e., 1/1360) were coated with anti-human IFNγ mAb diluted in coating buffer, 50. Mu.L/well (i.e., 3.7. Mu.L antibody was added to 5mL of coating buffer to a full 96-well plate) and incubated overnight at 4 ℃. Blocking buffer 200 μl/well was added for 2 hours to block excess protein binding capacity. Recombinant IFNγ dilutions were prepared as standard solutions and double dilutions were performed from 8000pg/mL to 125pg/mL with complete medium, plus the case with complete medium only. The plates were washed, standard solution and test supernatant (100. Mu.L/well) were added and incubated for 2-4 hours. Biotinylated anti-IFNγ 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 showed the presence or absence of 1.7.3hAb, 1In the case of antibodies 49.9hAb, 1.103.11hAb, 1.139.15hAb and 1.153.7hAb, human CD4 was stimulated with allogeneic DCs + T cells. Ifnγ levels were determined by ELISA. The results showed that fully human PD-1 antibodies increased ifnγ secretion in a dose dependent manner.
3.5 in vitro Effect of human PD-1 on Interleukin 2 (IL-2) production: CD4 + T cells and iDC/mDC were mixed in a T:1 and 100:1 ratio of T to DC. The culture is performed in the presence or absence of the human PD-1 antibody and the reference antibody. After 5 days, the supernatant from each culture was collected and assayed for cytokines. IL-2 levels in supernatants were determined by ELISA testing.
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 showed that fully human PD-1 antibodies increased ifnγ secretion in a dose dependent manner. The results show that anti-PD-1 antibodies increase IL-2 secretion in a dose dependent manner.
3.6 Effect of human PD-1 antibodies on cell proliferation and cytokine production by autoantigen-specific immune responses: in this assay, T cells and DC cells are from the same donor. Briefly, CD4 purification from PBMC + T cells were cultured in CMV pp65 peptide and low dose IL-2 (20U/mL) while DC were generated from monocytes cultured in PBMC of the same donor as in GM-CSF and IL-4. After 5 days, CD4 to be treated with CMV pp65 peptide was used + T cells were co-cultured with DCs pulsed with pp65 peptide in the presence or absence of human PD-1 antibody and reference antibody (as controls).
On day 5, 100. Mu.L of supernatant from each culture was used to determine the cytokines IFNγ and IL-2. Levels of ifnγ and IL-2 production were detected by ELISA assays. Specific T cell proliferation passage against pulsed addition of CMV pp65 peptide DC [ 3 H]And (3) measuring the incorporation of the thymidine.
The results show that PD-1 antibodies increased concentration-dependent CMV stimulated by autologous DCs loaded with CMV pp65 peptide + -CD4 + Proliferation of T cells.
3.7 Effect of human PD-1 antibodies on regulatory T-cell (Tregs) inhibition function: tregs are a subset of T cells that are critical immunomodulators, playing a critical role in maintaining self tolerance.
CD4 + CD25 + Regulatory T cells are associated with tumors because increased numbers of Tregs are found in various cancer patients and are associated with a poor prognosis. To directly assess the effect of human PD-1 antibodies on the immunosuppressive response, we performed Tregs experiments. CD4 was isolated 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 manufacturer's instructions (Stemsep) + PBMC were incubated with T cell concentrate mixture and magnetic colloid for purification of CD4 by negative selection + T cells. Thereafter, CD4 was resuspended in MACS buffer + T cells, on ice and CD25 + The beads were incubated for 30 minutes, washed and loaded onto a column. Collecting CD4 from the effluent solution that does not bind to the column + CD25 - T cells and washed prior to use. Subsequent recovery of CD4 from the column + CD25 + T cells were washed prior to use. Tregs are combined with CD4 in the presence or absence of a 10. Mu.g/mL concentration of human PD-1 antibody + CD25 - T cells and DCs (Treg: teff ratio 1:1) were cultured. No antibody or isotype antibody was used as negative control. Supernatants from the cultures were taken on day 5 for ELISA to detect cytokines by adding [3H ] at a concentration of 1 uCi/well]Thymidine and further incubation for 18 hours was used to detect cell proliferation. [ 3 H]Thymidine incorporation was by scintillation counting. The results showed that the PD-1 antibody removed Treg inhibitory function and restored responsive T cell proliferation and ifnγ secretion.
3.8ADCC/CDC assay: to minimize unwanted toxicity of healthy PD-1+ cells, selected anti-PD-1 fully human antibodies were confirmed to be free of ADCC and CDC functions.
3.9ADCC: pre-incubation with different concentrations of fully human antibodies in 96-well plates for 30 min using activated T cells expressing high levels of cell surface PD-1 as target cells followed by a 50:1 effectTarget cell ratio IL-2 activated PBMC (used as Natural Killer (NK) cell source, i.e.effector cells) were added. At 37℃with 5% CO 2 The plates were incubated for 6 hours in an incubator. Target cell lysis was determined by cytotoxicity detection kit (Roche). The optical density was determined by a Molecular Devices SpectraMax M e microplate reader. The results show that the fully human anti-PD-1 antibodies tested did not mediate ADCC.
CDC: target cells (activated T cells), diluted human serum complement (Quidel-A112) and different concentrations of fully human PD-1 antibodies were mixed in 96-well plates. The plates were incubated for 4 hours at 37℃in a 5% CO2 incubator. Target cell lysis was determined by CellTiter glo (Promega-G7573). Rituxan (Roche) and human B lymphocyte fine Raji (CD 20 positive) served as positive controls. The data shows that the PD-1 antibodies do not mediate CDC.
EXAMPLE 4 pharmacodynamic evaluation of anti-PD-1 antibodies in the MiXeno model of subcutaneous transplantation of HCC827 human non-small cell lung carcinoma
The purpose is as follows: anti-PD-1 antibody injection has anti-tumor effect in HCC827 human non-small cell lung cancer cell strain xenograft NCG mouse Mixeno model.
The method comprises the following steps: HCC827 is a human non-small cell lung cancer cell line, and the test uses NCG mice as the test system. NCG mice are severely immunodeficiency transgenic mice lacking NK cells, B cells, and T cells, lacking complement activity. The right back of the animalLeather sheetLower inoculation of 5X10 6 HCC827 cells when tumor volume reached 73mm on average 3 Grouping is performed at that time. Four groups of 16 animals each, a isotype IgG control group, an anti-PD-1 antibody injection 1mg/kg group, an anti-PD-1 antibody injection 5mg/kg group and a Keystuda 5mg/kg positive control group, each group being two subgroups, were inoculated with PBMC from different donors, respectively. Intraperitoneal administration was performed six times on days 6,9, 12, 15, 19, and 22 after tumor cell inoculation. According to the relative tumor inhibition (TGI) RTV ) Tumor Growth Inhibition (TGI) ΔTV ) Efficacy evaluation is carried out, and safety evaluation is carried out according to the weight change and death condition of animals.
Partially reconstituting human immune lines in immunodeficient mice by transplanting NCG mice with human PBMCTogether (mainly T cell function), the model re-established human T cells and HCC827 cells highly expressed human PD-L1. Based on the modeling data inside the detection mechanism, the proportion of human lymphocytes in the peripheral blood of mice 9 days after PBMC transplantation (hCD 45 + Cell%) can usually reach more than 5%, so the proportion of human lymphocytes in the peripheral blood of mice is not detected in this experiment.
The observation period and the administration period of the model are limited to a certain extent due to the influence of GVHD (Graft versus host disease) graft versus host reaction, and only the pharmacodynamic effect can be observed in a short time (15 days). However, the anti-PD-1 antibody injection phase was relatively higher even during the shorter administration periodStill shows more excellent efficacy. The tumor growth of each treatment group and the control group are shown in the following figures, and the pharmacodynamic analysis is shown in tables 1-3.
TABLE 1 analysis of drug efficacy-HCC 827 lung cancer Mixeno tumor model (PBMC: donor A & B)
Note that: a, vs. isotype control group (first group);
TABLE 2 analysis of drug efficacy-HCC 827 lung cancer Mixeno tumor model (PBMC: donor A)
Note that: a, vs. isotype control group (first group);
TABLE 3 analysis of drug efficacy-HCC 827 lung cancer Mixeno tumor model (PBMC: donor B)
Note that: a, vs. isotype control group (first group);
results
1) None of the treatment groups developed drug-treatment related toxic responses (such as severe weight loss or death) during the treatment period, indicating good tolerability of the mice to the product.
2) Referring to FIG. 1, anti-PD-1 antibody 5mg/kg showed significant tumor suppression at day 15 after the inoculation of tumor cells, relative to tumor suppression rate TGI RTV (%) 39% of the total weight of the composition was calculated as TGI ΔTV (%) was 66% and was statistically different from the tumor volume of animals of the isotype IgG control group.
Example 5: human body test research on efficacy and safety of anti-PD-1 antibody in treating refractory malignant tumors
The purpose is as follows: initial evaluation of anti-PD-1 antibodies for treatment of advanced refractory lymphomas and various solid tumors was performed with efficacy and safety.
The method comprises the following steps: the study inclusion criteria were age 18-75 years, ECOG 0-1, refractory solid tumors or lymphomas that did not receive PD-1/CTLA-4 treatment, as confirmed by the case study. The study included two phases, ia and Ib, with Ia being the up-dosing phase and Ib being the extended dosing phase. Patients enrolled in stage Ia received treatment with sirolimus at doses of 1, 4 or 10mg/kg once every 2 weeks (Q2W), or 240mg of Q3W or Q2W, respectively, with the primary purpose of assessing the Dose Limiting Toxicity (DLT) of anti-PD-1 antibodies to confirm RP2D. Stage ib 9 tumor patients received anti-PD-1 antibody RP2D single therapy and were evaluated for efficacy, once every 8 weeks (first 12 months) or 12 weeks (after 12 months), with evaluation criteria of RECIST 1.1 (solid tumor) or Lugano 2014 (lymphoma). Primary study endpoints were DLT (la) and efficacy (ib), secondary study endpoints included RP2D, maximum Tolerated Dose (MTD), and pharmacokinetics/pharmacodynamics (PK/PD), among others.
Results: by 18 months 4 of 2020, 289 patients (24 cases at Ia and 265 cases at Ib) were studied in the co-administration group. No DLT event occurred in stage Ia, determined RP2D to be 240mg Q2W; the tumor species incorporated in stage ib were gastric cancer (n=33), esophageal squamous carcinoma (n=33), hodgkin lymphoma (n=24), non-small cell lung cancer (n=40), nasopharyngeal carcinoma (n=38), peripheral NK/T cell lymphoma (n=14), urothelial carcinoma (n=36), liver cancer (n=21), and cholangiocarcinoma (n=31), respectively. Patients were evaluated for efficacy in stage Ib, and 62 patients achieved objective relief (either complete relief or partial relief) with an overall Objective Relief Rate (ORR) of 23.8% and a median progression-free survival (PFS) of 2.86 months (95% CI: 1.4-3.2). The median total survival (OS) was 13.01 months (95% CI:10.38,15.61) (Ia/Ib). The incidence of adverse events (TEAE) in any treatment during stage ib (265 cases) was 95.1%, and the incidence of TEAE grade 3 or above was 38.5%; the incidence of drug-related adverse reactions (TRAE) was 79.2%; the incidence of immune-related adverse reactions (irAE) was 33.6% (grade 3 or more, 4.9%). The efficacy of non-small cell lung cancer is detailed in Table 4 below.
Table 4: non-small cell lung cancer therapeutic effect table
Conclusion: 240mg of the anti-PD-1 antibody is used for treating the non-small cell lung cancer by intravenous drip (q 2 w) once every 2 weeks, and the curative effect is exact and the safety is good.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (8)
1. Use of an anti-PD-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for treating a patient with non-small cell lung cancer, wherein the antibody or antigen-binding fragment thereof comprises:
a heavy chain variable region comprising CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3; and a light chain variable region comprising CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6.
2. The use of claim 1, wherein the antibody or antigen-binding fragment thereof is a fully human monoclonal antibody.
3. The use of claim 1, the antibody or antigen binding fragment thereof, wherein the fully human monoclonal antibody is produced by a transgenic rat.
4. The use according to claim 1, said 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 + Induction of IL-2 production in T cells;
b) In CD4 + Induction of ifnγ production in T cells;
c) Induction of CD4 + Proliferation of T cells; and
d) Reversing Treg inhibition function.
5. The use of claim 1, wherein 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.
6. The use of claim 1, wherein the antibody or antigen binding fragment thereof, the bifunctional antibody is BsFv or ds bifunctional antibody (ds diabody).
7. The use of claim 1, wherein the antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region.
8. The use of claim 1, wherein the antibody or antigen-binding fragment thereof further comprises a conjugate.
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宋娜莎等: "PD-1抑制剂联合DP方案治疗中晚期非小细胞肺癌临床观察", 《社区医学杂志》, vol. 20, no. 14, 31 July 2022 (2022-07-31), pages 784 - 788 * |
郑大炜等: "调强放疗联合PD-1/PD-L1阻断疗法对中晚期非小细胞肺癌患者免疫状态及近期预后的影响", 《实用癌症杂志》, vol. 37, no. 7, 31 July 2022 (2022-07-31), pages 1165 - 1169 * |
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