CN116407629A - anti-PD-1 antibody and application thereof in preparation of medicines for treating liver cancer patients - Google Patents

anti-PD-1 antibody and application thereof in preparation of medicines for treating liver cancer patients Download PDF

Info

Publication number
CN116407629A
CN116407629A CN202310584061.6A CN202310584061A CN116407629A CN 116407629 A CN116407629 A CN 116407629A CN 202310584061 A CN202310584061 A CN 202310584061A CN 116407629 A CN116407629 A CN 116407629A
Authority
CN
China
Prior art keywords
antibody
cells
binding fragment
antigen
liver cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310584061.6A
Other languages
Chinese (zh)
Inventor
朱吉满
白莉惠
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Yuheng Biotechnology Co ltd
Original Assignee
Guangzhou Yuheng Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Yuheng Biotechnology Co ltd filed Critical Guangzhou Yuheng Biotechnology Co ltd
Publication of CN116407629A publication Critical patent/CN116407629A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Epidemiology (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 liver cancer. Provides a new way for treating liver cancer.

Description

anti-PD-1 antibody and application thereof in preparation of medicines for treating liver cancer patients
Technical Field
The invention belongs to the field of antibodies, and particularly relates to an anti-PD-1 antibody and application thereof in preparing medicines for treating liver cancer patients.
Background
Liver cancer is a malignant tumor of liver, and can be divided into primary and secondary two kinds. Primary liver malignant tumor originates from epithelial or mesenchymal tissue of liver, the former is called primary liver cancer, and is a malignant tumor with high incidence and great harm in China; the latter is called sarcoma, which is less common than primary liver cancer. Secondary or metastatic liver cancer refers to invasion of malignant tumors of multiple organ origins throughout the body into the liver. Liver metastasis is commonly observed in malignant tumors of organs such as stomach, biliary tract, pancreas, colorectal, ovary, uterus, lung and breast.
Primary liver cancer is classified into hepatocellular carcinoma, cholangiocarcinoma and mixed type according to pathological typing, wherein hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is the most common, abbreviated as liver cancer. As a common surgical malignancy in clinic, it has occult disease, early symptoms are not obvious, and most patients have advanced at the time of diagnosis. Symptoms of primary liver cancer include pain in the liver region, abnormal enlargement of the liver, systemic and digestive tract symptoms, and tumor cell metastasis and invasion. Although a great deal of research has been conducted on the pathogenesis and mechanism of tumors in human beings, the mechanism of occurrence and development of primary liver cancer still needs to be further elucidated. Primary liver cancer has a high incidence rate, and the incidence rate is still in the front of various malignant tumor diseases of human beings worldwide. The number of patients dying from primary liver cancer every year is the second leading cause of cancer death. The primary liver cancer is quite common in China which is the country with the highest population base and the cancer large country, and is high in southeast coastal areas, especially the number of liver cancer patients caused by virus infection, liver cirrhosis and aflatoxin intake is continuously increased, and the life and health of human beings are seriously threatened. Research shows that the incidence of primary liver cancer is different from male to female, and female incidence is lower than male incidence, and the eastern coastal region of inland region is most patients from 40 years old to 50 years old. Primary liver cancer has high morbidity and mortality, and metastatic invasion of tumor cells is a direct cause of treatment failure. In case of cancer in liver, cancer cells spread rapidly in the whole liver in a short period, and multiple metastasis of cancer cells from liver occurs, most patients already develop advanced stages in diagnosis, malignant tumor cells are already transferred to organs such as proximal lung through vein blood vessels, and a large number of organs such as brain and kidney of patients are also affected, and viscera tissues such as diaphragm can be directly invaded by cancer cells. Research indicates that early liver cancer is usually not easily detected, metastasis already occurs when symptoms seek medical attention, and liver cirrhosis usually accompanies the liver, the onset time of the liver cirrhosis is longer than that of other tumors, and the primary pathogenesis of most patients is at the hepatic portal part and part of the primary pathogenesis can also appear in the center of liver parenchyma.
Although research and development of early diagnosis, operation treatment and chemotherapy drugs of liver cancer have achieved a certain research result in recent years, mortality rate and recurrence rate of liver cancer within 5 years remain high, and on the other hand, the necessity of developing new drug treatment and treatment strategies has been proved.
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 medicines for treating liver 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 liver 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 (dsdiabody).
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 liver cancer.
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). At 37℃with 5% CO 2 The cells are cultured under conditions. 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. Transferring said hybridoma supernatant to said coated plate after blocking and washingAnd incubated at room temperature for 1 hour. The plates were then washed and then incubated with goat anti-rat IgG1 HRP (Bethyl) and goat anti-rat IgG2bHRP (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). The hybridoma supernatants were then 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 μl/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℃in 5% CO 2 . 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 μl):
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 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. The chips were regenerated after each run with H3PO4 at pH 1.7. 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 Cells/mlCHO-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 μl 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 isolated in the presence ofCulturing with GM-CSF (PeproTech, rocky Hill, N.J.; 800U/ml) and IL-4 (PeproTech; 500U/ml) in 10% FCS and antibiotic RPMI 1640 medium at a cell concentration of 2X10 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 for 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 + T cell concentration kit (CD 4) + T cell enrichment cocktail kit) negative selection of 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 overnight stimulation with LPS at 1 μg/ml. 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 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 bindingCapability. Recombinant IFNγ dilutions were prepared as standard solutions and double dilutions were made from 8000pg/ml to 125pg/ml with complete medium, plus the case with complete medium only. The plates were washed, added with standard solution and test supernatant (100. Mu.l/well), 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 show that human CD4 was stimulated with allogeneic DCs 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. 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 of IL-2 (20U/ml) while DC were generated from monocytes cultured in PBMC of the same donor 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 a 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 addition at 1 uCi/well concentration [ 3 H]Thymidine and further incubation for 18 hours was used to detect cell proliferation. [ 3 H]Thymidine incorporationPass-through 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: activated T cells expressing high levels of cell surface PD-1 were used as target cells and pre-incubated with different concentrations of fully human antibodies in 96-well plates for 30 min followed by addition of IL-2 activated PBMCs (used as Natural Killer (NK) cell sources, i.e. effector cells) at 50:1 effector/target cell ratio. [ the plates were incubated for 6 hours at 37℃in a 5% CO2 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. At 37℃with 5% CO 2 The plates were incubated for 4 hours in an 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: human body test research on efficacy and safety of anti-PD-1 antibody in treating refractory malignant tumor
The purpose is as follows: the efficacy and safety of anti-PD-1 anti-treatment advanced refractory lymphomas and various solid tumors were initially assessed.
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 anti-PD-1 antibody treatment at a dose 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 antibody 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 was liver cancer (260 patients with n=21ibs stage curative effect evaluation, 62 patients obtained objective relief (complete relief or partial relief), overall Objective Relief Rate (ORR) was 23.8%, median Progression Free Survival (PFS) was 2.86 months (95% CI: 1.4-3.2), median total survival (OS) was 13.01 months (95% CI:10.38,15.61) (stage ia/ibs). Stage ib (265) incidence of adverse events (TEAE) was 95.1%, stage 3 or more TEAE incidence 38.5%, drug-related adverse reaction (TRAE) incidence was 79.2%, immune-related adverse reaction (irAE) incidence 33.6% (stage 3 or more, 4.9%) liver cancer curative effect details are shown in table 1).
Figure BDA0004242633470000081
Figure BDA0004242633470000091
TABLE 1 therapeutic efficacy of anti-PD-1 antibodies for treating liver cancer
Conclusion: 240mg of the anti-PD-1 antibody is used for treating liver cancer by intravenous drip (q 2 w) once every 2 weeks, and the curative effect is definite 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 liver 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.
CN202310584061.6A 2022-12-28 2023-05-23 anti-PD-1 antibody and application thereof in preparation of medicines for treating liver cancer patients Pending CN116407629A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2022116935672 2022-12-28
CN202211693567.2A CN115925953A (en) 2022-12-28 2022-12-28 anti-PD-1 antibody and application thereof in preparation of medicine for treating liver cancer patient

Publications (1)

Publication Number Publication Date
CN116407629A true CN116407629A (en) 2023-07-11

Family

ID=86649101

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202211693567.2A Withdrawn CN115925953A (en) 2022-12-28 2022-12-28 anti-PD-1 antibody and application thereof in preparation of medicine for treating liver cancer patient
CN202310584061.6A Pending CN116407629A (en) 2022-12-28 2023-05-23 anti-PD-1 antibody and application thereof in preparation of medicines for treating liver cancer patients

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202211693567.2A Withdrawn CN115925953A (en) 2022-12-28 2022-12-28 anti-PD-1 antibody and application thereof in preparation of medicine for treating liver cancer patient

Country Status (1)

Country Link
CN (2) CN115925953A (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103242448A (en) * 2013-05-27 2013-08-14 郑州大学 Full-humanized anti-PD-1 monoclonal antibody and preparation method and application thereof
CN108079292A (en) * 2016-11-23 2018-05-29 苏州盛迪亚生物医药有限公司 A kind of purposes of anti-PD-1 antibody in the drug for preparing treatment liver cancer
EP3600426A1 (en) * 2017-03-31 2020-02-05 Merck Sharp & Dohme Corp. Compositions and methods for treating cancer with a combination of an antagonist of pd-1 and an anti-ctla4 antibody
CN110790839A (en) * 2018-08-03 2020-02-14 江苏恒瑞医药股份有限公司 anti-PD-1 antibody, antigen binding fragment thereof and medical application
CN113893343A (en) * 2021-10-16 2022-01-07 广州誉衡生物科技有限公司 anti-PD-1 antibody and application thereof in preparation of medicine for treating cervical cancer patient

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103242448A (en) * 2013-05-27 2013-08-14 郑州大学 Full-humanized anti-PD-1 monoclonal antibody and preparation method and application thereof
CN108079292A (en) * 2016-11-23 2018-05-29 苏州盛迪亚生物医药有限公司 A kind of purposes of anti-PD-1 antibody in the drug for preparing treatment liver cancer
EP3600426A1 (en) * 2017-03-31 2020-02-05 Merck Sharp & Dohme Corp. Compositions and methods for treating cancer with a combination of an antagonist of pd-1 and an anti-ctla4 antibody
CN110790839A (en) * 2018-08-03 2020-02-14 江苏恒瑞医药股份有限公司 anti-PD-1 antibody, antigen binding fragment thereof and medical application
CN113893343A (en) * 2021-10-16 2022-01-07 广州誉衡生物科技有限公司 anti-PD-1 antibody and application thereof in preparation of medicine for treating cervical cancer patient

Also Published As

Publication number Publication date
CN115925953A (en) 2023-04-07

Similar Documents

Publication Publication Date Title
US11932696B2 (en) Method of treating cancer with an anti-CCR8 that binds tumor infiltrating cells
WO2020135201A1 (en) Antibody and use thereof
EP3604338A1 (en) Anti-ox40 antibody and use thereof
TW202320852A (en) Anti-PD-1 antibody and application thereof to preparation of medicine for treating cervical cancer patients
CN115998861A (en) anti-PD-1 antibodies and their use in the preparation of a medicament for treating gastric cancer patients
CN115135344A (en) Antibodies for use in therapy
CN110944665A (en) AMHRII-binding compounds for preventing or treating lung cancer
JP7165855B2 (en) Use for prevention and treatment of myeloid-derived suppressor cell-related diseases
US20230227558A1 (en) Selection of responders for anti-btn3a treatment
Torchia et al. Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy
US20230312709A1 (en) Antibody targeting CD47 and application thereof
WO2022121846A1 (en) Pd-l1 antibody and application thereof
CN116407629A (en) anti-PD-1 antibody and application thereof in preparation of medicines for treating liver cancer patients
CN116549633A (en) anti-PD-1 antibodies and their use in the preparation of a medicament for the treatment of patients with nasopharyngeal carcinoma
CN116077645A (en) anti-PD-1 antibodies and their use in the preparation of a medicament for treating non-small cell lung cancer patients
CN116003605A (en) anti-PD-1 antibodies and their use in the preparation of a medicament for treating cholangiocarcinoma patients
CN116370622A (en) anti-PD-1 antibodies and their use in the preparation of a medicament for treating patients with urothelial cancer
CN116585467A (en) anti-PD-1 antibodies and their use in the preparation of a medicament for the treatment of esophageal squamous carcinoma patients
CN114258402A (en) anti-CD123 antibodies, anti-CD123 chimeric antigen receptors, and anti-CD123 chimeric antigen receptor T cells
CN116036266A (en) anti-PD-1 antibodies and their use in the preparation of a medicament for the treatment of colon cancer patients
EP4353746A1 (en) Anti-pd-1 humanized antibody or antigen-binding fragment thereof and application thereof
US20240092934A1 (en) Assessment of ceacam1 expression on tumor infiltrating lymphocytes
WO2020171171A1 (en) Anti-hla-dr antibody, and use thereof for cancer therapy
WO2023087016A1 (en) Methods to reverse treml1-induced immune suppression
CN116549629A (en) Application of CD45 as biomarker in screening effectiveness and accuracy of CD26 antibody or derivative thereof for treating tumor

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination