CN115466329A - anti-PD-1 humanized antibody and application thereof - Google Patents

anti-PD-1 humanized antibody and application thereof Download PDF

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CN115466329A
CN115466329A CN202210643983.5A CN202210643983A CN115466329A CN 115466329 A CN115466329 A CN 115466329A CN 202210643983 A CN202210643983 A CN 202210643983A CN 115466329 A CN115466329 A CN 115466329A
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antibody
seq
antigen
cells
amino acid
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霍永庭
符俊
张喆
芦迪
路力生
李凡
龚春喜
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Guangdong Fapon Biopharma Inc
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    • 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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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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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    • 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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    • 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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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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Abstract

The invention relates to the technical field of biological medicines, and particularly relates to an anti-PD-1 humanized antibody and application thereof. The antibody can be efficiently and specifically combined with PD-1, and can effectively block the combination of PD-1 with PD-L1 and PD-L2, and the antibody or the antigen binding fragment thereof, and related nucleic acid, vector, cell or pharmaceutical composition thereof can be used for preparing a medicament for treating PD-1 mediated diseases or symptoms.

Description

anti-PD-1 humanized antibody and application thereof
Technical Field
The invention belongs to the technical field of biological medicines. More particularly, it relates to an anti-PD-1 humanized antibody and its application.
Background
Apoptosis factor-1 (PD 1) is a member of the CD28 family and is expressed on activated B cells, T cells and myeloid cells. Human PD1 is coded by gene Pdcd1, located at 2q37.3, with 9.6kb total length, composed of 5 exons and 4 introns, and contains 663bp promoter upstream. PD1 is type I transmembrane protein of 55KDa, the molecular structure is composed of an extracellular region, a transmembrane region and an intracellular region, the extracellular region contains an immunoglobulin variable region IgV structural domain, and the intracellular region contains an Immunoreceptor Tyrosine Inhibition Motif (ITIM) and an immunoreceptor tyrosine transformation module (ITSM). The amino acid sequence of the PD-1 extracellular region has 24 percent of homology with CTLA-4 and 28 percent of homology with CD 28. Upon T cell activation, PD-1 recruits tyrosine phospholipase SHP2 primarily through ITIM, leading to dephosphorylation of downstream effector molecules.
PD-1 has two ligands: PD-L1 and PD-L2.PD-L1 and PD-L2 are both B7 homologues, and the PDL gene is located at position 9P24.2 of human chromosome, and has a size of 42kb, and its molecular structure comprises an immunoglobulin-like variable region domain, a constant region-like domain, a transmembrane region and a short cytoplasmic tail.
Binding of PD-1 to PD-L1 and PD-L2 down-regulates activation of T cells. PD-L1 is expressed on the surface of a variety of tumor cells, including: lung cancer, gastric cancer, liver cancer, esophageal cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, skin cancer, colon cancer, bladder cancer, glioma cancer, head and neck cancer, oral squamous cell carcinoma. Moreover, a large number of CD8+ T cells expressing PD-L1 are found in the periphery of the cancers, and the statistics of clinical results show that the high expression level of PD-L1 on tumor cells is related to poor prognosis of cancer patients.
Disclosure of Invention
The invention aims to solve the technical problems that the existing antibody is not high in affinity and specificity when being combined with PD-1 and cannot effectively block the combination of PD-1 with PD-L1 and PD-L2, and provides an anti-PD-1 humanized antibody which can effectively combine PD-1 and can effectively block the combination of PD-1 with PD-L1 and PD-L2.
The invention aims to provide an anti-PD-1 humanized antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain CDR region and a heavy chain CDR region, the heavy chain CDR region consists of HCDR1, HCDR2 and HCDR3, the light chain CDR region consists of LCDR1, LCDR2 and LCDR3, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are sequentially shown as SEQ ID NO. 9-11, the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are sequentially shown as SEQ ID NO. 12-14, and the amino acid sequence of the heavy chain variable region of the antibody is shown as any one of SEQ ID NO. 3-5; the amino acid sequence of the variable region of the light chain of the antibody is shown in any one of SEQ ID NO 6-8.
It is another object of the invention to provide nucleic acids, vectors, cells or pharmaceutical compositions related to said antibodies or antigen binding fragments thereof.
The invention also relates to the use of the antibody or antigen binding fragment thereof, and nucleic acids, vectors, cells, or pharmaceutical compositions related thereto, in the manufacture of a medicament for treating a PD-1 mediated disease or disorder.
Drawings
FIG. 1 is a graph showing the results of the effect of different concentrations of PD-1-76-C2 on IL-2/IFN-. Gamma.secretion.
FIG. 2 is a graph showing the effect of different concentrations of PD-1-76-C2 on T cell proliferation and secretion of the cytokine IL-2 by T cells.
FIG. 3 is a graph showing the effect of different concentrations of PD-1-76-C2 on T cell proliferation and secretion of the cytokine IFN-. Gamma.by T cells.
FIG. 4 is a graph showing the effect of the anti-PD-1 humanized antibodies h31, h61, and h43 on tumor volume.
FIG. 5 is a graph showing the effect of the anti-PD-1 humanized antibodies h31, h61, and h43 on the survival time of mice.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The invention relates to an anti-PD-1 humanized antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain CDR region and a heavy chain CDR region, the heavy chain CDR region consists of HCDR1, HCDR2 and HCDR3, the light chain CDR region consists of LCDR1, LCDR2 and LCDR3, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are sequentially shown as SEQ ID NO. 9-11, the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are sequentially shown as SEQ ID NO. 12-14, and the amino acid sequence of a heavy chain variable region of the antibody is shown as any one of SEQ ID NO. 3-5; the amino acid sequence of the variable region of the light chain of the antibody is shown in any one of SEQ ID NO 6-8.
The invention adopts Kabat numbering system to mark CDR area, but other methods to mark CDR area also belong to the protection scope of the invention.
In the present invention, the term "specifically binds" or the like refers to the binding of an antibody or antigen-binding fragment thereof to an epitope on a predetermined antigen. Typically, the antibody or antigen-binding fragment thereof is present in an amount of about less than 10 -6 M, e.g. less than about 10 -7 M、10 -8 M、10 -9 M or 10 -10 M or less affinity (K) D ) And (4) combining. KD refers to the ratio of off-rate to on-rate (koff/kon), which can be measured by methods familiar to those skilled in the art.
In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID No. 3, the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 6, or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID No. 4, the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 6, or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID No. 5, the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 7, or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID No. 5, the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 8.
In some embodiments, the antibody comprises a heavy chain constant region that is any one or more of IgG1, igG2, igG3, igG4, igA, igD, igE, or IgM; the light chain constant region is a kappa chain or a lambda chain.
In some embodiments, the species source of the heavy and light chain constant regions is selected from human, murine, or monkey.
In some embodiments, the antibody is a chimeric antibody or a multispecific antibody (e.g., a bispecific antibody).
In the present invention, the term "multispecific antibody" is an antigen binding protein or antibody that targets more than one antigen or epitope.
In the present invention, the term "bispecific antibody" is a multispecific antigen-binding protein or multispecific antibody, and may be produced by a variety of methods, including, but not limited to, fusion of hybridomas or ligation of Fab' fragments. See, e.g., songsivilai and Lachmann,1990, clin. Exp. Immunol.79:315 to 321; kostelny et al, 1992, J.Immunol.148:1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes that are present on the same or different protein targets.
In some embodiments, the antigen-binding fragment is F (ab') 2 Any one or more of Fab, scFv, fv and single domain antibody.
In the present invention, the term "F (ab') 2 "heavy chains comprising two light chains and two portions comprising a constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. F (ab') 2 The fragment thus consists of two Fab' fragments held together by the disulfide bond between the two heavy chains.
In the present invention, the term "Fab" consists of one light chain and CH1 and one heavy chain variable region. The heavy chain of a Fab molecule is unable to form a disulfide bond with another heavy chain molecule.
In the present invention, the term "scFv" is an Fv molecule in which the heavy and light chain variable regions are joined by a flexible linker to form a single polypeptide chain (which forms the antigen binding region) (see, e.g., bird et al, science.242:423-426 (1988) and Huston et al, proc. Natl. Acad. Sci. USA.90:5879-5883 (1988)).
In the present invention, the term "Fv" comprises the variable regions from the heavy and light chains, but lacks the constant regions.
In the present invention, the term "single domain antibody" comprises only one heavy chain variable domain (VHH) and two conventional CH2 and CH3 domains, but does not adhere to each other or even aggregate into a single chain antibody (scFv) as easily as an artificially engineered antibody. More importantly, the structure of the VHH which is cloned and expressed independently has the structural stability which is equivalent to that of the original heavy chain antibody and the binding activity with the antigen, and is the minimum unit which is known to be combined with the target antigen.
The invention also relates to a nucleic acid encoding the anti-PD-1 humanized antibody or an antigen-binding fragment thereof.
In a preferred embodiment, the nucleic acid comprises: a first nucleic acid encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof, and/or, a second nucleic acid encoding the light chain variable region of the antibody or antigen-binding fragment thereof.
In the present invention, the nucleic acid is usually RNA or DNA, and the nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA is preferably used when it is ligated into a vector. In addition, since antibodies are membrane proteins, nucleic acids typically carry a signal peptide sequence.
The invention also relates to a vector carrying said nucleic acid.
In the present invention, the term "vector" is a nucleic acid carrier into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), or artificial chromosomes of P1 origin (PACs); bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV 40).
The invention also relates to a cell carrying said nucleic acid, containing said vector or capable of expressing said antibody or antigen-binding fragment thereof.
The invention also relates to a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, said nucleic acid, said vector or said cell.
In the present invention, the term "pharmaceutical composition" is in a form that allows the biological activity of the active ingredient to be effective and does not comprise additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.
In the present invention, the term "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible for extending the shelf life or efficacy of the antibody.
In addition, the use of the antibody or antigen binding fragment thereof, the nucleic acid, the vector, the cell or the pharmaceutical composition for the preparation of a medicament for treating a PD-1 mediated disease or disorder is also within the scope of the present invention.
In some embodiments, the pharmaceutical composition or medicament is in a form suitable for injection.
In a preferred embodiment, the pharmaceutical composition or medicament is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.
The invention has the following beneficial effects:
the invention provides an anti-PD-1 humanized antibody or an antigen binding fragment thereof, which has high affinity and strong specificity, can be combined with CHO-hPD cells, CHO-cyno cells and activated PBMCs with high affinity, can be efficiently and specifically combined with PD-1, effectively blocks the combination of a ligand PD-L1/PD-L2 and CHO-hPD, can block the combination of the PD-1 and the ligand in MLR, and inhibits a PD-1 signal channel, thereby promoting the proliferation of T cells and secreting IL-2 and IFN-gamma cytokines; therefore, the antibody or the antigen binding fragment thereof, and the related nucleic acid, vector, cell or pharmaceutical composition thereof have wide application prospects in preparation of medicines for treating PD-1-mediated diseases or disorders.
EXAMPLE 1 preparation of anti-PD-1 antibody
1. Immunogens
Human PD-1 sequence (NCBI NP 005009) was synthesized by human, upstream primer: 5'-CCGCAAGCTTGCCGCCACCATG-3' (SEQ ID NO: 1), downstream primer: 5'-CCGGAATTCTCATTAATGGTGATGGTGATGATGCTGGAACTGGCCGGCA GGTC-3' (SEQ ID NO: 2), PCR amplified extracellular end, double digested with Hind III and EcoRI cloned into pCDNA3.4A eukaryotic expression system, this plasmid was used to transfect 293 cells, after harvesting supernatant, purification was performed to obtain human PD-1 recombinant protein (hPD-1).
2. Immunizing animals
125ug hPD-1 recombinant protein with concentration of 1.23mg/ml was used as antigen and mixed with equal amount of Freund's adjuvant (Sigma-Aldrich F5881), 5 female BAL b/C mice were immunized subcutaneously with 25ug of antigen per mouse, 6 weeks. Following the primary immunization, boosters of the same dose were administered weekly. After a total of 5 immunizations, the immune response was monitored by collecting mouse tail blood. Fusion was performed by FACS screening (described below) using mice with sufficient titers of anti-hPD-1 immunoglobulin. 3 days after the intraperitoneal booster with antigen, mice were sacrificed and spleens were removed for cell fusion.
3. Selection of BAL b/C mice that produce anti-hPD-1 antibodies
To select BAL b/C mice that produce anti-hPD-1 antibodies, the immunized mouse sera were tested by FACS. Serum dilutions from hPD-1 recombinant protein immunized mice were incubated with hPD transfected CHO cells for 30 min at 4 deg.C, washed 3 times with PBS, and after addition of 0.4ug/ml PE goat anti-mouse IgG (Biolegend 405307) and incubated for 30 min at 4 deg.C. After washing 3 times with PBS, the samples were placed into a Beckman Coulter flow cytometer (CytoFLEX A00-1-1102) to check if they could bind to hPD transfected CHO cells, BAL b/C mice producing anti-hPD-1 antibody were screened, and then cell fusion was performed.
4. Generation of murine monoclonal antibody hybridomas for hPD-1
Spleen cells of immunized BAL b/C mice are fused with mouse myeloma cells, and the resulting hybridomas are then screened for antigen-specific antibodies. Single cell suspensions from splenocytes from immunized mice were cell fused with PEG 1500 (Roche 10783641001) with one fifth of the mouse myeloma cells that did not secrete immunoglobulin (SP 2/0, ATCC CRL1581). The fused cells were cultured at about 1X 10 5 One/well was plated on a 96-well cell culture plate, which was placed in an incubator (Panasonic MCO-18 AIC) at 37 ℃ with 5% CO 2 . Followed by approximately one week of culture in HAT selective medium containing 1 Xstreptomycin diabody (Gibco 15140122), 1 XHAT (Sigma CRLP-7185) and 20% fetal bovine serum (Royacel RY-F11-01) in 1640 medium. After 1 week, HAT was replaced with HT medium (1640 medium containing 1 Xpenicillin diabody (gibco 15140122), 1 XHT (gibco 11067030) and 20% fetal bovine serum (Royacel RY-F11-01)) and the cell culture supernatant of the fusion plates was examined by FACS to screen out hybridomas secreting antibodies that bind hPD-1 protein. Hybridomas secreting antibodies that bind hPD-1 protein are replated and screened again. Hybridoma positive for screened hPD-1 protein binding antibodySubcloning is performed at least twice by the limited release method. The stable subclones were then cultured in vitro and small amounts of antibody were generated for further analysis. Hybridoma clone PD-76-C2 was selected for further analysis.
Example 2 anti-PD-1 murine mAb affinity characterization
According to conventional methods, a CHO (Chinese hamster ovary) cell line (CHO-hPD) expressing recombinant human PD-1 on the cell surface, a CHO cell line (CHO-cynoPD 1) expressing monkey PD1 (Uniprot: B0LAJ 2), a CHO cell line (CHO-mousPD 1) expressing mouse PD1 (Uniprot: Q02242) were prepared using recombinant techniques, and the cell lines were characterized for binding of anti-PD-1 murine mAb PD-1-76-C2 by Flow Cytometry (FCM).
To evaluate the binding of anti-PD-1 murine mAb to CHO-hPD1, 2X 10 wells were added to 96-well plates 5 CHO-hPD1 cells and anti-PD-1 murine monoclonal antibody diluted in a concentration gradient (initial concentration 10. Mu.g/ml, dilution three times) were incubated at 4 ℃ for 30 minutes, after washing the cells once with buffer (PBS containing 3% BSA), anti-murine IgG (Fc) Ab (Biolegend) fluorescent secondary antibody labeled with PE was added, after incubation at 4 ℃ for 30 minutes, after washing the cells once with buffer, the PBS was resuspended, and then the cell suspension was subjected to flow cytometry analysis by Cytoflex (Beckmann flow cytometer), and the amount of antibody bound to the cells was measured according to the Mean Fluorescence Intensity (MFI) of staining; the same method was used to evaluate the binding of this anti-PD-1 murine mAb to CHO-cyno cells, CHO-mousePD1 (sometimes abbreviated as "CHO-mPD1" in the present invention) cells.
The results are shown in Table 1, and the data show that the anti-PD-1 mouse monoclonal antibody PD-1-76-C2 can be combined with CHO-hPD1 cells and CHO-cyno cells with higher affinity; meanwhile, the murine mAb did not bind to CHO-mousePD1 cells.
Example 3 binding of anti-PD-1 antibodies to activated PBMCs
Fresh human Peripheral Blood Mononuclear Cells (PBMC) stimulated by PHA (Sigma) can activate and proliferate lymphocytes and express PD1 at the highest abundance on the third day, and can be used for carrying out a binding experiment of PD-1 antibody and activated lymph naturally expressing PD 1.
PBMC is obtained from fresh human peripheral blood by gradient centrifugation with lymph separation liquid, and density is adjusted to 1 × 10 6 Cells/ml were seeded into T75 while adding PHA-L (Sigma) at a final concentration of 1. Mu.g/ml to stimulate lymphocyte proliferation, 37 ℃,5% CO 2 After standing in the incubator for 3 days, the cell suspension was taken out, the supernatant was centrifuged off, the buffer (PBS containing 3% BSA) was resuspended, and the suspension was adjusted to 2X 10 5 Adding one of the antibodies per well into a 96-well U-shaped plate, adding 10 anti-PD 1 antibodies with concentration gradient starting from 30 mu g/ml and diluted by 3 times gradient, incubating for 30 minutes at 4 ℃, centrifuging for 5 minutes at 300g, washing the cells once by using a buffer solution, adding a PE-labeled goat anti-human IgG fluorescent antibody (Biolegend), and incubating for 30 minutes at 4 ℃; PBS resuspended cells after washing the cells once by centrifugation, and then subjected to CytoFlex flow cytometry for detection of the amount of antibody bound to PBMC.
The results are shown in Table 1, and anti-PD 1 antibodies bind to activated lymphocytes with high affinity.
TABLE 1
Figure BDA0003685205740000081
Example 4 anti-PD-1 murine mAb binding specificity
anti-PD-1 murine mab was conjugated to four different CD28 family member proteins to verify the specificity of antibody binding to PD-1. Using a standard ELISA method, PD-1, CD28, CTLA-4, ICOS (ACRO) was immobilized on an ELISA plate at a concentration of 1. Mu.g/ml, and anti-human PD-1 mouse monoclonal antibody (mAb) was added at a concentration of 10. Mu.g/ml, and anti-mouse IgG conjugated with peroxidase (HRP) was used as a secondary antibody (Sigma). TMB color development, after termination, the plate reader read OD450 value.
The results are shown in Table 2, where anti-PD-1 murine mAb PD-1-76-C2 specifically binds to PD-1, but not to other family members of CD 28.
TABLE 2
Figure BDA0003685205740000082
Example 5 determination of affinity of anti-human PD-1 murine mAb by the biolayer interferometry (BLI) method
ForteBio (Octet Qke) affinity assay: the association rate was measured by loading PD-1-his (ACRO) recombinant protein at a concentration of 5. Mu.g/ml on the HISIK biosensor for 120 seconds, then equilibrating the loaded sensor in standard buffer (PBST, PBS +0.02% Tuween 20) for 120 seconds, after which the sensor was transferred to anti-PD-1 murine mAb dilution for 180 seconds and transferred to standard buffer for 20 minutes to measure the dissociation rate. Finally, analysis is performed by using a kinetic model.
The data processing results are shown in table 3.
TABLE 3
Antibodies to be tested kon(1/Ms) kdis(1/s) KD(M)
Opdivo(ABA0333) 1.38E+06 3.63E-06 2.62E-12
PD-1-76-C2 7.71E+05 <1.0E-07 <1.0E-12
Example 6 anti-PD-1 murine mAb blocks binding of ligands PD-L1/PD-L2 to CHO-hPD1
The binding capacity of the anti-PD-1 murine mAb to blocking ligand and to stably expressed PD-1 on the surface of transfected CHO cells was analyzed by flow cytometry. The ligand protein used in the experiment is recombinant PD-L1/PD-L2 extracellular segment connected with human IgG1 Fc segment fusion protein: PD-L1-hFc (ACRO), PD-L2-hFc (ACRO).
CHO-PD1 cells were resuspended in buffer (PBS 3% BSA) adjusted to a density of 2X 10 6 Cells/ml, 100. Mu.l/well of cell suspension were added to a 96-well U-plate, and after centrifugation at 300g for 5 minutes, the supernatant was removed.
The subsequent process can be divided into two blocking modes: mode one, adding PD-L1-hFc/PD-L2-hFc with the concentration of 3 mug/ml into a cell hole, incubating for 30 minutes at 4 ℃, then adding anti-PD-1 mouse monoclonal antibody which is diluted by 3 times of gradient from 30 mug/ml, and is released in total of 10 concentration gradients, and incubating for 30 minutes at 4 ℃; mode two, add from 30 u g/ml, 3 times the gradient dilution, total 10 concentration gradient anti PD-1 mouse monoclonal antibody, 4 degrees C were incubated for 30 minutes, then add 3 u g/ml concentration of PD-L1-hFc/PD-L2-hFc protein, 4 degrees C were incubated for 30 minutes.
After centrifugation at 300g for 5 minutes, the cells were washed once with buffer, and PE-labeled goat anti-human IgG fluorescent antibody (Biolegend) was added and incubated at 4 ℃ for 30 minutes. After the cells are washed by centrifugation once, PBS is used for resuspending the cells, then CytoFlex flow cytometry analysis is carried out, the amount of ligand protein bound to the cells is detected, and the IC of PD-1 antibody binding blocking is calculated 50 The value is obtained.
Results are shown in table 4, anti-PD-1 murine mab: PD-1-76-C2 was effective in blocking the binding of PD-L1/PD-L2 to the cell CHO-PD1 in both modes.
TABLE 4
Figure BDA0003685205740000091
Example 7 Effect of anti-PD-1 antibodies on cytokine release from SEB-stimulated PBMC cells
In this example, overnight cultured Peripheral Blood Mononuclear Cells (PBMC) were tested for the effect of cytokine secretion in the presence or absence of anti-PD-1 antibodies when stimulated by the addition of the superantigen Staphylococcus aureus enterotoxin B (SEB).
Fresh peripheral mononuclear cells (PBMC) were used10% of FBS X-VIVO 15 medium (LONZA) resuspended, then added to T25 flask, 37 ℃,5% CO 2 Standing overnight, the next day, taking the suspension cells, centrifuging, resuspending fresh X-VIVO (containing 10% FBS) medium, adding SEB superantigen (Toxin technology) at a final concentration of 200ng/ml, and performing 1X 10 per well 5 Cells were added to 96-well plates, and anti-PD-1 antibodies were added at different concentrations, and isotype control antibodies (mIgG 1 isotype control antibody (Biolegend); hIgG4 isotype control antibody (Biolegend)) were added, without antibody control wells. After 3 days, the wells were sampled and IL-2/IFN-. Gamma.levels were measured using the IL 2/IFN-. Gamma.human Uncoated ELISA Kit (eBioscience) Kit.
The effect of different concentrations of PD-1-76-C2 on IL-2/IFN- γ secretion the results are shown in FIG. 1, with anti-PD-1 antibodies increasing IL-2/IFN- γ secretion in a concentration-dependent manner. The results show that in PBMC stimulated by SEB superantigen, anti-PD-1 antibodies: PD-1-76-C2 can further promote the secretion of cytokines by T cells.
Example 8 Effect of anti-PD-1 antibodies in Mixed lymphocyte reaction
In Mixed Lymphocyte Reaction (MLR), the presence or absence of anti-PD-1 antibodies can demonstrate T cell proliferation and high levels of T cell secreted cytokines in the case of blocked PD1 signaling.
CD14 was isolated from fresh PBMC using CD14 MicroBeads, human (Miltenyi) + Monocytes (monocytes) induced in the presence of GM-CSF/IL-4 for 6 days, followed by TNF- α addition, followed by 3 days before maturation of the DCs; on the day of experiment, easySep was used TM Purification of T cells in PBMC, 1X 10 from Human T Cell Enrichment Kit (StemCell) 4 DC cell of (1X 10) 5 The mixed cells were cultured in a mixed manner, and anti-PD-1 antibodies were added to the mixed cells at different concentration gradients, and isotype control antibodies (mIgG 1 isotype control antibody and hIgG4 isotype control antibody (Biolegend)) were additionally provided, without antibody control wells. After mixed culture for 3 days, taking the supernatant to detect IL-2, and after culturing for 2 days, taking the supernatant to detect IFN-gamma.
The effect of different concentrations of PD-1-76-C2 on T cell proliferation and cytokine IL-2 secretion by T cells is shown in FIG. 2, the effect of different concentrations of PD-1-76-C2 on T cell proliferation and cytokine IFN- γ secretion by T cells is shown in FIG. 3, and the results of FIGS. 2 and 3 indicate that anti-PD-1 antibody: PD-1-76-C2 can block the combination of PD1 and ligand in an antibody concentration-dependent mode in an MLR experiment, and inhibit a PD1 signal channel, thereby promoting the proliferation of T cells and promoting the secretion of IL-2, IFN-gamma cell factors by the T cells.
Example 9 humanization of anti-PD-1 murine mAb
The obtained anti-PD-1 mouse monoclonal antibody PD-1-76-C2 (the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO: 9-11 in sequence, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO: 12-14 in sequence, the heavy chain variable region sequence is shown as SEQ ID NO:15, and the light chain variable region sequence is shown as SEQ ID NO: 16) is humanized, and the specific method comprises the following steps:
human PD-1 sequence (NCBI NP 005009) is artificially synthesized and cloned to PCDNA3.4A eukaryotic expression system, 293 cells are transfected by the plasmid, and human PD-1 recombinant protein is obtained by purification after supernatant fluid is harvested. The obtained human PD-1 recombinant protein is used for subcutaneous immunization of female BAL b/C mice, spleen cells of the immunized BAL b/C mice are fused with mouse myeloma cells, and then the obtained hybridomas are screened for antigen-specific antibodies. The screened hybrid tumor combining with hPD-1 protein antibody positivity is subcloned for at least two times by a limiting dilution method, and then is cultured in vitro to be stably subcloned and generate a small amount of antibody, and then is further screened to obtain PD-1-76-C2 clone.
SEQ ID NO:9:NYGMN
SEQ ID NO:10:WINTHTGEPTYADDFKG
SEQ ID NO:11:EGEGIGFAY
SEQ ID NO:12:RSSQSIVYSNGKTYLE
SEQ ID NO:13:KVSNRFS
SEQ ID NO:14:FQGSHVPNT
SEQ ID NO:15：
QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQIKNLKNEDVATYFCTKEGEGIGFAYWGQGTLVTVSA
SEQ ID NO:16：
DVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGKTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPNTFGGGTKLEIKR
Selecting a humanized template which is best matched with a non-CDR region of the humanized template in a Germine database according to SEQ ID NO. 15 and SEQ ID NO. 16, wherein the template of an antibody heavy chain is IGHV1/7, and the template of an antibody light chain is IGKV2; under the principle that the structural stability of an antibody is not influenced, the combination of the antibody and an antigen is not influenced, protein modification sites such as glycosylation and phosphorylation are not introduced, sites such as oxidized amination are not introduced, and the structural stability is enhanced, a heavy chain humanized sequence is designed to be VH1-6, a light chain humanized sequence is designed to be VL1-3, and a light and heavy chain pairing form is designed to be IGHV1/IGKV2 and is a common pairing form, so that the humanized antibody is obtained.
Genes were synthesized based on the respective humanized antibody light and heavy chain amino acid sequences, and after double cleavage with Hind III (NEB) and EcoRI (NEB), the gene fragments were inserted into PCDNA3.4A expression vector (Invitrogen) through Hind III (NEB)/EcoRI (NEB) cleavage sites using T4 DNA ligase (TAKARA 2011A). Expression vectors and transfection reagent PEI (Poly science, inc. Cat. NO. 23966) were transfected into HEK293 cells (Life technologies Cat. NO. 11625019) at a ratio of 1:2 and placed in CO 2 Culturing in an incubator for 5-7 days. The expressed antibody was recovered by centrifugation and then purified by a conventional method to obtain an anti-PD-1 humanized antibody (h 11, h21, h31, h41, h51, h61, h12, h42, h13, h23, h33, h43, h53, h 63); among them, the amino acid sequences of 4 anti-PD-1 humanized antibodies (h 31, h61, h42, h 43) are shown in Table 5.
TABLE 5 amino acid sequence of anti-PD-1 humanized antibody (h 31, h61, h42, h 43) of Strain 4
Figure BDA0003685205740000121
SEQ ID NO:3：
QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTKEGEGIGFAYWGQGTTVTVSS
SEQ ID NO:4：
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYAQKFQGRVTMTLDTSISTAYMELSRLRSDDTAVYYCTKEGEGIGFAYWGQGTTVTVSS
SEQ ID NO:5：
QIQLVQSGAEVKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTHTGEPTYADDFKGRFTFTLDTSISTAYLEISRLRSDDTAVYYCTKEGEGIGFAYWGQGTTVTVSS
SEQ ID NO:6：
DVVMTQTPLSLSVTPGQPASISCKSSQSIVYSNGKTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR
SEQ ID NO:7：
DIVMTQTPLSLSVTPGQPASISCKSSQSIVYSNGKTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR
SEQ ID NO:8：
DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGKTYLEWYLQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPNTFGQGTKLEIKR
EXAMPLE 10 determination of the binding Capacity of anti-PD-1 humanized antibodies to CHO-hPD cells and CHO-cyno cells
1. Experimental methods
CHO (Chinese hamster ovary) cell line (CHO-hPD) expressing recombinant human PD-1 and CHO cell line (CHO-cynoPD 1) expressing monkey PD1 were used in recombinant technology to express recombinant human PD-1 on the cell surface, and these two cell lines were used in Flow Cytometry (FCM) assays for binding characterization of anti-PD-1 humanized candidate monoclonal antibodies. The specific method comprises the following steps:
to assess binding of anti-PD-1 humanized antibody to CHO-hPD1 cells, 2X 10 was added to 96-well plates 5 CHO-hPD1 cells and humanized antibody diluted by concentration gradient (initial concentration 30. Mu.g/ml, triple gradient dilution) were incubated at 4 ℃ for 30 minutes, after washing the cells once with buffer (PBS containing 3 BSA), a PE-labeled anti-human IgG (Fc) Ab (Biolegend) fluorescent secondary antibody was added, after incubation at 4 ℃ for 30 minutes, after washing the cells once with buffer and resuspending the PBS, and the cell suspension was subjected to flow cytometry analysis by Cytoflex (Beckmann flow cytometer), and the amount of antibody bound to the cells was measured based on the Mean Fluorescence Intensity (MFI) of staining. The binding of the anti-PD-1 humanized antibody to CHO-cyno cells was evaluated in the same manner.
2. Results of the experiment
The results of measuring the binding ability of the anti-PD-1 humanized antibody to CHO-hPD cells and CHO-cyno cells are shown in Table 6, and the results show that the anti-PD-1 humanized antibody of the present invention can bind to CHO-hPD cells and CHO-cyno cells with high affinity.
Example 11 determination of the binding Capacity of an anti-PD-1 humanized antibody to activated PBMCs
1. Experimental methods
Fresh human Peripheral Blood Mononuclear Cells (PBMC) stimulated by PHA (Sigma) can activate the proliferation of lymphocytes and express PD-1 at the highest abundance on the third day, and can be used for the determination of the binding capacity of the anti-PD-1 humanized antibody and activated lymphocytes naturally expressing PD-1. The specific method comprises the following steps:
PBMC is obtained from fresh human peripheral blood by gradient centrifugation of lymph separation liquid, and density is adjusted to 1 × 10 6 cells/ml were inoculated into T75, while adding PHA-L (Sigma) at a final concentration of 1. Mu.g/ml to stimulate lymphocyte proliferation, 37 ℃,5% 2 After standing in an incubator for 3 days, taking out a cell suspension, centrifuging to remove a supernatant, resuspending in a buffer solution (PBS containing 3% BSA), adding the suspension into a 96-well U-shaped plate according to 2E 5/well, adding anti-PD-1 humanized antibodies with different concentration gradients, incubating at 4 ℃ for 30 minutes, centrifuging at 300g for 5 minutes, washing the cells once by using the buffer solution, adding a PE-labeled goat anti-human IgG fluorescent antibody (Biolegend), and incubating at 4 ℃ for 30 minutes; PBS resuspended cells after washing the cells once by centrifugation, and then subjected to CytoFlex flow cytometry for detection of the amount of antibody bound to PBMC.
2. Results of the experiment
The results of the binding ability assay of the anti-PD-1 humanized antibody to activated PBMCs are shown in table 6, which shows that the anti-PD-1 humanized antibody of the present invention can bind to activated PBMCs with high affinity.
The binding capacity of the anti-PD-1 humanized antibody of the invention to CHO-hPD cells, CHO-cyno cells and activated PBMCs was comparable to that of the anti-PD-1 murine mAb PD-1-76-C2 (Table 1) of examples 2 and 3.
TABLE 6 binding Capacity assay results of anti-PD-1 humanized antibodies to CHO-hPD cells, CHO-cyno cells and activated PBMCs
Figure BDA0003685205740000141
Figure BDA0003685205740000151
Example 12 binding specificity of anti-PD-1 humanized antibodies
1. Experimental methods
The specificity of binding of the anti-PD-1 humanized antibody to PD-1 was verified by binding of the anti-PD-1 humanized antibody of the invention to four different CD28 family member proteins. PD-1, CD28, CTLA-4, ICOS was immobilized on an ELISA plate at a concentration of 1. Mu.g/ml by a standard ELISA method, anti-PD-1 humanized antibodies (h 31, h61, h42, h 43) were added at a concentration of 10. Mu.g/ml, and anti-human IgG (Fab) conjugated with peroxidase (HRP) was used as a secondary antibody. TMB color development, after termination, the plate reader read OD450 value.
2. Results of the experiment
The results of the binding specificity of the anti-PD-1 humanized antibody are shown in table 7, which shows that the anti-PD-1 humanized antibody can specifically bind to PD-1, but not to other proteins of the CD28 family members; thus, the anti-PD-1 humanized antibody of the invention has high binding specificity with PD-1.
TABLE 7 results of binding specificity of anti-PD-1 humanized antibodies
Figure BDA0003685205740000152
Example 13 affinity assay for anti-PD-1 humanized antibodies
1. Experimental method
According to the biofilm interference technique (BLI), using Fortebio
Figure BDA0003685205740000153
The detection instrument is used for detecting the affinity of the antibody, and the specific method comprises the following steps:
the binding rate was measured by loading PD-1-his recombinant protein at a concentration of 5. Mu.g/ml onto the HISIK biosensor for 120 seconds, then equilibrating the loaded sensor in standard buffer (PBST, PBS + 0.02%. Tuween 20) for 120 seconds, after which the sensor was transferred to an anti-PD-1 humanized antibody (h 31, h61, h42, h 43) dilution for 180 seconds and transferred to standard buffer for 20 minutes to measure the dissociation rate. And finally, analyzing and processing data by using a dynamic model. Opdivo (ABA 0333) was used as a positive control.
2. Results of the experiment
The results of affinity assay of the anti-PD-1 humanized antibodies are shown in table 8, and show that the anti-PD-1 humanized antibodies of the present invention all bind to PD-1 with high affinity.
The affinity of the anti-PD-1 humanized antibody of the invention was comparable to that of the anti-PD-1 murine mAb PD-1-76-C2 of example 5 (Table 3); the affinity of the anti-PD-1 humanized antibody of the invention was significantly improved compared to the positive control, opdivo (table 3).
TABLE 8 affinity assay results for anti-PD-1 humanized antibodies
Antibodies to be tested kon(1/Ms) kdis(1/s) KD(M)
h31 8.53E+05 <1.0E-07 <1.0E-12
h61 8.92E+05 <1.0E-07 <1.0E-12
h42 8.85E+05 <1.0E-07 <1.0E-12
h43 8.20E+05 <1.0E-07 <1.0E-12
Example 14 determination of the ability of an anti-PD-1 humanized antibody to block binding of ligand PD-L1/PD-L2 to CHO-hPD1
1. Experimental methods
The ability of the anti-PD-1 humanized antibody to block ligand binding to stably expressed PD-1 on the surface of transfected CHO cells was analyzed by flow cytometry. The ligand protein is recombinant PD-L1/PD-L2 extracellular domain connected with mouse IgG1 Fc domain fusion protein: PD-L1-mFc, PD-L2-mFc.
CHO-PD1 cells were resuspended in buffer (PBS 3% BSA) adjusted to a density of 2X 10 6 cells/ml, 100. Mu.l/well cell suspension were added to a 96-well U-plate, and after centrifugation at 300g for 5 minutes, the supernatant was removed. PD-L1-mFc/PD-L2-mFc was added to the wells at a concentration of 0.2. Mu.g/ml, and after incubation at 4 ℃ for 30 minutes, anti-PD-1 humanized antibody (h 31, h61, h42, h 43) diluted in a concentration gradient was added and incubated at 4 ℃ for 30 minutes.
After centrifugation at 300g for 5 minutes, the cells were washed once with buffer, and PE-labeled goat anti-mouse IgG fluorescent antibody (Biolegend) was added and incubated at 4 ℃ for 30 minutes. After the cells are centrifugally washed once, PBS (phosphate buffer solution) is used for resuspending the cells, then CytoFlex flow cytometry analysis is carried out, the amount of ligand protein bound to the cells is detected, and anti-PD-1 human is obtained by calculationIC for binding blocking of humanized antibody 50 The value is obtained.
2. Results of the experiment
The results of the determination of the binding capacity of the anti-PD-1 humanized antibody blocking ligand PD-L1/PD-L2 and CHO-hPD1 are shown in Table 9, and the results show that the anti-PD-1 humanized antibody of the present invention can effectively block the binding of the ligand PD-L1/PD-L2 and CHO-hPD.
TABLE 9 determination of the ability of anti-PD-1 humanized antibodies to block the binding of the ligand PD-L1/PD-L2 to CHO-hPD1
Figure BDA0003685205740000171
Example 15 Effect of anti-PD-1 humanized antibodies in Mixed lymphocyte reaction
1. Experimental method
The presence or absence of anti-PD-1 humanized antibodies in Mixed Lymphocyte Reaction (MLR) demonstrates T cell proliferation and high levels of T cell secreted cytokines in the presence of blocked PD-1 signaling. The specific method comprises the following steps:
CD14 was isolated from fresh PBMC using CD14 MicroBeads, human (Miltenyi) + Monocytes (monocytes) induced in the presence of GM-CSF/IL-4 for 6 days, followed by TNF- α addition, followed by 3 days before maturation of the DCs; on the day of experiment, easySep was used TM Purification of T cells from PBMC with Human T Cell Enrichment Kit (StemCell), 1X 10 4 cells/well of DC cells with 1X 10 5 cells/well of T cells mixed culture, and to the mixed cells added different concentration gradient of anti PD-1 humanized antibody (h 31, h61, h42, h 43), in addition to set up isotype control antibody, no antibody control hole. After 3 days of mixed culture, the supernatant was collected and subjected to IL-2 detection, and after 2 days of mixed culture, the supernatant was collected and subjected to IFN-. Gamma.detection.
2. Results of the experiment
The results of the effect of the anti-PD-1 humanized antibody on the mixed lymphocyte reaction are shown in Table 10, and the results show that the anti-PD-1 humanized antibody can block the binding of PD-1 and a ligand and inhibit a PD-1 signal channel in MLR, thereby promoting the proliferation of T cells and promoting the secretion of IL-2 and IFN-gamma cytokines by the T cells.
TABLE 10 results of Effect of anti-PD-1 humanized antibody on Mixed lymphocyte reaction
Figure BDA0003685205740000172
Figure BDA0003685205740000181
Example 16 evaluation of antitumor Effect of anti-PD-1 humanized antibody on mouse Colon cancer cells in vivo 1, experimental method
Purpose of the experiment: in vivo antitumor activity of the anti-PD-1 humanized antibodies (h 31, h61, h 43) against mouse colon cancer cells (MC 38 cells) was measured while an Isotype control group (Isotype group) was set.
Experimental materials: hPD1 Knock in mice, female, 6-8 weeks (C57 BL/6 background, source: beijing Wintoda Biotechnology, inc.); MC38 cells (national laboratory cell shared resource platform); FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), DMSO (Sigma, D2650), DPBS (Hyclone, SH 30028.02), fetal bovine serum (Gibco), glutamine (Gibco), penicillin-streptomycin (Gibco, 15140122), DMEM high sugar medium (Gibco, 11965084).
The instrument equipment comprises: electronic balance (Shanghai Shunheng Hengping scientific instruments Co., ltd., JA 12002), vernier caliper (Shanghai Meinaite practice Co., ltd., MNT-150T), microscope (Chongqing Ott optical instruments Co., ltd., BDS 200), medical centrifuge (Hunan Xiang instruments laboratory development Co., ltd., L530R), digital display thermostat water bath (Pures machines Co., ltd., HH-S), carbon dioxide incubator (Nippon Pan health medical instruments Co., ltd., MCO-18 AC), double-person vertical type super clean bench (tin-free easy purification apparatus Co., ltd., SW-CJ-VS 2).
The experimental steps are as follows:
cell culture: MC38 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, 1% glutamine and 1% penicillin-streptomycin (1:1).
Inoculation: collecting MC38 cells in logarithmic growth phase, and regulating cell concentration to 3 × 10 6 and/mL. 40 female hPD mice were inoculated subcutaneously with MC38 cells in a volume of 0.1 mL/mouse, i.e., 3X 10 5 Mice.
Administration: on the day of vaccination, day 0 (D0), day 7, mice were randomized into 4 groups of 8 mice by tumor volume and dosing was started (MC 38 tumor model dosing dose, pattern and frequency are shown in table 11).
Recording: d7 tumor volume was measured and recorded initially, after which tumor major and minor diameters were measured 2 times per week with a vernier caliper. According to the formula: (1/2). Times.Long diameter × (short diameter) 2 Tumor volume was calculated. When each mouse reached the end of the experiment (tumor volume over 2000 mm) 3 To the crinis Citrifis endpoint), mice were sacrificed by cervical dislocation and survival curves were recorded.
TABLE 11 MC38 tumor model dosing dose, regimen and frequency
Figure BDA0003685205740000182
Figure BDA0003685205740000191
2. Results of the experiment
The results of the effect of the anti-PD-1 humanized antibody on tumor volume are shown in table 12 and fig. 4, and it can be seen that the anti-PD-1 humanized antibody (h 31, h61, h 43) has significant tumor-suppressing effect on the tumor growth of the MC38 tumor model compared to the Isotype group (TGI of 100.85%,94.77%,99.05% in sequence; and 6, 5, and 7 tumor-free mice, respectively).
The effect of the anti-PD-1 humanized antibody on the survival of mice is shown in FIG. 5, and it can be seen that the anti-PD-1 humanized antibody (h 31, h61, h 43) can significantly prolong the survival of mice compared to the Isotype group.
TABLE 12 results of the effect of anti-PD-1 humanized antibody on tumor volume (mm) 3 )
Figure BDA0003685205740000192
The above results show that: the anti-PD-1 humanized antibody (h 31, h61 and h 43) provided by the invention can obviously inhibit the growth of MC38 cells, effectively prolongs the survival time of mice and has obvious curative effect on treating colon cancer of the mice.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Guangdong Fengcong pharmaceutical Co Ltd
<120> anti-PD-1 humanized antibody and application thereof
<130> 2021
<160> 16
<170> PatentIn version 3.5
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Arg
<210> 9
<211> 5
<212> PRT
<213> Artificial sequence
<400> 9
Asn Tyr Gly Met Asn
1 5
<210> 10
<211> 17
<212> PRT
<213> Artificial sequence
<400> 10
Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys
1 5 10 15
Gly
<210> 11
<211> 9
<212> PRT
<213> Artificial sequence
<400> 11
Glu Gly Glu Gly Ile Gly Phe Ala Tyr
1 5
<210> 12
<211> 16
<212> PRT
<213> Artificial sequence
<400> 12
Arg Ser Ser Gln Ser Ile Val Tyr Ser Asn Gly Lys Thr Tyr Leu Glu
1 5 10 15
<210> 13
<211> 7
<212> PRT
<213> Artificial sequence
<400> 13
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 14
<211> 9
<212> PRT
<213> Artificial sequence
<400> 14
Phe Gln Gly Ser His Val Pro Asn Thr
1 5
<210> 15
<211> 118
<212> PRT
<213> Artificial sequence
<400> 15
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Lys Asn Leu Lys Asn Glu Asp Val Ala Thr Tyr Phe Cys
85 90 95
Thr Lys Glu Gly Glu Gly Ile Gly Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 16
<211> 113
<212> PRT
<213> Artificial sequence
<400> 16
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser
20 25 30
Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Asn Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg

Claims (10)

1. An anti-PD-1 humanized antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain CDR region and a heavy chain CDR region, the heavy chain CDR region consists of HCDR1, HCDR2 and HCDR3, the light chain CDR region consists of LCDR1, LCDR2 and LCDR3, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO 9-11 in sequence, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO 12-14 in sequence, and the antibody is characterized in that the amino acid sequence of the heavy chain variable region of the antibody is shown as any one of SEQ ID NO 3-5.
2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the variable region in the light chain of the antibody has an amino acid sequence as set forth in any one of SEQ ID NOS 6 to 8.
3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO. 3 and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 6, or the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO. 4 and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 6, or the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO. 5 and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 7, or the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO. 5 and the amino acid sequence of the light chain variable region of the antibody is represented by SEQ ID NO. 8.
4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is any one or more of IgG1, igG2, igG3, igG4, igA, igD, igE, or IgM; the light chain constant region is a kappa chain or a lambda chain.
5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is a chimeric antibody or a multispecific antibody; the antigen binding fragment is F (ab') 2 Any one or more of Fab, scFv, fv and single domain antibody.
6. A nucleic acid encoding the anti-PD-1 humanized antibody or an antigen-binding fragment thereof;
preferably, the nucleic acid comprises: a first nucleic acid encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof, and/or, a second nucleic acid encoding the light chain variable region of the antibody or antigen-binding fragment thereof.
7. A vector carrying the nucleic acid of claim 6.
8. A cell carrying the nucleic acid of claim 6, comprising the vector of claim 7, or capable of expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 5.
9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid of claim 6, the vector of claim 7, or the cell of claim 8.
10. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid of claim 6, the vector of claim 7, the cell of claim 8, or the pharmaceutical composition of claim 9 in the manufacture of a medicament for treating a PD-1 mediated disease or disorder.
CN202210643983.5A 2021-06-11 2022-06-09 anti-PD-1 humanized antibody and application thereof Pending CN115466329A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110655666 2021-06-11
CN2021106556660 2021-06-11

Publications (1)

Publication Number Publication Date
CN115466329A true CN115466329A (en) 2022-12-13

Family

ID=84364777

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210643983.5A Pending CN115466329A (en) 2021-06-11 2022-06-09 anti-PD-1 humanized antibody and application thereof

Country Status (1)

Country Link
CN (1) CN115466329A (en)

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