WO2020108463A1 - 人源化抗人ox40单克隆抗体及其制备方法和用途 - Google Patents

人源化抗人ox40单克隆抗体及其制备方法和用途 Download PDF

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WO2020108463A1
WO2020108463A1 PCT/CN2019/120808 CN2019120808W WO2020108463A1 WO 2020108463 A1 WO2020108463 A1 WO 2020108463A1 CN 2019120808 W CN2019120808 W CN 2019120808W WO 2020108463 A1 WO2020108463 A1 WO 2020108463A1
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seq
variable region
chain variable
human
amino acid
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French (fr)
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殷刘松
周铁林
蒋忻坡
方卓
米艳玲
吴瑃辰
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南京金斯瑞生物科技有限公司
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Priority to US17/297,386 priority Critical patent/US20220119540A1/en
Priority to CN201980077176.8A priority patent/CN113166248B/zh
Priority to EP19891274.3A priority patent/EP3889180A4/en
Publication of WO2020108463A1 publication Critical patent/WO2020108463A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/90—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of tumor immunotherapy and molecular immunology, and particularly relates to a humanized anti-human OX40 monoclonal antibody.
  • the invention also relates to the preparation method and use of the humanized anti-human OX40 monoclonal antibody.
  • tumor immunotherapy In the treatment of tumors, tumor immunotherapy is widely used. It has become the third important method in cancer treatment other than radiotherapy and chemotherapy, and has gradually entered the foreground.
  • the immune system is a host defense system. In order to function properly, the immune system must be able to sensitively detect the invasion of foreign pathogens and distinguish them from the organism's own healthy tissues. Tumor immunotherapy uses this feature to regulate the function of the body's immune cells, so that human T cells can better recognize and act on cancerous cells, thereby playing the role of removing diseased tissue.
  • the immune system of vertebrates is a functional system composed of various organs, tissues, cells, and molecules. It is the most effective mechanism for the body to defend against the invasion of foreign substances. These immune organs, tissues, cells, and molecules work together to balance each other. In order to protect the body from external infection and maintain the balance in the body. Among them, cellular immunity is caused by the recognition of the antigen presented by the major histocompatibility complex (MHC) on the antigen-presenting cell (APC) through the T cell receptor (TCR), which is the first-level signal of T cell activation,
  • MHC major histocompatibility complex
  • APC antigen-presenting cell
  • TCR T cell receptor
  • T cell activation is not only accomplished by the first-level signal, but also requires an antigen-independent second-level signal.
  • the latter is achieved by the interaction of receptors or ligands from the surface of T cells with corresponding costimulatory factors from APC.
  • This mutual cooperation and checks and balances require the coordinated participation of many immune checkpoint proteins.
  • Activated immune checkpoint proteins can enhance the defense response of the immune system, and inhibitory immune checkpoint proteins can control the excessive immune system and prevent the generation of self-targeted immune response.
  • agonists or activating antibodies must be developed for activated immune checkpoint proteins, and inhibitors or inhibitory antibodies must be developed for inhibitory immune checkpoint proteins.
  • Antibody-mediated antagonism against inhibitory immune checkpoint proteins can inhibit the activity of the immune checkpoint protein pathway and enhance the function of T cells to kill tumor cells.
  • the regulation of inhibitory immune checkpoint proteins alone is not enough to eliminate tumors.
  • To enhance the function of T cells to kill tumor cells not only to weaken the inhibitory pathway on T cells, but also To strengthen the activation pathway on T cells.
  • the activation pathways on T cells are stimulating immune checkpoint proteins, such as 4-1BB, GITR, and OX40.
  • activated immune checkpoint proteins can activate PKB/AKT, NF-kB, NFAT and other pathways, promote the expansion of auxiliary CD4+ T cells and cytotoxic CD8+ T cells, and the production of cytokines.
  • OX40 (CD134; TNFRSF4) is mainly expressed on the surface of activated T cells, such as CD4, CD8 T cells, helper T cells (Th1, Th2, Th17), CD4+Foxp3+ regulatory T cells; in natural killer cells (NK), natural Killer T cells (NKT), and neutrophils also have a small amount of expression. Unlike CD28 and CD27, they are not expressed in unactivated T cells.
  • OX40 and its ligand OX40L (CD252) are members of the tumor necrosis factor superfamily, and belong to a large family with 4-1BB, CD27, CD40, GITR, etc.
  • OX40/OX40L is a second-level costimulatory immune checkpoint molecule, which is involved in the activation, proliferation and survival of T cells, and plays a key role in the formation of germinal centers and the differentiation and maturation of dendritic cells.
  • the present invention provides stimulating antibodies to OX40, enhances the immune response of T cells to various antigens, and performs tumor immunotherapy.
  • the present invention provides a humanized anti-human OX40 monoclonal antibody or functional fragment thereof, which comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region is contained in the following HCDR1, HCDR2, and HCDR3 sequences
  • the amino acid sequence of 1, 2, or 3 amino acid residues is substituted, inserted, or deleted, and the light chain variable region includes 1, 2, or 3 substitutions, insertions, or deletions in the LCDR1, LCDR2, and LCDR3 sequences, respectively, as follows Amino acid sequence of several amino acid residues:
  • amino acid sequence of HCDR1 is DYSMH
  • HCDR2 The amino acid sequence of HCDR2 is WISTETGEPTYADDFKG;
  • amino acid sequence of HCDR3 is VRPWYLAV
  • amino acid sequence of LCDR1 is RASQDISNYLN;
  • LCDR2 The amino acid sequence of LCDR2 is YTSRLYS;
  • the amino acid sequence of LCDR3 is QQANTLPLT.
  • the present invention provides a humanized anti-human OX40 monoclonal antibody or functional fragment thereof, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the following HCDR1, HCDR2, respectively An amino acid sequence having at least 80% identity with the HCDR3 sequence, the light chain variable region comprising an amino acid sequence having at least 80% identity with the following LCDR1, LCDR2, and LCDR3 sequences, respectively:
  • amino acid sequence of HCDR1 is DYSMH
  • HCDR2 The amino acid sequence of HCDR2 is WISTETGEPTYADDFKG;
  • amino acid sequence of HCDR3 is VRPWYLAV
  • amino acid sequence of LCDR1 is RASQDISNYLN;
  • LCDR2 The amino acid sequence of LCDR2 is YTSRLYS;
  • the amino acid sequence of LCDR3 is QQANTLPLT.
  • the heavy chain variable region comprises at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 with the above-mentioned HCDR1, HCDR2 and HCDR3 sequences, respectively Amino acid sequences of %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the light chain variable region comprises at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, and LCDR1, LCDR2, and LCDR3 sequences, respectively.
  • Amino acid sequences with 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the present invention provides a humanized anti-human OX40 monoclonal antibody or functional fragment thereof, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the following HCDR1, HCDR2 And the amino acid sequence shown in the HCDR3 sequence, the light chain variable region includes the amino acid sequences shown in the LCDR1, LCDR2, and LCDR3 sequences as follows:
  • amino acid sequence of HCDR1 is DYSMH
  • HCDR2 The amino acid sequence of HCDR2 is WISTETGEPTYADDFKG;
  • amino acid sequence of HCDR3 is VRPWYLAV
  • amino acid sequence of LCDR1 is RASQDISNYLN;
  • LCDR2 The amino acid sequence of LCDR2 is YTSRLYS;
  • the amino acid sequence of LCDR3 is QQANTLPLT.
  • amino acid sequence of the heavy chain variable region is selected from: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.
  • the light chain variable region amino acid sequence is selected from: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.
  • the humanized anti-human OX40 monoclonal antibody or functional fragment thereof has a heavy chain variable region and a light chain variable region selected from the following combinations:
  • the heavy chain variable region is SEQ ID NO: 7 and the light chain variable region is SEQ ID NO: 17;
  • the heavy chain variable region is SEQ ID NO: 8 and the light chain variable region is SEQ ID NO: 18;
  • the heavy chain variable region is SEQ ID NO: 9 and the light chain variable region is SEQ ID NO: 19;
  • the heavy chain variable region is SEQ ID NO: 10 and the light chain variable region is SEQ ID NO: 20;
  • the heavy chain variable region is SEQ ID NO: 11 and the light chain variable region is SEQ ID NO: 21;
  • the heavy chain variable region is SEQ ID NO: 12 and the light chain variable region is SEQ ID NO: 22
  • the heavy chain variable region is SEQ ID NO: 13 and the light chain variable region is SEQ ID NO: 23;
  • the heavy chain variable region is SEQ ID NO: 14 and the light chain variable region is SEQ ID NO: 24;
  • the heavy chain variable region is SEQ ID NO: 15 and the light chain variable region is SEQ ID NO: 25; or
  • the heavy chain variable region is SEQ ID NO: 16 and the light chain variable region is SEQ ID NO: 26.
  • the humanized anti-human OX40 monoclonal antibody or functional fragment thereof has a heavy chain variable region and a light chain variable region selected from the following combinations:
  • the heavy chain variable region is SEQ ID NO: 8 and the light chain variable region is SEQ ID NO: 18;
  • the heavy chain variable region is SEQ ID NO: 10 and the light chain variable region is SEQ ID NO: 20;
  • the heavy chain variable region is SEQ ID NO: 12 and the light chain variable region is SEQ ID NO: 22
  • the heavy chain variable region is SEQ ID NO: 13 and the light chain variable region is SEQ ID NO: 23; or
  • the heavy chain variable region is SEQ ID NO: 15 and the light chain variable region is SEQ ID NO: 25.
  • the humanized anti-human OX40 monoclonal antibody or functional fragment thereof has a heavy chain variable region and a light chain variable region selected from the following combinations:
  • the heavy chain variable region is SEQ ID NO: 12 and the light chain variable region is SEQ ID NO: 22; or
  • the heavy chain variable region is SEQ ID NO: 13 and the light chain variable region is SEQ ID NO: 23.
  • the dissociation constant KD between the humanized anti-human OX40 monoclonal antibody or functional fragment thereof and OX40 of the present invention is less than 3 nM.
  • the humanized anti-human OX40 monoclonal antibody or functional fragment thereof of the present invention specifically activates the positive immune regulation of OX40 and activates the secretion of cytokines by T cells.
  • the present invention provides an isolated polynucleotide encoding the humanized anti-human OX40 monoclonal antibody of the present invention or a functional fragment thereof.
  • the polynucleotide comprises a heavy chain coding sequence encoding the heavy chain variable region of the humanized anti-human OX40 monoclonal antibody of the present invention, and encodes the humanized anti-human OX40 monoclonal antibody of the present invention
  • the light chain coding sequence of the light chain variable region of an antibody is not limited to humanized anti-human OX40 monoclonal antibody of the present invention.
  • the present invention provides an expression vector comprising the polynucleotide.
  • the invention provides a host cell comprising the expression vector.
  • the host cell is a HEK293-6E cell.
  • the present invention provides the use of the humanized anti-human OX40 monoclonal antibody or functional fragment thereof, the polynucleotide, the expression vector or the host cell in the preparation of anti-tumor drugs .
  • the present invention provides the use of the humanized anti-human OX40 monoclonal antibody or functional fragment thereof, the polynucleotide, the expression vector, or the host cell for the treatment of tumors.
  • the present invention provides the humanized anti-human OX40 monoclonal antibody or functional fragment thereof, the polynucleotide, the expression vector, or the host cell for treating tumors.
  • the present invention provides an anti-tumor pharmaceutical composition
  • an anti-tumor pharmaceutical composition comprising an effective amount of the humanized anti-human OX40 monoclonal antibody or functional fragment thereof, and a pharmaceutically acceptable carrier.
  • the present invention provides a method for preparing the humanized anti-human OX40 monoclonal antibody or functional fragment thereof, including:
  • variable region coding sequence for recombinant antibody production to obtain the functionalized humanized anti-human OX40 monoclonal antibody or functional fragment thereof.
  • the humanized anti-human OX40 monoclonal antibody provided by the present invention has high affinity and high specificity for OX40, and can stimulate T cells to secrete cytokines, for example, specifically activate the positive immune regulation of OX40 and activate T cells to secrete cytokines. Therefore, the functional humanized anti-human OX40 monoclonal antibody provided by the present invention can activate T cells by activating the OX40 signaling pathway, thereby achieving the purpose of tumor immunotherapy.
  • Figure 1 Humanized anti-human OX40 monoclonal antibody affinity determination: specifically AH02906 (Figure 1A), AH02913 (Figure 1B), AH02915 (Figure 1C), AH02916 (Figure 1D), AH02917 (Figure 1E), AH02919 ( Figure 1F), AH02921 ( Figure 1G), AH02922 ( Figure 1H), AH02923 ( Figure 1I), AH02925 ( Figure 1J) and chimeric antibody (Figure 1K).
  • FIG. 3A is an ELISA test of each monoclonal antibody and human-derived OX-40 protein
  • Figure 3B is the ELISA detection of monoclonal antibody and monkey-derived OX-40 protein
  • FIG. 4A Thermal stability analysis of purified monoclonal antibodies, specifically SEC-HPLC analysis of thermal stability of humanized anti-human OX40 monoclonal antibody (treatment at 40°C for 2 weeks): AH02913-0/14 days-SEC-HPLC (FIG. 4A ), AH02916-0/14 days-SEC-HPLC (Figure 4B) and AH02919-0/14 days-SEC-HPLC ( Figure 4C), AH02921-0/14 days-SEC-HPLC ( Figure 4D), AH02923-0 /14 days-SEC-HPLC ( Figure 4E);
  • antibody refers to an immunoglobulin molecule, which is generally a tetramer composed of 2 identical heavy chains and 2 identical light chains connected to each other by disulfide bonds. According to the conservative differences in amino acid sequences, the heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. Within the variable regions of the heavy and light chains, there are three local regions with higher degrees of variation in the amino acid composition and arrangement sequence, which are the key positions for the binding of antibodies to antigens, and are therefore also called complementarity determining regions (CDRs).
  • V variable region
  • C constant region
  • variable regions of a heavy chain and a light chain interact to form an antigen binding site (Fv).
  • Fv antigen binding site
  • antibodies can be divided into different classes. There are five main types of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses, for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the subunit structure and three-dimensional conformation of different classes of immunoglobulins are known in the art.
  • the present invention is intended to include any of the aforementioned classes or subclasses of antibodies.
  • antibody as used herein is also intended to encompass digested fragments or functional variants thereof, for example, antibody fragments capable of binding OX40 or a portion thereof, including but not limited to Fab (eg, antibodies obtained by papain digestion), F (ab')2 (for example, by pepsin digestion), Fv or scFv (for example, by molecular biology techniques).
  • Fab eg, antibodies obtained by papain digestion
  • F (ab')2 for example, by pepsin digestion
  • Fv or scFv for example, by molecular biology techniques.
  • the term "monoclonal antibody” as used herein refers to an antibody that is uniform against only a specific antigen epitope. In contrast to common polyclonal antibody preparations that typically include different antibodies against different epitopes (epitopes), each monoclonal antibody is directed against a single epitope on the antigen.
  • the modifier “monoclonal” refers to the uniform characteristics of antibodies and is not to be interpreted as requiring the production of antibodies by any particular method.
  • the monoclonal antibodies of the invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.
  • isolated polynucleotide refers to a polynucleotide that occurs naturally in a non-natural world, including polynucleotides isolated from nature (including living organisms) by biological techniques, and also includes synthetic polynucleotides .
  • the isolated polynucleotide may be genomic DNA, cDNA, mRNA, or other RNA synthesized, or a combination thereof. This article provides multiple nucleotide sequences for encoding the heavy chain variable region and light chain variable region of the humanized anti-OX40 monoclonal antibody.
  • variable region of the chain and variable region of the light chain are designed based on the degeneracy of codons to design a nucleotide sequence that is not completely identical to the nucleotide sequence provided above, but all encode the same amino acid sequence. These modified nucleotide sequences are also included in the scope of the present invention.
  • modification refers to a primary amino acid sequence change relative to the original amino acid sequence, where the change results from a change in the sequence involving the amino acid residue/position.
  • typical modifications include substitution of another amino acid (e.g., conservative or non-conservative substitution) of the residue (at the position), insertion of one or more (generally fewer At 5 or 3) amino acids, and deletion of the residue/position.
  • Amino acid substitution or a change thereof, refers to replacing existing amino acid residues with different amino acid residues in a predetermined (initial) amino acid sequence.
  • the modification generally preferably produces a change in at least one physiological and biochemical activity of the variant polypeptide.
  • the altered physiological and biochemical activity may be the binding affinity, binding capacity and/or binding effect against the target molecule.
  • percent (%) amino acid sequence identity is defined as the comparison of sequences and the introduction of gaps as necessary to obtain the maximum percentage sequence identity, and does not consider any conservative substitutions as part of sequence identity, candidates The percentage of amino acid residues in the sequence that are the same as the amino acid residues in the specific peptide or polypeptide sequence. Sequence comparisons can be performed to determine percent amino acid sequence identity in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximum alignment over the full length of the sequences being compared.
  • vector refers to any molecule (eg, nucleic acid, plasmid or virus, etc.) used to transfer nucleotide encoding information into a host cell.
  • expression vector or “expression cassette” refers to a vector suitable for expressing a gene of interest (nucleotide sequence to be expressed) in a host cell, and usually includes a gene of interest, a promoter, a terminator, a marker gene and the like.
  • host cell refers to a cell that has been or can be transformed with a nucleic acid sequence and thereby expresses a selected gene of interest.
  • the term includes the progeny of the parent cell, regardless of whether the progeny and the original parent cell are identical in morphology or genetic composition, as long as the selected gene exists in the progeny.
  • Commonly used host cells include bacteria, yeast, mammalian cells, etc.
  • transfection refers to the uptake of foreign or foreign DNA into a cell. This technique can be used to introduce one or more foreign DNA portions into a suitable host cell. Cells can be induced by physical and chemical methods (for example, by calcium chloride treatment) to be in a physiological state that is optimal for uptake and containment of foreign DNA, that is, "competent state”.
  • the term "effective amount” as used herein refers to an amount that can produce a function or activity in humans and/or animals and that is acceptable to humans and/or animals.
  • “Pharmaceutically acceptable carrier” refers to a carrier used for administration, including various excipients, diluents and buffers, etc. These substances are suitable for administration to humans and/or animals without excessive adverse side effects, while It is suitable for maintaining the vitality of drugs or active agents located in it.
  • IMGThuman V gene+F+ORF+in-frameP
  • select the human Germline antibody sequence with the highest homology as the humanized receiving vector according to the alignment and use the three heavy chain complementarity determining regions HCDR1 and HCDR2 in the mouse antibody
  • HCDR3 the three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 were transferred into corresponding positions, and the post-translational modification sites (PTM) were analyzed as shown in Table 1.
  • PTM post-translational modification sites
  • Table 2 Humanized back mutation screening of monoclonal antibodies, ranking the affinity of antibodies with the highest affinity
  • the 10 antibody sequences showing the highest affinity are as follows (see for example SEQ ID NO: 7-26):
  • the selected antibody VH and VL sequences are codon-optimized, connected to the 5'end secretion signal peptide, and connected to the human antibody IgG1 heavy chain and kappa light chain constant region sequences, and cloned into pTT5 expression vectors respectively.
  • DNA sequences of human antibodies expressed and secreted in mammalian cells The plasmid was co-transfected with HEK293-6E suspension culture cells with PEI for transient expression. During transfection, the cell density was maintained at 1 ⁇ 10 6 cells/mL, and the PEI:DNA ratio was 3:1. The cells were cultured with shaking at 105 rpm in a 37°C 5% CO 2 incubator. 24 hours after transfection, 0.5% Trypton N-1 was added.
  • the cell culture supernatant was collected for antibody purification.
  • the tubing and protein A column were depyrogenated with 0.2M NaOH.
  • the column was re-equilibrated with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0).
  • the harvested cell culture supernatant was diluted 1:1 using 2 ⁇ the above buffer and sterilized by filtration.
  • the filtered supernatant and the Protein A column were incubated at room temperature for 2 hours.
  • IgG was eluted with sterile 0.1M sodium citrate (pH 3.5). Neutralize with a volume of sterile 1M Tris-HCl (pH 9).
  • the product buffer was exchanged with PBS (pH 7.4) to remove any elution buffer and concentrate the sample.
  • the antibody was quantified by OD280nm using 1.43 extinction coefficient Ec (0.1%).
  • the purified antibody was analyzed by SDS-PAGE using the BioRad electrophoresis system with 10% preformed gel (GenScript).
  • the gel was stained with Estain2.0 (GenScript) and the molecular size and purity were estimated by comparing the stained band with Protein Ladder (GenScript) (Table 3).
  • HBS-EP buffer Perform three pre-cycles with HBS-EP buffer as a sample to equilibrate the chip to stabilize the baseline. Inject low-concentration antibody at a flow rate of 30 ⁇ l/min for 200 seconds, and the antigen will bind to the antibody. Afterwards, the buffer is injected at a flow rate of 30 ⁇ l/min for 400 seconds for dissociation, and 10 mM Gly-HCl, pH 2.0 at a flow rate of 30 ⁇ l/min for three times each Regeneration takes place in 30 seconds, and one cycle ends.
  • the monoclonal antibodies specific to human OX40 (AH02913, AH02916, AH02919, AH02921, AH02922, AH02923 have an affinity of OX40-HIS measured by Biacore of nM. These results indicate that this The antibodies screened by the invention have a very high affinity.
  • OX40 overexpressing functional cell lines were used for functional testing of anti-OX40 monoclonal antibodies.
  • OX40L was used as a positive control for anti-OX40 monoclonal antibody.
  • 100 ⁇ l of supernatant was taken from each well to detect IL-8 content (Cisbio's detection kit).
  • OX40 agonistic antibodies directly act on the OX40 protein on the cell membrane to activate the cells to secrete IL-8. The more IL-8 secretion, the greater the activation of T cells.
  • Recombinant human OX-40 protein and recombinant monkey OX-40 protein were used to detect the human/monkey OX-40 protein cross-recognition ability.
  • ELISA plates (Nunc) were coated with 100 ⁇ l/well of 1 ⁇ g/ml recombinant OX40-His protein in PBS at 4°C overnight. The plate was washed with PBS-T (0.05% Tween), and blocked with 200 ⁇ l/well of PBST containing 1% BSA at 37° C. for 0.5 hour. Subsequently, the blocking solution was discarded, and 100 ⁇ l of purified antibody of 1000 ng/ml was added to the first well, and diluted in a 3-fold gradient, for a total of 11 test concentration gradients. Then incubate at room temperature for 1 hour.
  • the plate was washed three times with PBST, and incubated with 100 ⁇ l/well of horseradish peroxidase-conjugated goat anti-human Fab fragment-IgG (Fab-specific) (GenScript) at 37° C. for 0.5 hour.
  • the plate was washed five times with PBST, and then TMB color developing solution (GenScript) was added and incubated for 15 minutes in the dark at room temperature.
  • the reaction was stopped by adding 50 ⁇ l of 1M HCl stop solution (Sigma).
  • the plate was read at 450nm using a microplate reader.
  • FIG. 3A is the ELISA detection of each monoclonal antibody and human-derived OX-40 protein
  • FIG. 3B is the ELISA detection of monoclonal antibody and monkey-derived OX-40 protein.
  • the six antibodies of AH02913, AH02916, AH02919, AH02921 and AH02923, and chimeric antibodies were expressed in 200ml system to obtain purified antibody samples of more than 5mg and endotoxin control at 3EU/mg level for subsequent experiments.
  • test results show (Table 7), AH02913, AH02919, AH02921, AH02923 Tm are above 70 °C, only AH02916 Tm is shown at 62.9 °C.
  • the antibody samples were treated separately at 40°C, then centrifuged to remove the precipitate, and the amount of remaining antibody was evaluated by ELISA. (Test at 40°C for 7 days and test at 14 days respectively, and use unprocessed samples stored at -80°C as controls for each test)
  • the processed samples were sent to SEC-HPLC and nr-SDS.
  • Oxidation pressurization test The antibody molecule was transferred into 20 mM ammonium acetate solution (pH5.0), and AAPH (2,2'-azobis (2-amidinopropane)) (50:1) was added in the dark at 40°C for 24 hours.
  • the processed antibody samples were detected by mass spectrometry to determine the proportion of chemical changes in the corresponding amino acid molecules, and SEC-HPLC to determine the changes in the degree of polymerization of antibody molecules.
  • the mass spectrometric analysis of the deamidation pressurization experiment detected that no deamidation modification occurred in the AH02913, AH02916 and AH02923 CDR sequences, which is consistent with the conclusion of the sequence analysis.

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Abstract

本发明涉及一种人源化抗人OX40单克隆抗体及其制备方法和用途。本发明提供的人源化抗人OX40单克隆抗体对OX40具有高亲和性、高特异性，能够刺激T细胞分泌细胞因子，例如特异地激活OX40的免疫正调节，激活T细胞分泌细胞因子。因而，本发明提供的功能性人源化抗人OX40单克隆抗体，可通过激活OX40信号通路来激活T细胞，进而实现肿瘤免疫治疗的目的。

Description

人源化抗人OX40单克隆抗体及其制备方法和用途 技术领域
本发明属于肿瘤免疫疗法和分子免疫学领域，尤其是涉及一种人源化抗人OX40单克隆抗体。本发明还涉及该人源化抗人OX40单克隆抗体的制备方法及其用途。
背景技术
在肿瘤治疗中，肿瘤免疫治疗被广泛运用，它已成为肿瘤治疗中既放疗、化疗以外的第三种重要的手段，并逐步走入前台。
免疫系统是一种宿主防御系统，为了正常地发挥作用，免疫系统必须能够灵敏的检测到外来病原体的入侵，并将它们与生物体自身的健康组织区分开来。肿瘤免疫治疗正是利用这一特性，通过调控人体的免疫细胞功能，使人体T细胞能更好的识别并作用到癌变细胞，从而起到清除病变组织的作用。
脊椎动物的免疫系统是一个由多种器官、组织、细胞和分子组成的功能性系统，是机体防卫外源物入侵的最有效的机制，这些免疫器官、组织、细胞和分子相互协作，相互制衡，从而达到保护机体免受外来侵染和维持体内平衡的作用。其中，细胞免疫是通过T细胞受体(TCR)识别抗原呈递细胞(APC)上主要组织相容性复合体(MHC)所呈递的抗原来引起的，这是T细胞激活的第一级信号，然而，T细胞激活并不是仅仅由第一级信号作用就能完成的，还需要抗原非依赖性的第二级信号。后者通过来自T细胞表面的受体或配体与来自APC上的相应的协同刺激因子相互作用来实现。这种相互协作，相互制衡需要众多免疫检查点蛋白的协调参与，激活性免疫检查点蛋白可增强免疫系统的防御反应，抑制性免疫检查点蛋白可控制过强的免疫系统，防止产生针对自身的免疫反应。在研发免疫检查点蛋白的治疗性药物时，对激活性免疫检查点蛋白，要研制激动剂或激活性抗体，对抑制性免疫检查点蛋白，要研制抑制剂或抑制性抗体。
对抑制性免疫检查点，如PD-1和CTLA-4的研究比较多。由抗体介导的针对抑制性免疫检查点蛋白的拮抗作用可以抑制该免疫检查点蛋白通路的活性，达到增强T细胞杀死肿瘤细胞的功能。然而，对绝大多数的癌症患者，仅仅对抑制性免疫检查点蛋白进行调控是不足于消除肿瘤的，要增强T细胞的杀死肿瘤细胞的功能，不仅要减弱T细胞上的抑制通路，还要加强T细胞上的激活通路。T细胞上的激活通路就是刺激性免疫检查点蛋白，如4-1BB、GITR和OX40。通过细胞内信号传导，激活性免疫检查点蛋白可激活PKB/AKT、NF-kB、NFAT等通路，促进辅助性CD4+T细胞和细胞毒性CD8+T细胞的扩增和细胞因子的产生，提升了免疫系统抗肿瘤的能力。
OX40(CD134；TNFRSF4)主要表达于活化的T细胞表面,如CD4、CD8T细胞，辅助性T细胞(Th1,Th2,Th17)，CD4+Foxp3+调控性T细胞；在自然杀伤细胞(NK)，自然杀伤T细胞(NKT)，和中性粒细胞上也有少量表达。与CD28、CD27不同，在未活化T细胞中不表达。OX40及其配体OX40L(CD252)同属于肿瘤坏死因子超家族成员,与4-1BB，CD27,CD40，GITR等等同属于一个大家族。OX40/OX40L是第二级共刺激免疫检查点分子，参与了T细胞的活化、增殖以及存活的过程，对生发中心的形成和树突状细胞的分化成熟起 到关键性作用。
本发明提供对OX40的刺激性抗体，增强T细胞对各种抗原的免疫反应来进行肿瘤免疫治疗。
发明内容
本发明提供了人源化抗人OX40单克隆抗体或其功能片段，其包含重链可变区和轻链可变区，所述重链可变区包含分别在如下HCDR1、HCDR2和HCDR3序列中替换、插入或缺失1个、2个或3个氨基酸残基的氨基酸序列，所述轻链可变区包含分别在如下LCDR1、LCDR2和LCDR3序列中替换、插入或缺失1个、2个或3个氨基酸残基的氨基酸序列：
HCDR1的氨基酸序列为DYSMH；
HCDR2的氨基酸序列为WISTETGEPTYADDFKG；
HCDR3的氨基酸序列为VRPWYLAV；
LCDR1的氨基酸序列为RASQDISNYLN；
LCDR2的氨基酸序列为YTSRLYS；
LCDR3的氨基酸序列为QQANTLPLT。
在一方面，本发明提供了人源化抗人OX40单克隆抗体或其功能片段，其包含重链可变区和轻链可变区，所述重链可变区包含分别与如下HCDR1、HCDR2和HCDR3序列具有至少80％同一性的氨基酸序列，所述轻链可变区包含分别与如下LCDR1、LCDR2和LCDR3序列具有至少80％同一性的氨基酸序列：
HCDR1的氨基酸序列为DYSMH；
HCDR2的氨基酸序列为WISTETGEPTYADDFKG；
HCDR3的氨基酸序列为VRPWYLAV；
LCDR1的氨基酸序列为RASQDISNYLN；
LCDR2的氨基酸序列为YTSRLYS；
LCDR3的氨基酸序列为QQANTLPLT。
在一个实施方案中，所述重链可变区包含分别与上述HCDR1、HCDR2和HCDR3序列具有至少81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。
在一个实施方案中，所述轻链可变区包含分别与如上LCDR1、LCDR2和LCDR3序列具有至少至少81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。
在一个实施方案中，本发明提供了人源化抗人OX40单克隆抗体或其功能片段，其包含重链可变区和轻链可变区，所述重链可变区包含如下HCDR1、HCDR2和HCDR3序列所示的氨基酸序列，所述轻链可变区包含如下LCDR1、LCDR2和LCDR3序列所示的氨基酸序列：
HCDR1的氨基酸序列为DYSMH；
HCDR2的氨基酸序列为WISTETGEPTYADDFKG；
HCDR3的氨基酸序列为VRPWYLAV；
LCDR1的氨基酸序列为RASQDISNYLN；
LCDR2的氨基酸序列为YTSRLYS；
LCDR3的氨基酸序列为QQANTLPLT。
在一个实施方案中，所述重链可变区氨基酸序列选自：SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15或SEQ ID NO:16。
在一个实施方案中，所述轻链可变区氨基酸序列选自：SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25或SEQ ID NO:26。
在一个实施方案中，所述人源化抗人OX40单克隆抗体或其功能片段，其重链可变区和轻链可变区选自以下组合：
所述重链可变区为SEQ ID NO:7以及所述轻链可变区为SEQ ID NO:17；
所述重链可变区为SEQ ID NO:8以及所述轻链可变区为SEQ ID NO:18；
所述重链可变区为SEQ ID NO:9以及所述轻链可变区为SEQ ID NO:19；
所述重链可变区为SEQ ID NO:10以及所述轻链可变区为SEQ ID NO:20；
所述重链可变区为SEQ ID NO:11以及所述轻链可变区为SEQ ID NO:21；
所述重链可变区为SEQ ID NO:12以及所述轻链可变区为SEQ ID NO:22
所述重链可变区为SEQ ID NO:13以及所述轻链可变区为SEQ ID NO:23；
所述重链可变区为SEQ ID NO:14以及所述轻链可变区为SEQ ID NO:24；
所述重链可变区为SEQ ID NO:15以及所述轻链可变区为SEQ ID NO:25；或
所述重链可变区为SEQ ID NO:16以及所述轻链可变区为SEQ ID NO:26。
在一个优选的实施方案中，所述人源化抗人OX40单克隆抗体或其功能片段，其重链可变区和轻链可变区选自以下组合：
所述重链可变区为SEQ ID NO:8以及所述轻链可变区为SEQ ID NO:18；
所述重链可变区为SEQ ID NO:10以及所述轻链可变区为SEQ ID NO:20；
所述重链可变区为SEQ ID NO:12以及所述轻链可变区为SEQ ID NO:22
所述重链可变区为SEQ ID NO:13以及所述轻链可变区为SEQ ID NO:23；或
所述重链可变区为SEQ ID NO:15以及所述轻链可变区为SEQ ID NO:25。
在一个更优选的实施方案中，所述人源化抗人OX40单克隆抗体或其功能片段，其重链可变区和轻链可变区选自以下组合：
所述重链可变区为SEQ ID NO:12以及所述轻链可变区为SEQ ID NO:22；或
所述重链可变区为SEQ ID NO:13以及所述轻链可变区为SEQ ID NO:23。
在一个实施方案中，本发明的人源化抗人OX40单克隆抗体或其功能片段与OX40之间的解离常数KD小于3nM。
在一个实施方案中，本发明的人源化抗人OX40单克隆抗体或其功能片段特异性地激活OX40的免疫正调节，激活T细胞分泌细胞因子。
在一个实施方案中，本发明提供了分离的多核苷酸，其编码本发明的人源化抗人OX40单克隆抗体或其功能片段。
在一个实施方案中，多核苷酸包含编码本发明所述人源化抗人OX40单克隆抗体的重链可变区的重链编码序列，和编码本发明所述人源化抗人OX40单克隆抗体的轻链可变区的轻链编码序列。
在另一方面，本发明提供了表达载体，包含所述的多核苷酸。
在另一方面，本发明提供了宿主细胞，其包含所述表达载体。
在一个实施方案中，所述宿主细胞为HEK293-6E细胞。
在另一方面，本发明提供所述人源化抗人OX40单克隆抗体或其功能片段、所述多核苷酸、所述表达载体或所述宿主细胞在制备用于抗肿瘤的药物中的用途。
在另一方面，本发明提供了所述人源化抗人OX40单克隆抗体或其功能片段、所述多核苷酸、所述表达载体或所述宿主细胞用于治疗肿瘤的用途。
在另一方面，本发明提供了所述人源化抗人OX40单克隆抗体或其功能片段、所述多核苷酸、所述表达载体或所述宿主细胞用于治疗肿瘤。
在另一方面，本发明提供了抗肿瘤药物组合物，其包含有效量的所述人源化抗人OX40单克隆抗体或其功能片段，和药学上可接受的载体。
在另一方面，本发明提供了一种制备所述人源化抗人OX40单克隆抗体或其功能片段的方法，包括：
(1)对鼠源抗体进行人源化，获得所述人源化抗人OX40单克隆抗体或其功能片段的轻链和重链的可变区编码序列；以及
(2)用所述可变区编码序列进行重组抗体生产，获得功能性所述人源化抗人OX40单克隆抗体或其功能片段。
本发明提供的人源化抗人OX40单克隆抗体对OX40具有高亲和性、高特异性，能够刺激T细胞分泌细胞因子，例如特异地激活OX40的免疫正调节，激活T细胞分泌细胞因子。因而，本发明提供的功能性人源化抗人OX40单克隆抗体，可通过激活OX40信号通路来激活T细胞，进而实现肿瘤免疫治疗的目的。
附图简要说明
图1.人源化抗人OX40的单克隆抗体亲和力测定:具体为AH02906(图1A)、AH02913(图1B)、AH02915(图1C)、AH02916(图1D)、AH02917(图1E)、AH02919(图1F)、AH02921(图1G)、AH02922(图1H)、AH02923(图1I)、AH02925(图1J)和嵌合抗体(图1K)。
图2.人源化抗人OX40单克隆抗体细胞活性功能检测：具体为AH02913(图2A)、AH02916(图2B)、AH02919(图2C)、AH02921(图2D)、AH02923(图2E)、OX40IgG(图2F)和；OX40配体(图2G)。
图3.人源化抗人OX40单克隆抗体与细胞表面过表达人源/猴源OX-40蛋白的交叉反应测定；其中图3A是各个单克隆抗体与人源OX-40蛋白的ELISA检测，图3B是单克隆抗体与猴源OX-40蛋白的ELISA检测
图4.纯化单克隆抗体热稳定性分析，具体为人源化抗人OX40单克隆抗体热稳定性SEC-HPLC分析(40℃处理2周)：AH02913-0/14天-SEC-HPLC(图4A)、AH02916-0/14天-SEC-HPLC(图4B)和AH02919-0/14天-SEC-HPLC(图4C)、AH02921-0/14天-SEC-HPLC(图4D)、AH02923-0/14天-SEC-HPLC(图4E)；
图5.纯化单克隆抗体热稳定性分析，具体为人源化抗人OX40单克隆抗体热稳定性ELISA分析(40℃处理2周)：AH02913(图5A)、AH02916(图5B)、AH02919(图5C)、AH02921(图5D)和AH02923(图5E)；
图6.纯化单克隆抗体成药性检测分析，具体为人源化抗人OX40单克隆抗体氧化加压/脱酰胺加压试验后ELISA分析：AH02913(图6A)、AH02916(图6B)、AH02919(图6C)、AH02921(图6D)和AH02923(图6E)。
具体实施方式
除非另有说明，本发明所用的技术和科学术语具有与本发明所属领域的普通技术员通常所理解的含义。
本文所用的术语“抗体”指免疫球蛋白分子，其通常为由2个相同重链和2个相同轻链通过二硫键相互连接组成的四聚体。根据氨基酸序列的保守性差异，将重链和轻链分为位于氨基端的可变区(V)和位于羧基端的恒定区(C)。在重链和轻链的可变区内，分别有三个局部区域的氨基酸组成和排列顺序具有更高的变异程度，为抗体与抗原结合的关键位置，因而也称为互补决定区(CDR)。在本文中，三个重链互补决定区分别称为HCDR1、HCDR2和HCDR3，三个轻链互补决定区分别称为LCDR1、LCDR2和LCDR3。一条重链和一条轻链的可变区相互作用形成了抗原结合部位(Fv)。根据它们重链恒定区的氨基酸序列，可将抗体分为不同类别。有五种主要类型的完整抗体：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可进一步分为亚类，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。不同类别的免疫球蛋白的亚单位结构和三维构象在本领域内是已知的。本发明旨在包括任何前述类或亚类的抗体。
本文使用的术语“抗体”还旨在涵盖其消化片段或功能性变体，例如，能够结合OX40或其部分的抗体片段，包括但不限于Fab(例如，抗体经木瓜蛋白酶消化而得到)、F(ab’)2(例如，通过胃蛋白酶消化得到)、Fv或scFv(例如通过分子生物学技术得到)。
本文使用的术语“单克隆抗体”指均一的仅针对某一特定抗原表位的抗体。与典型地包括针对不同抗原决定簇(表位)的不同抗体的普通多克隆抗体制剂相比，每种单克隆抗体针对抗原上的单个抗原决定簇。修饰语“单克隆”表示抗体的均一特征，不解释为需要通过任何特定方法产生的抗体。本发明的单克隆抗体优选通过重组DNA方法产生，或通过本文其它地方描述的筛选方法获得。
本文使用的术语“分离的多核苷酸”指非自然界中天然存在状态的多核苷酸，包括通过生物学技术从自然界(包括生物体内)分离出的多核苷酸，也包括人工合成的多核苷酸。分离的多核苷酸可以是基因组DNA、cDNA、mRNA或合成的其它RNA，或者它们的组合。本文提供了多个用于编码人源化抗OX40单克隆抗体的重链可变区和轻链可变区的核苷酸序列，需要指出的是，本领域技术人员可以根据本文所提供的重链可变区和轻链可变区的氨基酸序列，基于密码子简并性，设计出与以上提供的核苷酸序列不完全相同的核苷酸序列，但都编码相同的氨基酸序列。这些经改动的核苷酸序列也包括在本发明的范围内。
本文中所用的氨基酸残基/位置“修饰”，指相对于初始氨基酸序列的一级氨基酸序列变化，其中变化来自于涉及所述氨基酸残基/位置的序列的改变。例如，典型的修饰包括用另一个氨基酸替换(如，保守或非保守替换)(在所述位置上的)残基、在所述残基/位置相邻位上插入一个或多个(一般少于5或3个)氨基酸、和缺失所述残基/位置。“氨基酸替换”、或其变化，指在预先确定的(初始)氨基酸序列中，用不同的氨基酸残基代替现有的氨基酸残基。相对于含初始(或“野生型”)氨基酸序列的多肽，修饰一般优选会产生变体多肽的 至少一种生理生化活性的改变。例如，对于抗体，改变的生理生化活性可以是针对靶分子的结合亲和力、结合能力和/或结合效果。
关于肽或多肽序列的“百分比(％)氨基酸序列同一性”定义为对比序列并在必要时引入缺口以获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分率。可以本领域技术范围内的多种方式进行序列对比以测定百分比氨基酸序列同一性，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定测量对比的适宜参数，包括对所比较的序列全长获得最大对比所需的任何算法。
当涉及多核苷酸时，本文所用的术语“载体”指用于将核苷酸编码信息转移到宿主细胞内的任一种分子(例如，核酸、质粒或病毒等)。术语“表达载体”或“表达盒”指适于在宿主细胞内表达目的基因(待表达核苷酸序列)的载体，通常包括目的基因、启动子、终止子、标记基因等部分。
本文所用的术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等。
本文所用的术语“转染”指外来或外源DNA被细胞摄入，该技术可用于将一种或多种外源DNA部分导入适宜的宿主细胞。可通过理化方法(例如通过氯化钙处理)诱导细胞，使其处于最适摄取和容纳外来DNA的生理状态，即“感受态”。
提及药物组合物时，本文所使用的术语“有效量的”指可对人和/或动物产生功能或活性且可被人和/或动物所接受的量。“药学上可接受的载体”指用于给药的载体，包括各种赋形剂、稀释剂和缓冲剂等，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。
下面将结合具体实施例对本发明的一些方面进行详细描述。除非另有说明，下文描述的实施例的方法和材料均为可以通过市场购买获得的常规产品。
实施例
实施例1鼠源抗人OX40抗体人源化
1)从杂交瘤细胞中分离得到多核苷酸序列，测序得到鼠源抗人OX40抗体99A2A8D4E8抗体序列(CDR区域采用下划线显示)(参见例如SEQ ID NO:1-2)
Figure PCTCN2019120808-appb-000001
SEQ ID NO:2,99A2A8D4E8-VL
DIQMTQTTSSLSASLGDRVTISC RASQDISNYLNWYQQKPDGTVKLLIY YTSRLYSGVPSRFSGSG
SGADYSLTVSNLEQEDIATYFC QQANTLPLTFGAGTKLELK
2)抗人OX40抗体CDR移植质粒构建
选择IMGT human V gene(F+ORF+in-frameP)数据库，根据比对选择同源性最高的人Germline抗体序列作为人源化接收载体，将鼠抗体中三个重链互补决定区HCDR1、HCDR2和HCDR3，三个轻链互补决定区LCDR1、LCDR2和LCDR3分别转入相应位置，并且分析翻译后修饰位点(PTM)见表1。序列分析显示M34，W50，W102三个位点是翻译后氧化修饰的热点(参见例如SEQ ID NO:3-4)。
Figure PCTCN2019120808-appb-000002
Figure PCTCN2019120808-appb-000003
表1：翻译后修饰位点风险分析
Figure PCTCN2019120808-appb-000004
3)设计噬菌体文库CBM(灰色底纹)构建抗人OX40抗体99A2A8D4E8VH-VL的Phage-Fab和FASEBA-Fab质粒，筛选人源化抗体回复突变位点(参见例如SEQ ID NO:5-6)。
99A2A8D4E8 CBM
SEQ ID NO:5,99A2A8D4E8-VH-CBM
QVQLVQSGPEVKKPGASVKISCKASGYTFT DYSMHWVKQAPGQGLEWMG WISTETGEPTYADDFKG
RFTFTLDTSASTAYLEISSLRSEDTAVYFCAR VRPWYLAVWGQGTLVTVSS
Figure PCTCN2019120808-appb-000005
4)原核表达抗体产物亲和力排序及其VH/VL序列(表2)，选择抗人OX40抗体亲和力最高的序列进行真核系统表达。
表2：单克隆抗体人源化回复突变筛选，具有最高亲和力的抗体亲和力排序
Figure PCTCN2019120808-appb-000006
呈现最高亲和力的10条抗体序列如下(参见例如SEQ ID NO:7-26)：
Figure PCTCN2019120808-appb-000007
Figure PCTCN2019120808-appb-000008
Figure PCTCN2019120808-appb-000009
Figure PCTCN2019120808-appb-000010
Figure PCTCN2019120808-appb-000011
Figure PCTCN2019120808-appb-000012
Figure PCTCN2019120808-appb-000013
Figure PCTCN2019120808-appb-000014
Figure PCTCN2019120808-appb-000015
Figure PCTCN2019120808-appb-000016
Figure PCTCN2019120808-appb-000017
Figure PCTCN2019120808-appb-000018
Figure PCTCN2019120808-appb-000019
Figure PCTCN2019120808-appb-000020
Figure PCTCN2019120808-appb-000021
Figure PCTCN2019120808-appb-000022
Figure PCTCN2019120808-appb-000023
Figure PCTCN2019120808-appb-000024
Figure PCTCN2019120808-appb-000025
Figure PCTCN2019120808-appb-000026
实施例2：人源化抗体重组生产
选中的抗体VH、VL序列经密码子优化后，连接在5’端分泌信号肽后，与人抗体IgG1重链、kappa轻链恒定区序列相连接，分别克隆到pTT5表达载体中制备成为可以在哺乳动物细胞内表达并分泌的人抗体DNA序列。质粒采用PEI共转染HEK293-6E悬浮培养细胞，进行瞬时表达。转染时，细胞密度维持在1×10 6细胞/mL，PEI:DNA比例为3:1。细胞在37℃ 5％CO 2培养箱中105转/分钟震荡培养。转染24小时后，加入0.5％Trypton N-1。5天后，收集细胞培养上清用于抗体纯化。纯化之前，将管道和蛋白A柱用0.2M NaOH去热原。将柱用含有0.05M Tris和1.5M NaCl(pH8.0)的缓冲液重新平衡。随后将收获的细胞培养物上清液，使用2×上述缓冲液1:1稀释并过滤除菌。将过滤的上清液和蛋白A柱室温孵育2小时，用并1×上述缓冲液洗涤柱后，使用无菌0.1M柠檬酸钠(pH3.5)洗脱IgG，收集了洗脱液并用九分之一体积的无菌1M Tris-HCl(pH9)中和。在无菌条件下，将所述产品缓冲液交换为PBS(pH7.4)以除去任何的洗脱缓冲液并浓缩所述样品。浓缩之后，使用1.43的消光系数Ec(0.1％)通过OD280nm对抗体进行定量。
纯化的抗体通过BioRad电泳系统用10％预制胶(GenScript)通过SDS-PAGE来分析。将所述凝胶用Estain2.0(GenScript)染色并通过比较染色带与Protein Ladder(GenScript)来估计分子大小和纯度(表3)。
表3：人源化抗体重组生产
Figure PCTCN2019120808-appb-000027
实施例3：人源化单克隆抗体的亲和力测定
以10μl/min流速的HBS-EP缓冲液平衡芯片表面5分钟，随后以10μl/min的流速注射“NHS+EDC”的1:1混合液100秒来活化芯片，将稀释在10mM醋酸钠缓冲液中的抗原 蛋白(OX40-HIS)以10μl/min流速注射约180秒进行偶联，最后以10μl/min的流速注射乙醇胺200秒进行表面封闭。
以HBS-EP缓冲液作为样品进行三个预循环来平衡芯片使基线稳定。以30μl/min流速注射低浓度抗体200秒，抗原与抗体发生结合，之后30μl/min流速注射缓冲液400秒进行解离，30μl/min流速注射10mM Gly-HCl,pH2.0共三次，每次30秒进行再生，一次循环结束。
改变抗体浓度进行下一个梯度浓度的循环测定直到所有梯度浓度(2.5nM、5nM、10nM、20nM、40nM、80mM)及重复浓度(如20nM)测定结束。实验数据经过双扣减(对照通道及零浓度)后，在Biacore 8K evaluation software中进行“1:1结合”模型的拟合。使用Biacore 8K测定抗体针对OX40-HIS重组蛋白的亲和力。
如图1和表4所示，特异性针对人OX40的单克隆抗体(AH02913,AH02916,AH02919,AH02921,AH02922,AH02923对OX40‐HIS的亲和力由Biacore测得均达到nM级。这些结果表明，本发明筛选到的抗体具有非常高的亲和力。
表4人源化抗人OX40-HIS的单克隆抗体亲和力测定
Figure PCTCN2019120808-appb-000028
实施例4：人源化抗人OX40单克隆抗体的细胞活性功能验证
OX40过表达的功能细胞系被用来做抗OX40的单克隆抗体的功能性检测。把OX40过表达的功能细胞系铺在384空板上，37℃及5％CO 2孵育过夜，把不同浓度的抗体样品加入到每个孔内，无抗体孔作为背景对照，人的IgG1作为阴性对照，OX40L作为抗OX40的单克隆抗体的阳性对照，在37℃及5％CO 2条件下孵育24小时后，从每孔中取100μl上清检测IL-8含量(Cisbio的检测试剂盒)。在对OX40激动性抗体的检测中，OX40激动性抗体直接作用于细胞膜上的OX40蛋白，激活了细胞，使之分泌IL-8,IL-8分泌越多，表明对T细胞的激活越大。
实验结果显示，人源化抗人OX40单克隆抗体(AH02913、AH02916、AH02919、AH02921、AH02923)能够特异性地激活OX40的免疫正调节，激活T细胞分泌细胞因子，对应EC 50分别为1.62nM,3.00nM,1.03nM,2.14nM,1.55nM(图2和表5)。
表5人源化抗人OX40单克隆细胞细胞激活反应
Figure PCTCN2019120808-appb-000029
实施例5：人源化单克隆抗体的交叉识别能力测定
采用重组人OX-40蛋白和重组猴OX-40蛋白分别检测人源化单克隆抗体对于人/猴OX-40蛋白的交叉识别能力。
将ELISA板(Nunc)用100μl/孔的PBS中1μg/ml的重组OX40-His蛋白在4℃下包被过夜。用PBS-T(0.05％吐温)洗涤板，并将其用200μl/孔的含1％BSA的PBST在37℃封闭0.5小时。随后弃去封闭液，向首孔加入1000ng/ml的纯化抗体100μl，并按照3倍梯度稀释，共计11个测试浓度梯度。然后在室温下孵育1小时。将板用PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗人Fab片段-IgG(Fab-特异性)(GenScript)37℃孵育0.5小时。将板用PBST洗涤五次，然后加入TMB显色液(GenScript)并在室温下在黑暗中孵育15分钟。通过加入50μl的1MHCl终止液(Sigma)终止反应。使用酶标仪在450nm下读板。
结果显示重组表达的抗体能够共同识别重组人OX-40蛋白和重组猴OX-40蛋白(图3和表6)。其中，图3A是各个单克隆抗体与人源OX-40蛋白的ELISA检测，图3B是单克隆抗体与猴源OX-40蛋白的ELISA检测。
表6.各重组抗体识别人OX-40蛋白和猴OX-40蛋白的ELISA检测EC 50
Figure PCTCN2019120808-appb-000030
实施例6：人源化抗人OX40单克隆抗体的成药性评价
AH02913、AH02916、AH02919、AH02921和AH02923以及嵌合抗体六个抗体按200ml体系表达，得到5mg以上，内毒素控制在3EU/mg水平的纯化抗体样品供后续实验所需。
1.热稳定性检测
1.1差示扫描荧光DSF方法检测样品变性温度Tm值
表7人源化抗人OX40单克隆抗体Tm检测
Figure PCTCN2019120808-appb-000031
Figure PCTCN2019120808-appb-000032
检测结果显示(表7)，AH02913,AH02919,AH02921,AH02923的Tm都在70℃以上，只有AH02916的Tm显示在62.9℃.
1.2热稳定性检测实验设置
A.抗体样品浓度>5mg/ml进行耐久试验。
抗体样品在40℃分别处理，然后离心去除沉淀，再用ELISA评估残留的抗体量。(40℃处理7天，14天分别检测，每次检测同时用存放于-80℃未处理的样品做对照)
处理样品送测SEC-HPLC和nr-SDS。
结果如图4和表8所示，经过热处理的AH02913、AH02916、AH02919、AH02921和AH02923抗体样品没有出现大量聚集，同时呈现出稳定的抗体纯度。
表8人源化抗人OX40单克隆抗体热处理前后nr-SDS纯度检测
样品 抗体峰前(％) 抗体峰(％)
AH02913-D0 3.99 96.01
AH02913-D14 17.87 82.13
AH02916-D0 4.6 95.4
AH02916-D14 16.03 83.97
AH02919-D0 16.51 83.49
AH02919-D14 24.9 75.1
AH02921-D0 21.87 78.12
AH02921-D14 27.57 72.44
AH02923-D0 4.27 95.73
AH02923-D14 17.55 82.45
另外，人源化抗人OX40单克隆抗体热稳定加压试验后ELISA检测(图5)显示经过热处理的AH02913、AH02916、AH02919、AH02921和AH02923抗体样品与人源OX‐40蛋白的识别能力没有受到影响。
2.成药性实验
抗人OX40单克隆抗体CDR区域序列分析显示(表1)，VH区域预测存在重链可变区M34,W50,W102位点氧化修饰的热点。人源化后的抗人OX40单克隆抗体分别进行：
A.氧化加压试验：抗体分子转入20mM醋酸铵溶液中(pH5.0)，加入AAPH(2,2’-azobis(2-amidinopropane))(50:1)40℃避光处理24小时。
B.去酰胺化加压实验：抗体分子置于PBS溶液中(pH9)，48h，40℃
来判断氧化修饰/去酰胺修饰对抗体分子的抗原识别能力的影响。
处理后的抗体样品采用质谱检测判断相应氨基酸分子发生化学变化的比例，SEC-HPLC判断抗体分子的聚合度变化。
结果显示(表9-10)，质谱检测覆盖率都达到了95％左右，得到可信的结果。各个处理前、后的抗体分子在M34,W50都没有检测到有氧化修饰；处理前的抗体分子在W102也没有检测到氧化，AH02913在AAPH-24处理样品中，W102有31.43％氧化；AH02916在AAPH-24处理样品中，W102有54.33％氧化；AH02923在AAPH-24处理样品中，W102有35.78％氧化。同时检测到在恒定区位点M428,M252也发生氧化修饰。所以AH02913、AH02916和AH02923CDR序列中W102位点存在发生氧化修饰的可能性。
同时，去酰胺化加压实验质谱分析检测AH02913、AH02916和AH02923CDR序列中没有发生脱酰胺修饰，这与序列分析结论一致。
ELISA验证结果显示(图6)，AH02913、AH02916、AH02919、AH02921和AH02923抗体分子在氧化加压处理和脱酰胺处理条件下，仍然保持较好的抗原结合能力。
表9.成药性检测分析:人源化抗人OX40单克隆抗体氧化加压试验后质谱检测
Figure PCTCN2019120808-appb-000033
Figure PCTCN2019120808-appb-000034
表10.成药性检测分析:人源化抗人OX40单克隆抗体脱酰胺加压试验后质谱检测
Figure PCTCN2019120808-appb-000035

Claims (13)

  1. 人源化抗人OX40单克隆抗体或其功能片段，其包含重链可变区和轻链可变区，所述重链可变区包含分别与如下HCDR1、HCDR2和HCDR3序列具有至少80％同一性的氨基酸序列，所述轻链可变区包含分别与如下LCDR1、LCDR2和LCDR3序列具有至少80％同一性的氨基酸序列：
    HCDR1的氨基酸序列为DYSMH；
    HCDR2的氨基酸序列为WISTETGEPTYADDFKG；
    HCDR3的氨基酸序列为VRPWYLAV；
    LCDR1的氨基酸序列为RASQDISNYLN；
    LCDR2的氨基酸序列为YTSRLYS；
    LCDR3的氨基酸序列为QQANTLPLT。
  2. 如权利要求1所述的人源化抗人OX40单克隆抗体或其功能片段，其包含重链可变区和轻链可变区，所述重链可变区包含如下HCDR1、HCDR2和HCDR3序列所示的氨基酸序列，所述轻链可变区包含如下LCDR1、LCDR2和LCDR3序列所示的氨基酸序列：
    HCDR1的氨基酸序列为DYSMH；
    HCDR2的氨基酸序列为WISTETGEPTYADDFKG；
    HCDR3的氨基酸序列为VRPWYLAV；
    LCDR1的氨基酸序列为RASQDISNYLN；
    LCDR2的氨基酸序列为YTSRLYS；
    LCDR3的氨基酸序列为QQANTLPLT。
  3. 如权利要求2所述的人源化抗人OX40单克隆抗体或其功能片段，所述重链可变区氨基酸序列选自：SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15或SEQ ID NO:16。
  4. 如权利要求2所述的人源化抗人OX40单克隆抗体或其功能片段，所述轻链可变区氨基酸序列选自：SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25或SEQ ID NO:26。
  5. 如权利要求2所述的人源化抗人OX40单克隆抗体或其功能片段，其重链可变区和轻链可变区选自以下组合：
    所述重链可变区为SEQ ID NO:7以及所述轻链可变区为SEQ ID NO:17；
    所述重链可变区为SEQ ID NO:8以及所述轻链可变区为SEQ ID NO:18；
    所述重链可变区为SEQ ID NO:9以及所述轻链可变区为SEQ ID NO:19；
    所述重链可变区为SEQ ID NO:10以及所述轻链可变区为SEQ ID NO:20；
    所述重链可变区为SEQ ID NO:11以及所述轻链可变区为SEQ ID NO:21；
    所述重链可变区为SEQ ID NO:12以及所述轻链可变区为SEQ ID NO:22
    所述重链可变区为SEQ ID NO:13以及所述轻链可变区为SEQ ID NO:23；
    所述重链可变区为SEQ ID NO:14以及所述轻链可变区为SEQ ID NO:24；
    所述重链可变区为SEQ ID NO:15以及所述轻链可变区为SEQ ID NO:25；或
    所述重链可变区为SEQ ID NO:16以及所述轻链可变区为SEQ ID NO:26。
  6. 如权利要求1或2所述的人源化抗人OX40单克隆抗体或其功能片段，其与OX40之间的解离常数KD小于3nM。
  7. 分离的多核苷酸，其编码如权利要求1-6中任一项所述的人源化抗人OX40单克隆抗体或其功能片段。
  8. 如权利要求7所述的多核苷酸，其包含编码所述人源化抗人OX40单克隆抗体的重链可变区的重链编码序列，和编码所述人源化抗人OX40单克隆抗体的轻链可变区的轻链编码序列。
  9. 表达载体，其包含权利要求7或8所述的多核苷酸。
  10. 宿主细胞，其包含如权利要求9所述的表达载体。
  11. 如权利要求1-6中任一项所述的人源化抗人OX40单克隆抗体或其功能片段、权利要求7或8所述的多核苷酸、权利要求9所述的表达载体或权利要求10所述的宿主细胞在制备用于抗肿瘤的药物中的用途。
  12. 抗肿瘤药物组合物，其包含有效量的如权利要求1-6中任一项所述的人源化抗人OX40单克隆抗体或其功能片段，和药学上可接受的载体。
  13. 制备如权利要求1-6任一项所述的人源化抗人OX40单克隆抗体或其功能片段的方法，包括：
    (1)对鼠源抗体进行人源化，获得所述人源化抗人OX40单克隆抗体或其功能片段的轻链和重链的可变区编码序列；以及
    (2)用所述可变区编码序列进行重组抗体生产，获得功能性所述人源化抗人OX40单克隆抗体或其功能片段。
PCT/CN2019/120808 2018-11-26 2019-11-26 人源化抗人ox40单克隆抗体及其制备方法和用途 WO2020108463A1 (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112794907A (zh) * 2020-12-03 2021-05-14 安徽安科生物工程（集团）股份有限公司 一株全人源抗人huOX40单克隆抗体
WO2022042692A1 (zh) * 2020-08-27 2022-03-03 苏州景涞医疗科技有限公司 低毒性抗ox40抗体、其药物组合物及应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113651883B (zh) * 2021-08-04 2022-06-10 广州呼研所医药科技有限公司 人源化抗HAdV-B7单克隆中和抗体、制备方法及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106103486A (zh) * 2014-03-31 2016-11-09 豪夫迈·罗氏有限公司 抗ox40抗体和使用方法
CN107074953A (zh) * 2014-10-10 2017-08-18 免疫医疗有限责任公司 人源化抗ox40抗体及其用途
CN108623686A (zh) * 2017-03-25 2018-10-09 信达生物制药(苏州)有限公司 抗ox40抗体及其用途
CN108623685A (zh) * 2017-03-25 2018-10-09 信达生物制药(苏州)有限公司 抗ox40抗体及其用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2016303550B2 (en) * 2015-08-04 2019-06-13 Glaxosmithkline Intellectual Property Development Limited Combination treatments and uses and methods thereof
CN109232738B (zh) * 2017-07-11 2021-11-09 南京金斯瑞生物科技有限公司 一种抗人ox40单克隆抗体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106103486A (zh) * 2014-03-31 2016-11-09 豪夫迈·罗氏有限公司 抗ox40抗体和使用方法
CN107074953A (zh) * 2014-10-10 2017-08-18 免疫医疗有限责任公司 人源化抗ox40抗体及其用途
CN108623686A (zh) * 2017-03-25 2018-10-09 信达生物制药(苏州)有限公司 抗ox40抗体及其用途
CN108623685A (zh) * 2017-03-25 2018-10-09 信达生物制药(苏州)有限公司 抗ox40抗体及其用途

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022042692A1 (zh) * 2020-08-27 2022-03-03 苏州景涞医疗科技有限公司 低毒性抗ox40抗体、其药物组合物及应用
CN116419970A (zh) * 2020-08-27 2023-07-11 中山恒动生物制药有限公司 低毒性抗ox40抗体、其药物组合物及应用
CN116419970B (zh) * 2020-08-27 2024-03-22 中山恒动生物制药有限公司 低毒性抗ox40抗体、其药物组合物及应用
CN112794907A (zh) * 2020-12-03 2021-05-14 安徽安科生物工程（集团）股份有限公司 一株全人源抗人huOX40单克隆抗体
CN112794907B (zh) * 2020-12-03 2022-09-06 安徽安科生物工程（集团）股份有限公司 一株全人源抗人huOX40单克隆抗体

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