WO2020020275A1 - 人源化抗人ctla4单克隆抗体及其制备方法和用途 - Google Patents

人源化抗人ctla4单克隆抗体及其制备方法和用途 Download PDF

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WO2020020275A1
WO2020020275A1 PCT/CN2019/097643 CN2019097643W WO2020020275A1 WO 2020020275 A1 WO2020020275 A1 WO 2020020275A1 CN 2019097643 W CN2019097643 W CN 2019097643W WO 2020020275 A1 WO2020020275 A1 WO 2020020275A1
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seq
variable region
chain variable
monoclonal antibody
humanized anti
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French (fr)
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殷刘松
周铁林
方卓
米艳玲
吴瑃辰
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Nanjing Jinsirui Science and Technology Biology Corp
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Nanjing Jinsirui Science and Technology Biology Corp
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Priority to US17/263,148 priority patent/US12037396B2/en
Priority to CN201980049141.3A priority patent/CN112513084B/zh
Priority to CA3107524A priority patent/CA3107524C/en
Priority to CN202210894065.XA priority patent/CN115850475B/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K2317/50—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
    • 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
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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

Definitions

  • the invention belongs to the fields of tumor immunotherapy and molecular immunology, and particularly relates to a humanized anti-human CTLA4 monoclonal antibody.
  • the invention also relates to a preparation method of the humanized anti-human CTLA4 monoclonal antibody and use thereof.
  • the immune system of vertebrates is a functional system composed of a variety of organs, tissues, cells and molecules. It is the most effective mechanism for the body to defend against the invasion of foreign objects (Janeway et al., Immunology: The Immune System and Health and Disease New York: Garland Science (2005). Immune organs, tissues, and cells cooperate and balance each other. With the coordination of numerous immune checkpoint proteins and cytokines, it can protect the body from external infection and maintain the balance in the body.
  • the adaptive immune system against foreign pathogens consists of humoral immunity (mediated by B cells) and cellular immunity (mediated by T cells).
  • cellular immunity is caused by the recognition of antigens presented by the major histocompatibility complex (MHC) on antigen-presenting cells (APC) through the T cell receptor (TCR).
  • MHC major histocompatibility complex
  • APC antigen-presenting cells
  • TCR T cell receptor
  • B7-1 also known as B7, B7.1, or CD80
  • B7-2 also known as B7.2 or CD86
  • CTLA4 and CD28 are both members of the Ig superfamily containing a single extracellular Ig domain, and both can bind to the B7 protein, but the regulatory effect is opposite.
  • CTLA4 has a higher affinity than the binding of CD28 and B7 protein, and competes to form a more stable interaction, which makes T cells lack secondary stimulation signals and becomes anergic. At the same time, T cells can induce T cell decay after T cell activation. Perish. Thus, the immune system can be negatively regulated and maintain the homeostasis of T cells in the body. Therefore, the use of monoclonal antibodies to block negatively regulated signals transmitted by CTLA4 can provide new therapies for human diseases that benefit from immune stimulation, such as immunotherapy for cancer and infectious diseases.
  • CTLA4 monoclonal antibodies have been used in various clinical trials to treat a variety of human cancers, including melanoma, prostate cancer, bladder cancer, colorectal cancer, malignant mesothelioma, gastrointestinal cancer, liver cancer, non-small cell lung cancer (Grosso et al., Cancer Immunology 13: 5, 2013).
  • Ipilimumab (Keler et al., J. Immunol 171: 6251-6259 (2003)) and Tremelimumab (Ribas et al., Oncologist 12: 873-883 (2005)) are available.
  • CTLA4 monoclonal antibody that has been successfully marketed at present, Ipilimumab (trade name Yervoy) marks the practicality of tumor immunotherapy in the clinical stage. Moreover, with the pre-clinical experiments verifying the ability of monoclonal antibodies against different immunomodulatory factors to synergistically treat cancer, CTLA4 monoclonal antibodies have been combined with monoclonal antibodies or small molecule compounds of different immunosuppressive molecules, and are being targeted for Clinical trials for different cancers.
  • CTLA4 monoclonal antibody there is only one CTLA4 monoclonal antibody currently on the market, and CTLA4 monoclonal antibodies also have varying degrees of side effects, including inducing immunogenicity in some patients, excessive inhibition of CTLA4 signals may cause autoimmune diseases, and different CTLA4 Monoclonal antibodies have various degrees of developability.
  • CTLA4 Monoclonal antibodies have various degrees of developability.
  • the development of new humanized functional antibodies that can block the binding of CTLA4 and B7 proteins with higher affinity, specificity, functionality and diversity has become an urgent need for tumor immunotherapy. Problem solved.
  • the present invention provides a humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 and HCDR2, respectively, as follows
  • HCDR3 sequence having an amino acid sequence of at least 80% identity the light chain variable region comprises an amino acid sequence having at least 80% identity with the following LCDR1, LCDR2 and LCDR3 sequences, respectively:
  • amino acid sequence of HCDR1 is SYWIN
  • amino acid sequence of HCDR2 is RIAPGSGTTYYNEMFTG;
  • amino acid sequence of HCDR3 is GDYFDY
  • amino acid sequence of LCDR1 is SAKSVSYIH;
  • the amino acid sequence of LCDR2 is DTSTLAS
  • the amino acid sequence of LCDR3 is QQRTTYPLT.
  • the heavy chain variable region comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the HCDR1, HCDR2, and HCDR3 sequences described above, respectively.
  • the light chain variable region comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the following LCDR1, LCDR2, and LCDR3 sequences, respectively.
  • the present invention provides a humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising the following HCDR1, HCDR2 And HCDR3 sequence, the light chain variable region comprises the amino acid sequence shown in the following LCDR1, LCDR2 and LCDR3 sequences:
  • amino acid sequence of HCDR1 is SYWIN
  • amino acid sequence of HCDR2 is RIAPGSGTTYYNEMFTG;
  • amino acid sequence of HCDR3 is GDYFDY
  • amino acid sequence of LCDR1 is SAKSVSYIH;
  • the amino acid sequence of LCDR2 is DTSTLAS
  • the amino acid sequence of LCDR3 is QQRTTYPLT.
  • the present invention provides a humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, comprising a heavy chain variable region and a light chain variable region, said heavy chain variable regions being contained in the following HCDR1, HCDR2, and HCDR3 sequences, respectively.
  • the amino acid sequence of 1, 2, or 3 amino acid residues is replaced, inserted, or deleted, and the light chain variable region comprises 1, 2, or 3 in the LCDR1, LCDR2, and LCDR3 sequences, respectively.
  • amino acid sequence of HCDR1 is SYWIN
  • amino acid sequence of HCDR2 is RIAPGSGTTYYNEMFTG;
  • amino acid sequence of HCDR3 is GDYFDY
  • amino acid sequence of LCDR1 is SAKSVSYIH;
  • the amino acid sequence of LCDR2 is DTSTLAS
  • the amino acid sequence of LCDR3 is QQRTTYPLT.
  • amino acid sequence of the variable region of the heavy chain is selected from the group consisting of: 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.
  • amino acid sequence of the light chain variable region is selected from the group consisting of: 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 or SEQ ID NO: 24.
  • the heavy chain variable region is SEQ ID NO: 9 and the light chain variable region is SEQ ID NO: 17;
  • the heavy chain variable region is SEQ ID NO: 10 and the light chain variable region is SEQ ID NO: 18;
  • the heavy chain variable region is SEQ ID NO: 11 and the light chain variable region is SEQ ID NO: 19;
  • the heavy chain variable region is SEQ ID NO: 12 and the light chain variable region is SEQ ID NO: 20;
  • the heavy chain variable region is SEQ ID NO: 13 and the light chain variable region is SEQ ID NO: 21;
  • the heavy chain variable region is SEQ ID NO: 14 and the light chain variable region is SEQ ID NO: 22;
  • the heavy chain variable region is SEQ ID NO: 15 and the light chain variable region is SEQ ID NO: 23; or
  • the heavy chain variable region is SEQ ID NO: 16 and the light chain variable region is SEQ ID NO: 24.
  • the heavy chain variable region is SEQ ID NO: 10 and the light chain variable region is SEQ ID NO: 18;
  • the heavy chain variable region is SEQ ID NO: 11 and the light chain variable region is SEQ ID NO: 19;
  • the heavy chain variable region is SEQ ID NO: 12 and the light chain variable region is SEQ ID NO: 20; or
  • the heavy chain variable region is SEQ ID NO: 14 and the light chain variable region is SEQ ID NO: 22.
  • the heavy chain variable region is SEQ ID NO: 11 and the light chain variable region is SEQ ID NO: 19; or
  • the heavy chain variable region is SEQ ID NO: 14 and the light chain variable region is SEQ ID NO: 22.
  • the humanized anti-human CTLA4 monoclonal antibody or functional fragment thereof of the present invention comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 1, and a light chain having as shown in SEQ ID NO: 2 The amino acid sequence shown.
  • the dissociation constant KD between the humanized anti-human CTLA4 monoclonal antibody or functional fragment thereof of the present invention and CLTA4 is less than 0.02 nM.
  • the humanized anti-human CTLA4 monoclonal antibody or functional fragment thereof of the present invention specifically releases the negative immunoregulation of CTLA4 and activates T cells to secrete cytokines.
  • the present invention provides an isolated polynucleotide encoding the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof.
  • the polynucleotide of the present invention comprises a heavy chain coding sequence encoding a heavy chain variable region of the humanized anti-human CTLA4 monoclonal antibody, and the humanized anti-human CTLA4 monoclonal antibody The light chain coding sequence of the light chain variable region.
  • the 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 use of the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, the polynucleotide, the expression vector, or the host cell in the manufacture of a medicament for antitumor .
  • the present invention provides the use of the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, the polynucleotide, the expression vector, or the host cell for treating tumors.
  • the tumor is selected from multiple myeloma, non-small cell lung cancer, colorectal cancer, renal cell cancer, prostate cancer, breast cancer, and ovarian cancer.
  • the present invention provides the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, the polynucleotide, the expression vector, or the host cell for treating a tumor.
  • the tumor is selected from multiple myeloma, non-small cell lung cancer, colorectal cancer, renal cell cancer, prostate cancer, breast cancer, and ovarian cancer.
  • the present invention provides an antitumor pharmaceutical composition
  • an antitumor pharmaceutical composition comprising an effective amount of the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, and a pharmaceutically acceptable carrier.
  • the present invention provides a method for preparing the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, comprising transfecting competent cells with the expression vector as described above, and culturing the cells.
  • the present invention provides a method for preparing the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, comprising:
  • the humanized anti-human CTLA4 monoclonal antibody provided by the present invention has high affinity and specificity for CTLA4, and can stimulate T cells to secrete cytokines, such as specifically canceling the negative immune regulation of CTLA4 and activating T cells to secrete cytokines. Therefore, the functional humanized anti-human CTLA4 monoclonal antibody provided by the present invention can activate T cells by blocking the CTLA4 signal pathway, thereby achieving the purpose of tumor immunotherapy.
  • FIG. 3 Thermal stability analysis of purified monoclonal antibodies, specifically SEC-HPLC analysis of the thermal stability of humanized anti-human CTLA4 monoclonal antibodies (treated at 40 ° C for 2 weeks): chimeric antibodies-14 days-SEC-HPLC (Figure 3A) ), AH01674-14 days-SEC-HPLC ( Figure 3B), and AH01695-14 days-SEC-HPLC ( Figure 3C);
  • FIG 4 Thermal stability analysis of purified monoclonal antibody, specifically humanized anti-human CTLA4 monoclonal antibody thermal stability ELISA analysis (treatment at 40 ° C for 2 weeks): chimeric antibody (also referred to herein as " "Chimeric IgG", the two can be used interchangeably) ( Figure 4A), AH01674 ( Figure 4B) and AH01695 ( Figure 4C);
  • Figure 6 Analysis of the drug resistance of purified monoclonal antibodies, specifically the affinity detection after oxidative stress test of humanized anti-human CTLA4 monoclonal antibodies: chimeric antibody (Figure 6A), chimeric antibody-AAPH ( Figure 6B), AH01674 ( Figure 6C), AH01674-AAPH ( Figure 6D), AH01695 ( Figure 6E), and AH01695-AAPH ( Figure 6F);
  • Figure 7 Analysis of the drugability of purified monoclonal antibodies, specifically SEC detection after oxidative stress test of humanized anti-human CTLA4 monoclonal antibody: chimeric antibody (Figure 7A), chimeric antibody-AAPH ( Figure 7B), AH01674 ( Figure 7C), AH01674-AAPH ( Figure 7D), AH01695 ( Figure 7E), and AH01695-AAPH ( Figure 7F).
  • antibody refers to an immunoglobulin molecule, which is generally a tetramer consisting of two identical heavy chains and two identical light chains connected to each other via disulfide bonds. According to the conservative differences in amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxy terminus. In the variable regions of the heavy and light chains, there are three local regions, each of which has a higher degree of variation in the amino acid composition and arrangement order. It is a key location for the binding of antibodies to antigens, so it is also called the complementarity determining region (CDR).
  • CDR complementarity determining region
  • the three heavy chain complementarity determining regions are called HCDR1, HCDR2, and HCDR3, and the three light chain complementarity determining regions are called LCDR1, LCDR2, and LCDR3, respectively.
  • the variable regions of a heavy chain and a light chain interact to form an antigen binding site (Fv).
  • Antibodies can be classified into different classes based on the amino acid sequence of the constant region of their heavy chains. There are five main types of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the subunit structures and three-dimensional conformations of different classes of immunoglobulins are known in the art.
  • the invention is intended to include any of the aforementioned classes or subclasses of antibodies.
  • antibody as used herein is also intended to encompass its digested fragments or functional variants, for example, antibody fragments capable of binding CTLA4 or portions thereof, including but not limited to Fab (e.g., antibodies obtained by digestion with papain), F (ab ') 2 (for example, obtained by pepsin digestion), Fv, or scFv (for example, obtained by molecular biology techniques).
  • Fab e.g., antibodies obtained by digestion with papain
  • F (ab ') 2 for example, obtained by pepsin digestion
  • Fv for example, obtained by molecular biology techniques
  • the term "monoclonal antibody” as used herein refers to a homogeneous antibody that is directed against a particular epitope. In contrast to ordinary polyclonal antibody preparations that typically include different antibodies directed 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 an antibody and is not to be construed as an antibody that needs to be produced 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 exists in a naturally occurring state in a non-natural world, including a polynucleotide isolated from nature (including an organism) by biological techniques, and also includes a synthetic polynucleotide .
  • An isolated polynucleotide may be genomic DNA, cDNA, mRNA, or other synthetic RNA, or a combination thereof. Numerous nucleotide sequences for encoding the heavy chain variable region and the light chain variable region of a humanized anti-CTLA4 monoclonal antibody are provided herein.
  • amino acid sequences of the variable region of the light chain and the variable region of the light chain are designed to be not identical to the nucleotide sequence provided above, but both encode the same amino acid sequence. These modified nucleotide sequences are also included within the scope of the present invention.
  • modification refers to a primary amino acid sequence change relative to the original amino acid sequence, wherein the change results from a change in the sequence involving the amino acid residue / position.
  • typical modifications include substitution (e.g., conservative or non-conservative substitution) of a residue (at that position) with another amino acid, insertion of one or more (typically less At 5 or 3) amino acids, and the residues / positions are deleted.
  • Amino acid substitution or a change thereof, refers to the replacement of an existing amino acid residue with a different amino acid residue in a predetermined (initial) amino acid sequence.
  • the modification generally preferably results in a change in at least one physiological and biochemical activity of the variant polypeptide relative to the polypeptide containing the original (or "wild-type") amino acid sequence.
  • the altered physiological and biochemical activity may be the binding affinity, binding capacity, and / or binding effect on the target molecule.
  • Percent (%) amino acid sequence identity with respect to a peptide or polypeptide sequence is defined as a sequence that is compared and gaps are introduced where necessary to obtain the maximum percent sequence identity, without any conservative substitutions being considered as part of the sequence identity.
  • vector refers to any molecule (e.g., a nucleic acid, a plasmid, or a virus) 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 (a nucleotide sequence to be expressed) in a host cell, and generally 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 is capable of being 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 is the same in morphology or genetic composition as the original parent cell, as long as the selected gene of interest is present in the progeny.
  • Commonly used host cells include bacteria, yeast, mammalian cells, and the like.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell, and this technique can be used to introduce one or more exogenous DNA portions into a suitable host cell.
  • Cells can be induced by physicochemical methods (for example, by calcium chloride treatment) to bring them into a physiological state that is optimal for uptake and accommodation of foreign DNA, that is, "competent”.
  • the term "effective amount” as used herein refers to an amount that can produce a function or activity in a human and / or animal and can be accepted by a human and / or animal.
  • “Pharmaceutically acceptable carrier” refers to a carrier for administration, including various excipients, diluents, buffers, etc., which are suitable for human and / or animal administration without excessive adverse side effects, and Suitable for maintaining the vitality of the drug or active agent located therein.
  • the IMGT human gene (F + ORF + in-frameP) database was selected.
  • the human Germline antibody sequence with the highest homology was selected as the humanized receiving vector based on the alignment.
  • the three heavy chain complementarity determining regions HCDR1 and HCDR2 in the mouse antibody were selected.
  • HCDR3, the three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 were transferred to the corresponding positions, and the post-translational modification sites (PTM) were analyzed, as shown in Table 1. Sequence analysis revealed that the two sites, W33 and M63, are hot spots for post-translational oxidative modification (see, for example, SEQ ID NO: 3-4).
  • Table 2 Monoclonal antibody humanized back mutation screening, antibody affinity ranking with highest affinity
  • the antibody sequences of 3 CBM and 5 5BM exhibiting the highest affinity are as follows (see, for example, SEQ ID NO: 9-24):
  • the selected antibody VH and VL sequences were codon-optimized, linked to the 5 'end secretion signal peptide, and linked to the human antibody IgG1 heavy chain and kappa light chain constant region sequences. They were cloned into pTT5 expression vectors and prepared for use in Human antibody DNA sequence expressed and secreted in mammalian cells.
  • the plasmid was co-transfected with HEK293-6E suspension culture cells 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. Twenty-four hours after transfection, 0.5% Trypton N-1 was added. Five days later, the cell culture supernatant was collected, and the antibodies were purified using protein-A agarose gel to quantify and identify the purity (Table 3).
  • the chip surface was equilibrated with HBS-EP buffer at a flow rate of 10 ⁇ l / min for 5 minutes, and then a “1: 1 mixture of“ NHS + EDC ”was injected at a flow rate of 10 ⁇ l / min for 7 minutes to activate the chip.
  • the capture antibody Goat anti-mouse IgG
  • ethanolamine was injected at a flow rate of 10 ⁇ l / min for 7 minutes for surface blocking.
  • HBS-EP buffer was used as a sample to perform three pre-cycles to balance the chip to stabilize the baseline, and the antibody diluted in HBS-EP buffer was injected at a flow rate of 10 ⁇ l / min for 0 to 5 minutes (by adjusting the capture time to control antibody and antigen binding The signal is at ⁇ 100RU) and the buffer is equilibrated for 1 minute.
  • Inject low-level antigen 0.33nM CTLA4-Fc at a flow rate of 30 ⁇ l / min for 5 minutes.
  • the antigen binds to the antibody, and then dissociate by injecting buffer at a flow rate of 30 ⁇ l / min for 15 minutes.
  • Inject 50 mM HCl four times at a flow rate of 100 ⁇ l / min. Regeneration takes place in 10 seconds, and one cycle ends.
  • the anti-CTLA-4 antibody functional assay was performed using an anti-CTLA-4 blocking assay kit developed by Promega.
  • the kit contains two cell lines, CD80 / CD86aAPC / Raji-stimulated cells and functional cells expressing CTLA-4. Without the addition of anti-CTLA-4 antibodies, Raji cells bind to CTLA-4 of functional cells to inhibit immune signal transmission, and will not activate NF ⁇ B to bind to downstream promoter sequences to achieve the expression of reporter gene luc2.
  • the anti-human CTLA-4 antibody is added, the CTLA-4 protein is blocked, the immune response stimulated by Raji cells will be reactivated, the fluorescent enzymes in functional cells will be expressed and react with the substrate, and the fluorescent signal generated can be detected collect.
  • Raji cells expressing CD80 / CD86 and functional cells expressing CTLA-4 were cultured and counted.
  • Raji cells were plated at 50,000 cells / well into 96-well plates.
  • Sample antibodies and positive and negative control antibodies were added to Raji cells in a gradient, and then functional cells were added at 50,000 / well.
  • AH01674, AH01695 and chimeric antibodies were expressed in a 200ml system to obtain more than 5mg, purified antibody samples with endotoxin controlled at 3EU / mg level for subsequent experiments.
  • the antibody samples were processed separately at 40 ° C, then the pellet was removed by centrifugation, and the amount of residual antibody was evaluated by ELISA. (Processed at 40 °C for 7 days and 14 days respectively, and each test is performed with untreated samples stored at -80 °C as a control)
  • each antibody sample is more than 5mg / ml, and treated with five gradients at room temperature, 30, 40, 50, and 60 ° C for 20min, and then centrifuged to remove the precipitate, and then the amount of residual antibody is evaluated by ELISA. For each test, an untreated sample stored at -80 ° C was used as a control.
  • a / B treated samples were sent for SEC-HPLC.
  • Oxidative pressure test The antibody molecules were transferred to a 20 mM ammonium acetate solution (pH 5.0), and AAPH (2,2'-azobis (2-amidinopropane)) (50: 1) was added to the light-proof treatment at 40C for 24 hours.
  • the processed antibody samples were determined by mass spectrometry to determine the proportion of chemical changes in the corresponding amino acid molecules, Biocore detection to determine the change in affinity, and SEC-HPLC to determine the change in the degree of polymerization of the antibody molecules.
  • the deamidation pressure experiment mass spectrometry detected no deamidation in the AH01674 and AH01695CDR sequences, which was consistent with the conclusion of the sequence analysis.

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Abstract

本发明属于肿瘤免疫疗法和分子免疫学领域，提供了一种人源化抗人CTLA4单克隆抗体、及其制备方法和用途。本发明的人源化抗人CTLA4单克隆抗体能够特异地解除CTLA4的免疫负调节，激活T细胞分泌细胞因子，进而用于肿瘤治疗。

Description

人源化抗人CTLA4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CTLA4单克隆抗体。本发明还涉及该人源化抗人CTLA4单克隆抗体的制备方法及其的用途。
背景技术
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(Janeway et al.,Immunology:The Immune System in Health and Disease.New York:Garland Science,2005)。免疫器官、组织、细胞间相互协作、制衡，在众多免疫检查点蛋白和细胞因子的协调下，达到保护机体免受外来侵染和维持体内平衡的作用。针对外来病原体的获得性免疫系统由体液免疫(由B细胞介导)和细胞免疫(由T细胞介导)组成。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)呈递的抗原来引起的。这种激活还需要APC的共刺激。APC上的两种同源B7家族成员B7-1(也称为B7、B7.1或CD80)和B7-2(也称为B7.2或CD86)，都可在与T细胞上CD28抗原结合时递送共刺激信号，导致T细胞激活。CTLA4和CD28同是含有单个细胞外Ig结构域的Ig超家族的成员，均可结合B7蛋白，但调控效果相反。CTLA4比CD28与B7蛋白的结合有更高的亲和力，竞争形成更稳定相互作用，使T细胞缺乏第二级刺激信号，变得无能(anergic)；同时可以在T细胞激活后，诱导T细胞凋亡。从而对免疫系统进行负调节，维持体内T细胞稳态。因此，用单克隆抗体阻断CTLA4传导的负调节信号可以提供受益于免疫刺激的人类疾病的新疗法，比如癌症和传染性疾病的免疫治疗。目前CTLA4单克隆抗体已在不同的临床试验阶段用于治疗多种人类癌症，包括黑色素瘤、前列腺癌、膀胱癌、结肠直肠癌、恶性间皮瘤、胃肠道癌、肝癌、非小细胞肺癌(Grosso et al.,Cancer Immunology 13:5,2013)。现已有Ipilimumab(Keler et al.,J Immunol171:6251-6259(2003))和Tremelimumab(Ribas et al.,Oncologist12:873-883(2005))。目前已经成功上市的一种CTLA4单克隆抗体，Ipilimumab(商品名Yervoy)标志着肿瘤免疫疗法在临床阶段切实可行。而且，随着临床前实验验证了针对不同免疫调节因子的单克隆抗体在协同治疗癌症方面的能力，CTLA4单克隆抗体已与不同免疫抑制分子的单克隆抗体或者小分子化合物形成组合疗法，正在针对不同癌症进行临床实验。但目前上市的只有一种CTLA4单克隆抗体，而且CTLA4单克隆抗体也存在不同程度的副反应，包括可在某些患者中诱导免疫原性，过度抑制CTLA4信号可能引起自身免疫病，以及不同CTLA4单克隆抗体具有不同程度的可开发性。同时，为避免不同患者人群疗效差异，开发新的具有更高的亲和力、特异性、功能性以及多样性的能够阻断CTLA4与B7蛋白结合的人源化功能性抗体，成为肿瘤免疫治疗的亟待解决的课题。
发明内容
在一方面，本发明提供了人源化抗人CTLA4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SYWIN；
HCDR2的氨基酸序列为RIAPGSGTTYYNEMFTG；
HCDR3的氨基酸序列为GDYFDY；
LCDR1的氨基酸序列为SASKSVSYIH；
LCDR2的氨基酸序列为DTSTLAS；
LCDR3的氨基酸序列为QQRTTYPLT。
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70％、至少80％、至少85％、至少90％或至少95％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下LCDR1、LCDR2和LCDR3序列具有至少70％、至少80％、至少85％、至少90％或至少95％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CTLA4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SYWIN；
HCDR2的氨基酸序列为RIAPGSGTTYYNEMFTG；
HCDR3的氨基酸序列为GDYFDY；
LCDR1的氨基酸序列为SASKSVSYIH；
LCDR2的氨基酸序列为DTSTLAS；
LCDR3的氨基酸序列为QQRTTYPLT。
本发明提供了人源化抗人CTLA4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SYWIN；
HCDR2的氨基酸序列为RIAPGSGTTYYNEMFTG；
HCDR3的氨基酸序列为GDYFDY；
LCDR1的氨基酸序列为SASKSVSYIH；
LCDR2的氨基酸序列为DTSTLAS；
LCDR3的氨基酸序列为QQRTTYPLT。
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: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或SEQ ID NO:24。
在一个实施方案中，其中，
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:17；
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:18；
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:19；
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:20；
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:21；
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:22；
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:23；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:24。
在一个实施方案中，其中，
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:18；
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:19；
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:20；或
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:22。
在一个实施方案中，其中，
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:19；或
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:22。
在一个实施方案中，本发明的人源化抗人CTLA4单克隆抗体或其功能片段，其包含重链具有如SEQ ID NO:1所示的氨基酸序列，和轻链具有如SEQ ID NO:2所示的氨基酸序列。
在一个实施方案中，本发明的人源化抗人CTLA4单克隆抗体或其功能片段与CLTA4之间的解离常数KD小于0.02nM。
在一个实施方案中，本发明的人源化抗人CTLA4单克隆抗体或其功能片段特异性地解除CTLA4的免疫负调节，激活T细胞分泌细胞因子。
在另一方面，本发明提供了分离的多核苷酸，其编码所述人源化抗人CTLA4单克隆抗体或其功能片段。
在一个实施方案中，本发明的多核苷酸包含编码所述人源化抗人CTLA4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CTLA4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，其包含所述多核苷酸。
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CTLA4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CTLA4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。
在一个实施方案中，所述肿瘤选自多发性骨髓瘤、非小细胞肺癌、结肠直肠癌、肾细胞癌、前列腺癌、乳腺癌和卵巢癌。
在另一方面，本发明提供了所述人源化抗人CTLA4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。
在一个实施方案中，所述肿瘤选自多发性骨髓瘤、非小细胞肺癌、结肠直肠癌、肾细胞癌、前列腺癌、乳腺癌和卵巢癌。
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CTLA4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，本发明提供了制备所述人源化抗人CTLA4单克隆抗体或其功能片段的方法，包括以如所述表达载体转染感受态细胞，并对所述细胞进行培养。
在另一方面，本发明提供了一种制备所述人源化抗人CTLA4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CTLA4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CTLA4单克隆抗体或其功能片段。
本发明提供的人源化抗人CTLA4单克隆抗体对CTLA4具有高亲和性、高特异性，能够刺激T细胞分泌细胞因子，例如特异地解除CTLA4的免疫负调节，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CTLA4单克隆抗体，可通过阻断CTLA4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CTLA4单克隆抗体亲和力测定；
图2.纯化单克隆抗体能够解除CTLA4的免疫负调节；
图3.纯化单克隆抗体热稳定性分析，具体为人源化抗人CTLA4单克隆抗体热稳定性SEC-HPLC分析(40℃处理2周)：嵌合抗体-14天-SEC-HPLC(图3A)、AH01674-14天-SEC-HPLC(图3B)和AH01695-14天-SEC-HPLC(图3C)；
图4.纯化单克隆抗体热稳定性分析，具体为人源化抗人CTLA4单克隆抗体热稳定性ELISA分析(40℃处理2周)：嵌合抗体(在本文以及附图中也可称为“嵌合IgG”，二者可互换使用)(图4A)、AH01674(图4B)和AH01695(图4C)；
图5.纯化单克隆抗体热稳定性分析，具体为人源化抗人CTLA4单克隆抗体热稳定性ELISA分析(温度梯度)：AH01674(图5A)和AH01695(图5B)；
图6.纯化单克隆抗体成药性检测分析，具体为人源化抗人CTLA4单克隆抗体氧化加压试验后亲和力检测：嵌合抗体(图6A)、嵌合抗体-AAPH(图6B)、AH01674(图6C)、AH01674-AAPH(图6D)、AH01695(图6E)和AH01695-AAPH(图6F)；
图7.纯化单克隆抗体成药性检测分析，具体为人源化抗人CTLA4单克隆抗体氧化加压试验后SEC检测：嵌合抗体(图7A)、嵌合抗体-AAPH(图7B)、AH01674(图7C)、AH01674-AAPH(图7D)、AH01695(图7E)和AH01695-AAPH(图7F)。
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CTLA4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CTLA4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CTLA4抗体人源化
1)鼠源抗人CTLA4抗体42B11G12D3抗体序列(CDR区域采用下划线显示)(参见例如SEQ ID NO:1-2)
Figure PCTCN2019097643-appb-000001
2)抗人CTLA4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W33和M63两个位点是翻译后氧化修饰的热点(参见例如SEQ ID NO:3-4)。
SEQ ID NO:3,42B11G12D3-VH-GRAFTED
QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYWINWVRQAPGQGLEWMG RIAPGSGTTYYNEMFTG
RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAI GDYFDYWGQGTMVTVSS
Figure PCTCN2019097643-appb-000002
表1：PTM风险分析
Figure PCTCN2019097643-appb-000003
3)设计噬菌体文库CBM(灰色底纹)，5BM(加粗)，构建抗人CTLA4抗体42B11G12D3 VH-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:5-8)。
42B11G12D3_CBM
Figure PCTCN2019097643-appb-000004
42B11G12D3_5BM
Figure PCTCN2019097643-appb-000005
Figure PCTCN2019097643-appb-000006
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CTLA4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 PCTCN2019097643-appb-000007
呈现最高亲和力的3条CBM和5条5BM的抗体序列如下(参见例如SEQ ID NO:9-24)：
Figure PCTCN2019097643-appb-000008
Figure PCTCN2019097643-appb-000009
Figure PCTCN2019097643-appb-000010
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，抗体使用protein-A琼脂糖凝胶纯化，定量并鉴定纯度(表3)。
表3：人源化抗体重组生产
Figure PCTCN2019097643-appb-000011
Figure PCTCN2019097643-appb-000012
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7分钟来活化芯片，将稀释在10mM醋酸钠缓冲液中的捕获抗体(Goat anti-mouse IgG)以10μl/min流速注射约7分钟进行偶联，最后以10μl/min的流速注射乙醇胺7分钟进行表面封闭。
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，10μl/min流速注射稀释在HBS-EP缓冲液中的抗体0～5分钟(通过调整捕获时间来控制抗体和抗原结合信号在～100RU)，缓冲液平衡1分钟。以30μl/min流速注射低浓度抗原0.33nM CTLA4-Fc 5分钟，抗原与抗体发生结合，之后30μl/min流速注射缓冲液15分钟进行解离，100μl/min流速注射50mM HCl共四次，每次10秒进行再生，一次循环结束。
改变抗原浓度进行下一个梯度浓度的循环测定直到所有梯度浓度(1.25nM、2.5nM、5nM、10nM、20nM、40nM)及重复浓度(如5nM CTLA4-Fc)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CTLA4-Fc重组蛋白的亲和力。
如图1和表4所示，特异性针对人CTLA4-Fc的单克隆抗体(AH01672、AH01674、AH01679、AH01686、AH01695、AH01696、AH01704)对CTLA4-Fc的亲和力由Biacore测得均达到sub-nM级至pM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
Figure PCTCN2019097643-appb-000013
实施例4：人源化抗人CTLA4单克隆抗体的功能性验证
抗人CTLA-4抗体的功能检测分析使用的是Promega公司开发的抗CTLA-4阻断分析试剂盒。试剂盒包含两个细胞系，CD80/CD86aAPC/Raji刺激细胞和表达CTLA-4的功能细胞。在未加入抗CTLA-4抗体的情况下，Raji细胞与功能细胞的CTLA-4结合从而抑制免疫信号传递，不会激活NFκB去结合下游启动子序列实现报告基因luc2的表达。当抗人CTLA-4抗体加入后，CTLA-4蛋白被阻断，Raji细胞激发的免疫反应会被重新激活，功能细胞内的荧光酶会表达并与底物反应，产生的荧光信号可以被检测收集。
试验中，表达CD80/CD86的Raji细胞和表达CTLA-4的功能细胞被培养计数。Raji细胞按照50000细胞/孔铺备到96孔板中。样品抗体和阳性、阴性对照抗体按照梯度加入到 Raji细胞中，然后按照50000/孔加入功能细胞。在37℃5％CO 2环境中混合孵育6小时。加入荧光反应底物并室温避光反应10分钟，然后检测荧光强度。如果抗体有CTLA-4阻断效果，荧光强度会与增加的抗体浓度呈现反曲线。
实验结果显示，人源化抗人CTLA4单克隆抗体(AH01672、AH01674、AH01679、AH01695)能够特异性地解除CTLA4的免疫负调节，激活T细胞分泌细胞因子，对应EC50分别为7.310μg/ml、1.115μg/ml、17.10μg/ml、5.464μg/ml(图2和表5)。选择EC50较低的AH01674(1.115μg/ml)和AH01695(5.464μg/ml)两株人源化抗体往前推进做成药性及热稳定性的研究。
表5
抗体名称 Yervoy 嵌合抗体 AH1672 AH1674 AH1679 AH1695
EC50(μg/ml) 0.9334 3.960 7.310 1.115 17.10 5.464
实施例5：人源化抗人CTLA4单克隆抗体的成药性评价
AH01674和AH01695以及嵌合抗体三个抗体按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
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评估残留的抗体数量。(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)
B.各抗体样品浓度>5mg/ml，分别在室温、30、40、50、60℃五个梯度处理20min，然后离心去除沉淀，再用ELISA评估残留的抗体量。每次检测同时用存放-80度未处理的样品做对照。
A/B处理样品同时送测SEC-HPLC。
结果如图3-5所示，经过热处理的AH01674、AH01695、嵌合抗体三种抗体样品没有出现大量聚集，同时呈现出稳定的抗体与抗原的结合能力。
2.成药性实验
抗人CTLA4单克隆抗体CDR区域序列分析显示(表1)，VH区域预测存在W33、M63位点氧化修饰的热点。人源化后的抗人CTLA4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，Biocore检测判断亲和力的变化，SEC-HPLC判断抗体分子的聚合度变化。
结果显示(表6-8和图6-7)，质谱检测覆盖率都达到了95％左右，得到可信的结果。抗体AH01674对照样品中，检测到M@63有9.54％的氧化，在AAPH-24处理样品中，M@63有75.62％氧化。抗体AH01695对照样品中，检测到M@63有8.91％的氧化，M@250未检 测到氧化，在AAPH-24处理样品中，M@63有70.90％氧化，M@250有26.14％的氧化。M250位于抗体分子恒定区。所以AH01674和AH01695CDR序列中M63位点存在发生氧化修饰的可能性。
亲和力验证、SEC验证结果显示，AH01674和AH01695抗体分子氧化加压处理的没有影响抗体与抗原的亲和力，也没有影响抗体分子的均一性。
同时，去酰胺化加压实验质谱分析检测AH01674和AH01695CDR序列中没有发生脱酰胺修饰，这与序列分析结论一致。
表6.成药性检测分析:人源化抗人CTLA4单克隆抗体氧化加压试验后质谱检测
Figure PCTCN2019097643-appb-000014
表7.成药性检测分析:人源化抗人CTLA4单克隆抗体氧化加压试验后亲和力检测
Figure PCTCN2019097643-appb-000015
表8.成药性检测分析:人源化抗人CTLA4单克隆抗体脱酰胺加压试验后质谱检测
样品名称 肽段 修饰 修饰百分比
AH01674-对照 T41-43 脱酰胺@2(136) 3.245
AH01674-对照 T41-43    
AH01674-ph9-48h T42-43 脱酰胺@2(137) 2.84
AH01674-ph9-48h T42-43    
AH01674-对照 T42-43 脱酰胺@2(137) 10.597
AH01674-对照 T42-43    
AH01674-ph9-48h T42-43 脱酰胺@2(137) 11.833
AH01674-ph9-48h T42-43    
AH01674-对照 T40 脱酰胺@1(157) 3.83
AH01674-对照 T40    
AH01674-对照 T40-41 脱酰胺@1(157) 4.994
AH01674-对照 T40-41    
AH01674-ph9-48h T40-41 脱酰胺@1(157) 5.28
AH01674-ph9-48h T40-41    
AH01674-对照 T63-65 脱酰胺@11(284) 4.319
AH01674-对照 T63-65    
AH01674-ph9-48h T63-65 脱酰胺@11(284) 3.311
AH01674-ph9-48h T63-65    
AH01674-对照 T89 脱酰胺@1(359) 2.695
AH01674-对照 T89 脱酰胺@1(359)  
AH01674-对照 T89    
AH01674-ph9-48h T89 脱酰胺@1(359) 0.575
AH01674-ph9-48h T89    
AH01674-对照 T108-109 脲甲基化@2(423),脱酰胺@11(432) 4.156
AH01674-对照 T108-109 脲甲基化@2(423),脱酰胺@11(432)  
AH01674-对照 T108-109 脲甲基化@2(423)  
AH01674-ph9-48h T108-109 脲甲基化@2(423),脱酰胺@11(432) 4.176
AH01674-ph9-48h T108-109 脲甲基化@2(423),脱酰胺@11(432)  
AH01674-ph9-48h T108-109 脲甲基化@2(423)  
AH01674-对照 T71-74 脱酰胺@9(313) 2.1
AH01674-对照 T71-74    
AH01674-对照 T71-75 脱酰胺@9(313) 11.767
AH01674-对照 T71-75    
AH01674-ph9-48h T71-74 脱酰胺@9(313) 2.678
AH01674-ph9-48h T71-74    
AH01674-ph9-48h T71-75 脱酰胺@9(313) 9.999
AH01674-ph9-48h T71-75    
AH01674-对照 T92-96    
AH01674-ph9-48h T92-96 脱酰胺@19(387) 14.689
AH01674-ph9-48h T92-96    
AH01695-对照 T41-43 脱酰胺@2(136) 2.934
AH01695-对照 T41-43    
AH01695-ph9-48h T41-43 脱酰胺@2(136) 3.73
AH01695-ph9-48h T41-43    
AH01695-对照 T42-43 脱酰胺@2(137) 12.049
AH01695-对照 T42-43    
AH01695-ph9-48h T42-43 脱酰胺@2(137) 11.621
AH01695-ph9-48h T42-43    
AH01695-对照 T42-43 脱酰胺@1(157) 5.666
AH01695-对照 T42-43    
AH01695-ph9-48h T42-43 脱酰胺@1(157) 5.315
AH01695-ph9-48h T42-43    
AH01695-对照 T65-67 脱酰胺@11(284) 2.378
AH01695-对照 T65-67    
AH01695-ph9-48h T65-67 脱酰胺@11(284) 2.323
AH01695-ph9-48h T65-67    
AH01695-ph9-48h T91 脱酰胺@1(359) 0.501
AH01695-ph9-48h T91    
AH01695-对照 T110-111 脲甲基化@2(423),脱酰胺@11(432) 3.608
AH01695-对照 T110-111 脲甲基化@2(423),脱酰胺@11(432) 3.608
AH01695-对照 T110-111 脲甲基化@2(423)  
AH01695-ph9-48h T110-111 脲甲基化@2(423),脱酰胺@11(432) 3.776
AH01695-ph9-48h T110-111 脲甲基化@2(423),脱酰胺@11(432) 3.776
AH01695-ph9-48h T110-111 脲甲基化@2(423)  
AH01695-对照 T73-76 脱酰胺@9(313) 2.476
AH01695-对照 T73-76    
AH01695-ph9-48h T73-76 脱酰胺@9(313) 1.904
AH01695-ph9-48h T73-76    
AH01695-对照 T92-96    
AH01695-ph9-48h T92-96 脱酰胺@19(387) 15.33
AH01695-ph9-48h T92-96    
嵌合-0h T38-39    
嵌合-0h T38-39 脱酰胺@2(137) 9.531
嵌合-ph9-48h T37-39    
嵌合-ph9-48h T37-39 脱酰胺@2(136) 3.735
嵌合-ph9-48h T38-39    
嵌合-ph9-48h T38-39 脱酰胺@1(136) 10.057
嵌合-0h T93    
嵌合-0h T93 脱酰胺@1(359) 2.211
嵌合-ph9-48h T93    
嵌合-ph9-48h T93 脱酰胺@1(359) 2.672
嵌合-ph9-48h T93 脱酰胺@1(359) 2.672
嵌合-0h T44    
嵌合-0h T44 脱酰胺@1(157) 4.112
嵌合-0h T44-45    
嵌合-0h T44-45 脱酰胺@1(157) 5.232
嵌合-ph9-48h T44    
嵌合-ph9-48h T44 脱酰胺@1(157) 4.158
嵌合-ph9-48h T44-45    
嵌合-ph9-48h T44-45 脱酰胺@1(157) 5.457
嵌合-0h T66-69    
嵌合-0h T66-69 脱酰胺@12(284) 2.719
嵌合-ph9-48h T66-69    
嵌合-ph9-48h T66-69 脱酰胺@12(284) 1.989
嵌合-ph9-48h T67-69    
嵌合-ph9-48h T67-69 脱酰胺@11(284) 3.146
嵌合-0h T112-113 脲甲基化@2(423)  
嵌合-0h T112-113 脲甲基化@2(423),脱酰胺@11(432) 3.936
嵌合-0h T112-113 脲甲基化@2(423),脱酰胺@11(432) 3.936
嵌合-ph9-48h T112-113 脲甲基化@2(423)  
嵌合-ph9-48h T112-113 脲甲基化@2(423),脱酰胺@11(432) 4.317
嵌合-ph9-48h T112-113 脲甲基化@2(423),脱酰胺@11(432) 4.317
嵌合-0h T75-78    
嵌合-0h T75-78 脱酰胺@9(313) 2.021
嵌合-0h T75-79    
嵌合-0h T75-79 脱酰胺@9(313) 9.676
嵌合-ph9-48h T75-79    
嵌合-ph9-48h T75-79 脱酰胺@9(313) 8.446
嵌合-0h T96-100    
嵌合-ph9-48h T96-100 脱酰胺@19(387) 12.52
嵌合-ph9-48h T96-100    

Claims (18)

  1. 人源化抗人CTLA4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SYWIN；
    HCDR2的氨基酸序列为RIAPGSGTTYYNEMFTG；
    HCDR3的氨基酸序列为GDYFDY；
    LCDR1的氨基酸序列为SASKSVSYIH；
    LCDR2的氨基酸序列为DTSTLAS；
    LCDR3的氨基酸序列为QQRTTYPLT。
  2. 如权利要求1所述的人源化抗人CTLA4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SYWIN；
    HCDR2的氨基酸序列为RIAPGSGTTYYNEMFTG；
    HCDR3的氨基酸序列为GDYFDY；
    LCDR1的氨基酸序列为SASKSVSYIH；
    LCDR2的氨基酸序列为DTSTLAS；
    LCDR3的氨基酸序列为QQRTTYPLT。
  3. 如权利要求2所述的人源化抗人CTLA4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: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CTLA4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或SEQ ID NO:24。
  5. 如权利要求2所述的人源化抗人CTLA4单克隆抗体或其功能片段，其中，
    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:17；
    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:18；
    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:19；
    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:20；
    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:21；
    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:22；
    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:23；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:24。
  6. 如权利要求1或2所述的人源化抗人CTLA4单克隆抗体或其功能片段，其与CLTA4之间的解离常数KD小于0.02nM。
  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CTLA4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CTLA4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CTLA4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CTLA4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. 如权利要求1-6中任一项所述的人源化抗人CTLA4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所述的宿主细胞用于治疗肿瘤的用途。
  13. 如权利要求11或12所述的用途，其中所述肿瘤选自多发性骨髓瘤、非小细胞肺癌、结肠直肠癌、肾细胞癌、前列腺癌、乳腺癌和卵巢癌。
  14. 如权利要求1-6中任一项所述的人源化抗人CTLA4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，其用于治疗肿瘤。
  15. 如权利要求14中所述的人源化抗人CTLA4单克隆抗体或其功能片段、多核苷酸、表达载体或宿主细胞，其中所述肿瘤选自多发性骨髓瘤、非小细胞肺癌、结肠直肠癌、肾细胞癌、前列腺癌、乳腺癌和卵巢癌。
  16. 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CTLA4单克隆抗体或其功能片段，和药学上可接受的表达载体。
  17. 制备如权利要求1-6任一项所述的人源化抗人CTLA4单克隆抗体或其功能片段的方法，包括以如权利要求9所述的表达载体转染感受态细胞，并对所述感受态细胞进行培养。
  18. 制备如权利要求1-6任一项所述的人源化抗人CTLA4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CTLA4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CTLA4单克隆抗体或其功能片段。
PCT/CN2019/097643 2018-07-25 2019-07-25 人源化抗人ctla4单克隆抗体及其制备方法和用途 Ceased WO2020020275A1 (zh)

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US17/263,148 US12037396B2 (en) 2018-07-25 2019-07-25 Humanized anti-human CTLA4 monoclonal antibody, preparation method therefor and use thereof
CN201980049141.3A CN112513084B (zh) 2018-07-25 2019-07-25 人源化抗人ctla4单克隆抗体及其制备方法和用途
CA3107524A CA3107524C (en) 2018-07-25 2019-07-25 Humanized Monocolonial Antibody Against CTLA4: Preparation Method and Application
CN202210894065.XA CN115850475B (zh) 2018-07-25 2019-07-25 人源化抗人ctla4单克隆抗体及其制备方法和用途

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