Humanized anti-human CTLA4 monoclonal antibody, its preparation method and uses

Abstract

This invention relates to a humanized anti-human CTLA4 monoclonal antibody. The invention also relates to a method for preparing the humanized anti-human CTLA4 monoclonal antibody and its uses.

Description

Technical Field

[0001] This invention belongs to the fields of tumor immunotherapy and molecular immunology, and specifically relates to a humanized anti-human CTLA4 monoclonal antibody. This invention also relates to a method for preparing this humanized anti-human CTLA4 monoclonal antibody and its uses. Background Technology

[0002] The vertebrate immune system is a functional system composed of multiple organs, tissues, cells, and molecules, and is the most effective mechanism for defending against the invasion of foreign substances (Janeway et al., Immunology: The Immune System in Health and Disease. New York: Garland Science, 2005). Immune organs, tissues, and cells cooperate and maintain balance with each other, coordinating numerous immune checkpoint proteins and cytokines to protect the body from external infections and maintain homeostasis. The acquired immune system against foreign pathogens consists of humoral immunity (mediated by B cells) and cellular immunity (mediated by T cells). Cellular immunity is triggered by the recognition of antigens presented by the major histocompatibility complex (MHC) on antigen-presenting cells (APCs) by T cell receptors (TCRs). This activation also requires co-stimulation by APCs. Two homologous B7 family members on the APC, B7-1 (also known as B7, B7.1, or CD80) and B7-2 (also known as B7.2 or CD86), can both deliver co-stimulatory signals upon binding to the CD28 antigen on T cells, leading to T cell activation. CTLA4 and CD28 are both members of the Ig superfamily containing a single extracellular Ig domain and can both bind to B7 proteins, but their regulatory effects are opposite. CTLA4 has a higher affinity for binding to B7 proteins than CD28, competitively forming a more stable interaction, resulting in T cells lacking secondary stimulatory signals and becoming anergic; simultaneously, it can induce T cell apoptosis after T cell activation. This negatively regulates the immune system, maintaining T cell homeostasis in the body. Therefore, blocking the negative regulatory signaling transmitted by CTLA4 with monoclonal antibodies could provide new therapies for human diseases that benefit from immune stimulation, such as immunotherapy for cancer and infectious diseases. Currently, CTLA4 monoclonal antibodies are 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, and 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 already available. The successful market launch of Ipilimumab (trade name Yervoy) signifies the feasibility of tumor immunotherapy in the clinical stage.Furthermore, as preclinical trials have validated the ability of monoclonal antibodies targeting different immunomodulatory factors to synergistically treat cancer, CTLA4 monoclonal antibodies have been combined with monoclonal antibodies or small molecule compounds of different immunosuppressive molecules to form combination therapies, and are currently undergoing clinical trials for various cancers. However, only one CTLA4 monoclonal antibody is currently on the market, and CTLA4 monoclonal antibodies also have varying degrees of side effects, including the potential to induce immunogenicity in some patients, the possibility of excessive inhibition of CTLA4 signaling causing autoimmune diseases, and the varying degrees of development potential among different CTLA4 monoclonal antibodies. Meanwhile, to avoid differences in efficacy among different patient populations, developing new humanized functional antibodies with higher affinity, specificity, functionality, and diversity that can block the binding of CTLA4 to the B7 protein has become an urgent issue to be addressed in tumor immunotherapy. Summary of the Invention

[0003] In one aspect, 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, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the following HCDR1, HCDR2, and HCDR3 sequences, respectively, and the light chain variable region comprises an amino acid sequence having at least 80% identity with the following LCDR1, LCDR2, and LCDR3 sequences, respectively:

[0004] The amino acid sequence of HCDR1 is SYWIN;

[0005] The amino acid sequence of HCDR2 is RIAPGSGTTYYNEMFTG;

[0006] The amino acid sequence of HCDR3 is GDYFDY;

[0007] The amino acid sequence of LCDR1 is SASKSVSYIH;

[0008] The amino acid sequence of LCDR2 is DTSTLAS;

[0009] The amino acid sequence of LCDR3 is QQRTTYPLT.

[0010] In one embodiment, 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 with the HCDR1, HCDR2, and HCDR3 sequences described above, respectively.

[0011] In one embodiment, 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 with the following LCDR1, LCDR2, and LCDR3 sequences, respectively.

[0012] In one embodiment, 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, wherein the heavy chain variable region comprises the amino acid sequences shown in the HCDR1, HCDR2, and HCDR3 sequences below, and the light chain variable region comprises the amino acid sequences shown in the LCDR1, LCDR2, and LCDR3 sequences below:

[0013] The amino acid sequence of HCDR1 is SYWIN;

[0014] The amino acid sequence of HCDR2 is RIAPGSGTTYYNEMFTG;

[0015] The amino acid sequence of HCDR3 is GDYFDY;

[0016] The amino acid sequence of LCDR1 is SASKSVSYIH;

[0017] The amino acid sequence of LCDR2 is DTSTLAS;

[0018] The amino acid sequence of LCDR3 is QQRTTYPLT.

[0019] This 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 comprises amino acid sequences in which one, two, or three amino acid residues are substituted, inserted, or deleted in the following HCDR1, HCDR2, and HCDR3 sequences, respectively. The light chain variable region comprises amino acid sequences in which one, two, or three amino acid residues are substituted, inserted, or deleted in the following LCDR1, LCDR2, and LCDR3 sequences, respectively.

[0020] The amino acid sequence of HCDR1 is SYWIN;

[0021] The amino acid sequence of HCDR2 is RIAPGSGTTYYNEMFTG;

[0022] The amino acid sequence of HCDR3 is GDYFDY;

[0023] The amino acid sequence of LCDR1 is SASKSVSYIH;

[0024] The amino acid sequence of LCDR2 is DTSTLAS;

[0025] The amino acid sequence of LCDR3 is QQRTTYPLT.

[0026] In one embodiment, the amino acid sequence of the heavy chain variable region is selected from: 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.

[0027] In one embodiment, the amino acid sequence of the light chain variable region 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 or SEQ ID NO:24.

[0028] In one implementation scheme, wherein

[0029] The heavy chain variable region is SEQ ID NO:9 and the light chain variable region is SEQ ID NO:17;

[0030] The heavy chain variable region is SEQ ID NO:10 and the light chain variable region is SEQ ID NO:18;

[0031] The heavy chain variable region is SEQ ID NO:11 and the light chain variable region is SEQ ID NO:19;

[0032] The heavy chain variable region is SEQ ID NO:12 and the light chain variable region is SEQ ID NO:20;

[0033] The heavy chain variable region is SEQ ID NO:13 and the light chain variable region is SEQ ID NO:21;

[0034] The heavy chain variable region is SEQ ID NO:14 and the light chain variable region is SEQ ID NO:22;

[0035] The heavy chain variable region is SEQ ID NO:15 and the light chain variable region is SEQ ID NO:23; or

[0036] The variable region of the heavy chain is SEQ ID NO:16 and the variable region of the light chain is SEQ ID NO:24.

[0037] In one implementation scheme, wherein

[0038] The heavy chain variable region is SEQ ID NO:10 and the light chain variable region is SEQ ID NO:18;

[0039] The heavy chain variable region is SEQ ID NO:11 and the light chain variable region is SEQ ID NO:19;

[0040] The heavy chain variable region is SEQ ID NO:12 and the light chain variable region is SEQ ID NO:20; or

[0041] The variable region of the heavy chain is SEQ ID NO:14 and the variable region of the light chain is SEQ ID NO:22.

[0042] In one implementation scheme, wherein

[0043] The heavy chain variable region is SEQ ID NO:11 and the light chain variable region is SEQ ID NO:19; or

[0044] The variable region of the heavy chain is SEQ ID NO:14 and the variable region of the light chain is SEQ ID NO:22.

[0045] In one embodiment, the humanized anti-human CTLA4 monoclonal antibody or its functional fragment 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 an amino acid sequence as shown in SEQ ID NO:2.

[0046] In one embodiment, the dissociation constant KD between the humanized anti-human CTLA4 monoclonal antibody or its functional fragment and CLTA4 is less than 0.02 nM.

[0047] In one embodiment, the humanized anti-human CTLA4 monoclonal antibody or its functional fragment of the present invention specifically relieves the negative immune regulation of CTLA4 and activates T cells to secrete cytokines.

[0048] In another aspect, the present invention provides isolated polynucleotides encoding the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof.

[0049] In one embodiment, the polynucleotide of the present invention comprises a heavy chain coding sequence encoding the heavy chain variable region of the humanized anti-human CTLA4 monoclonal antibody, and a light chain coding sequence encoding the light chain variable region of the humanized anti-human CTLA4 monoclonal antibody.

[0050] In another aspect, the present invention provides an expression vector comprising the polynucleotide.

[0051] In another aspect, the present invention provides a host cell containing the expression vector.

[0052] In one embodiment, the host cell is a HEK293-6E cell.

[0053] In another aspect, 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 in the preparation of a medicament for antitumor purposes.

[0054] In another aspect, 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 the treatment of tumors.

[0055] In one embodiment, the tumor is selected from multiple myeloma, non-small cell lung cancer, colorectal cancer, renal cell carcinoma, prostate cancer, breast cancer, and ovarian cancer.

[0056] In another aspect, 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 the treatment of tumors.

[0057] In one embodiment, the tumor is selected from multiple myeloma, non-small cell lung cancer, colorectal cancer, renal cell carcinoma, prostate cancer, breast cancer, and ovarian cancer.

[0058] In another aspect, the present invention provides 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.

[0059] In another aspect, 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, and culturing the cells.

[0060] In another aspect, the present invention provides a method for preparing the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, comprising:

[0061] (1) Humanizing the mouse antibody to obtain the variable region coding sequences of the light and heavy chains of the humanized anti-human CTLA4 monoclonal antibody or its functional fragment; and

[0062] (2) Use the variable region coding sequence to produce recombinant antibodies to obtain functional humanized anti-human CTLA4 monoclonal antibody or its functional fragment.

[0063] The humanized anti-human CTLA4 monoclonal antibody provided by this invention exhibits high affinity and specificity for CTLA4, and can stimulate T cells to secrete cytokines, for example, specifically relieving the negative immune regulation of CTLA4 and activating T cells to secrete cytokines. Therefore, the functional humanized anti-human CTLA4 monoclonal antibody provided by this invention can activate T cells by blocking the CTLA4 signaling pathway, thereby achieving the goal of tumor immunotherapy. Brief description of the attached figures

[0064] Figure 1 Affinity assay for humanized anti-human CTLA4 monoclonal antibody;

[0065] Figure 2 Purified monoclonal antibodies can relieve the negative immune regulation of CTLA4;

[0066] Figure 3. Thermal stability analysis of purified monoclonal antibodies, specifically SEC-HPLC analysis of the thermal stability of humanized anti-human CTLA4 monoclonal antibody (treated at 40℃ for 2 weeks): chimeric antibody - 14 days - SEC-HPLC ( Figure 3A AH01674-14 days-SEC-HPLC Figure 3B ) and AH01695-14 days-SEC-HPLC ( Figure 3C );

[0067] Figure 4 Thermostability analysis of purified monoclonal antibodies, specifically ELISA analysis of the thermostability of humanized anti-human CTLA4 monoclonal antibodies (treated at 40°C for 2 weeks): chimeric antibodies (also referred to as "chimeric IgG" in this article and figures; the two terms are interchangeable) Figure 4 A), AH01674 Figure 4 B) and AH01695 Figure 4 C);

[0068] Figure 5 Thermostability analysis of purified monoclonal antibodies, specifically ELISA analysis (temperature gradient) of the thermostability of humanized anti-human CTLA4 monoclonal antibody: AH01674 ( Figure 5 A) and AH01695 Figure 5 B);

[0069] Figure 6 Purified monoclonal antibody drugability detection and analysis, specifically affinity detection of humanized anti-human CTLA4 monoclonal antibody after oxidative stress test: chimeric antibody ( Figure 6 A) Chimeric antibody-AAPH ( Figure 6 B), AH01674 Figure 6 C), AH01674-AAPH Figure 6D), AH01695 Figure 6 E) and AH01695-AAPH ( Figure 6 F);

[0070] Figure 7. Drugability analysis of purified monoclonal antibodies, specifically SEC detection of humanized anti-human CTLA4 monoclonal antibody after oxidative stress assay: chimeric antibody ( Figure 7A ), chimeric antibody-AAPH ( Figure 7B ), AH01674 ( Figure 7C ), AH01674-AAPH ( Figure 7D ), AH01695 ( Figure 7E ) and AH01695-AAPH ( Figure 7F ). Detailed Implementation

[0071] Unless otherwise stated, the technical and scientific terms used in this invention have the meanings commonly understood by a person skilled in the art to which this invention pertains.

[0072] The term "antibody" as used in this article refers to immunoglobulin molecules, which are typically tetramers composed of two identical heavy chains and two identical light chains linked together by disulfide bonds. Based on differences in the conservation of their amino acid sequences, the heavy and light chains are divided into variable regions (V) located at the amino terminus and constant regions (C) located at the carboxyl terminus. Within the variable regions of both the heavy and light chains, three local regions exhibit a higher degree of variability in their amino acid composition and sequence, serving as key sites for antibody-antigen binding; these are also known as complementarity-determining regions (CDRs). In this article, the three heavy chain CDRs are designated HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are designated LCDR1, LCDR2, and LCDR3. The interaction between the variable regions of one heavy chain and one light chain forms the antigen-binding site (Fv). Antibodies can be classified into different categories based on the amino acid sequence of their heavy chain constant regions. There are five main types of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided 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. This invention is intended to include antibodies of any of the aforementioned classes or subclasses.

[0073] The term “antibody” as used herein is also intended to cover its digestive fragments or functional variants, such as antibody fragments capable of binding to CTLA4 or portions thereof, including but not limited to Fab (e.g., antibodies obtained by papain digestion), F(ab')2 (e.g., obtained by pepsin digestion), Fv, or scFv (e.g., obtained by molecular biology techniques).

[0074] As used herein, the term "monoclonal antibody" refers to a homogeneous antibody that targets only a specific antigenic epitope. In contrast to conventional polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody targets a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous nature of the antibody and is not interpreted as requiring the antibody to be produced by any particular method. The monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.

[0075] As used herein, the term "isolated polynucleotide" refers to polynucleotides not naturally occurring in nature, including polynucleotides isolated from nature (including organisms) through biological techniques, as well as artificially synthesized polynucleotides. Isolated polynucleotides can be genomic DNA, cDNA, mRNA, or other synthetic RNA, or combinations thereof. This document provides several nucleotide sequences encoding the heavy chain variable region and light chain variable region of a humanized anti-CTLA4 monoclonal antibody. It should be noted that those skilled in the art can design nucleotide sequences that are not entirely identical to the nucleotide sequences provided above, based on codon degeneracy, using the amino acid sequences of the heavy chain variable region and light chain variable region provided herein, but all encoding the same amino acid sequence. These modified nucleotide sequences are also included within the scope of this invention.

[0076] As used herein, the term "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence relative to the initial amino acid sequence, where the change arises from a sequence alteration involving the amino acid residue / position. Typical modifications include replacing a residue (e.g., a conserved or non-conserved substitution) at a position with another amino acid, inserting one or more (generally fewer than 5 or 3) amino acids adjacent to the residue / position, and deleting the residue / position. "Amino acid substitution," or a variation thereof, refers to replacing an existing amino acid residue in a predetermined (initial) amino acid sequence with a different amino acid residue. Modifications generally preferably result in an alteration of at least one physiological or biochemical activity of the variant peptide relative to the initial (or "wild-type") amino acid sequence. For example, for antibodies, the altered physiological or biochemical activity could be binding affinity, binding capacity, and / or binding effect against a target molecule.

[0077] The "percentage (%) amino acid sequence identity" for a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence after comparing sequences and, where necessary, introducing gaps to obtain the maximum percentage sequence identity, and without considering any conserved substitutions as part of the sequence identity. Sequence comparisons can be performed in a variety of ways within the scope of the art to determine the percentage amino acid sequence identity, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the comparison, including any algorithms required to obtain maximum comparison across the full length of the sequences being compared.

[0078] When referring to polynucleotides, the term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer nucleotide-encoded information into a host cell. The terms "expression vector" or "expression cassette" refer to a vector suitable for expressing a target gene (the nucleotide sequence to be expressed) within a host cell, typically including the target gene, promoter, terminator, marker gene, etc.

[0079] The term "host cell" as used in this article refers to a cell that has been or can be transformed with a nucleic acid sequence to express the selected target gene. This term includes the offspring of the parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent cell, as long as the selected target gene is present in the offspring. Commonly used host cells include bacteria, yeast, and mammalian cells.

[0080] As used in this article, "transfection" refers to the uptake of foreign or exogenous DNA by cells. This technique can be used to introduce one or more portions of exogenous DNA into suitable host cells. Cells can be induced to a physiological state, i.e., "competent state," through physicochemical methods (e.g., treatment with calcium chloride).

[0081] When referring to pharmaceutical compositions, the term "effective amount" as used herein means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. "Pharmaceutically acceptable carrier" refers to a carrier for administration, including various excipients, diluents, and buffers, which are suitable for administration to humans and / or animals without excessive adverse side effects, and are suitable for maintaining the activity of the drug or active agent contained therein.

[0082] The present invention will now be described in detail with reference to specific embodiments. Unless otherwise stated, the methods and materials used in the embodiments described below are all conventional products that can be purchased commercially.

[0083] Example

[0084] Example 1: Humanization of mouse-derived anti-human CTLA4 antibody

[0085] 1) Mouse anti-human CTLA4 antibody 42B11G12D3 antibody sequence (CDR region is shown underlined) (see, for example, SEQ ID NO:1-2)

[0086]

[0087] 2) Construction of anti-human CTLA4 antibody CDR plasmid

[0088] The IMGT human V gene (F+ORF+in-frameP) database was selected, and the human Germline antibody sequence with the highest homology was chosen as the humanization receiving vector based on alignment. The three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and the three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) from the mouse antibody were transferred to their respective positions. Post-translational modification sites (PTMs) were analyzed, as shown in Table 1. Sequence analysis revealed that sites W33 and M63 are hotspots for post-translational oxidative modification (see, for example, SEQ ID NO: 3-4).

[0089]

[0090]

[0091] Table 1: PTM Risk Analysis

[0092]

[0093] 3) Design phage libraries CBM (gray background) and 5BM (bold), construct Phage-Fab and FASEBA-Fab plasmids for anti-human CTLA4 antibody 42B11G12D3VH-VL, and screen for humanized antibody reversion mutation sites (see, for example, SEQ ID NO:5-8).

[0094] 42B11G12D3_CBM

[0095]

[0096] 42B11G12D3_5BM

[0097]

[0098]

[0099] 4) The affinity ranking of prokaryotic expression antibody products and their VH / VL sequences (Table 2) were used to select the sequence with the highest affinity for anti-human CTLA4 antibody for eukaryotic system expression.

[0100] Table 2: Screening for humanization reversion mutations of monoclonal antibodies, ranking antibodies with the highest affinity.

[0101]

[0102] The antibody sequences exhibiting the highest affinity are the 3 CBM and 5 5BM sequences as follows (see, for example, SEQ ID NO:9-24):

[0103]

[0104]

[0105]

[0106] Example 2: Production of humanized antibody recombinant

[0107] The selected antibody VH and VL sequences, after codon optimization, were linked to the 5' end secretion signal peptide and then linked to the constant region sequences of the human antibody IgG1 heavy chain and kappa light chain. These sequences were then cloned into the pTT5 expression vector to prepare human antibody DNA sequences that can be expressed and secreted in mammalian cells. The plasmids were co-transfected with PEI into HEK293-6E suspension cells for transient expression. At transfection, the cell density was maintained at 1 × 10⁻⁶ cells / cells. 6 Cells / mL, PEI:DNA ratio 3:1. Cells were cultured at 37℃ with shaking at 105 rpm in a 5% CO2 incubator. 24 hours after transfection, 0.5% Trypton N-1 was added. 5 days later, the cell culture supernatant was collected, and the antibody was purified using protein-A agarose gel electrophoresis, quantified, and its purity was identified (Table 3).

[0108] Table 3: Production of recombinant humanized antibodies

[0109]

[0110]

[0111] Example 3: Affinity determination of humanized monoclonal antibodies

[0112] The chip surface was equilibrated with HBS-EP buffer at a flow rate of 10 μl / min for 5 minutes. Then, the chip was activated by injecting a 1:1 mixture of "NHS+EDC" at a flow rate of 10 μl / min for 7 minutes. The capture antibody (Goat anti-mouse IgG) diluted in 10 mM sodium acetate buffer was injected at a flow rate of 10 μl / min for about 7 minutes for conjugation. Finally, the surface was blocked by injecting ethanolamine at a flow rate of 10 μl / min for 7 minutes.

[0113] Three pre-cycles were performed using HBS-EP buffer as the sample to equilibrate the chip and stabilize the baseline. Antibody diluted in HBS-EP buffer was injected at a flow rate of 10 μl / min for 0–5 minutes (the antibody-antigen binding signal was controlled to be within ~100 RU by adjusting the capture time), followed by 1 minute of buffer equilibration. A low concentration of antigen (0.33 nM CTLA4-Fc) was injected at a flow rate of 30 μl / min for 5 minutes to allow antigen-antibody binding. Then, buffer was injected at a flow rate of 30 μl / min for 15 minutes for dissociation. Finally, 50 mM HCl was injected four times at a flow rate of 100 μl / min, each time for 10 seconds, to regenerate the antigen, completing one cycle.

[0114] The antigen concentration was varied and cycled through different concentration gradients until all gradient concentrations (1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, 40 nM) and a repeat concentration (e.g., 5 nM CTLA4-Fc) were measured. Experimental data were double-subtracted (control channel and zero concentration) and then fitted to a "1:1 binding" model using Biacore 8K evaluation software. The affinity of the antibody for the recombinant CTLA4-Fc protein was determined using Biacore 8K.

[0115] like Figure 1 As shown in Table 4, the specific monoclonal antibodies against human CTLA4-Fc (AH01672, AH01674, AH01679, AH01686, AH01695, AH01696, and AH01704) all exhibited sub-nM to pM affinity for CTLA4-Fc as measured by Biacore. These results demonstrate that the antibodies screened in this invention possess very high affinity.

[0116] Table 4

[0117]

[0118] Example 4: Functional validation of humanized anti-human CTLA4 monoclonal antibody

[0119] Functional assays of the anti-human CTLA-4 antibody were performed using the Promega Anti-CTLA-4 Blocking Assay Kit. The kit contains two cell lines: CD80 / CD86 aAPC / Raji stimulated cells and functional cells expressing CTLA-4. Without the anti-CTLA-4 antibody, Raji cells bind to CTLA-4 in functional cells, inhibiting immune signal transduction and preventing activation of NFκB to bind to downstream promoter sequences for reporter gene luc2 expression. Upon addition of the anti-human CTLA-4 antibody, CTLA-4 protein is blocked, reactivating the immune response elicited by Raji cells. A luciferase within the functional cells is expressed and reacts with its substrate, generating a fluorescent signal that can be detected and collected.

[0120] In the experiment, Raji cells expressing CD80 / CD86 and functional cells expressing CTLA-4 were cultured and counted. Raji cells were seeded at a rate of 50,000 cells / well in 96-well plates. Sample antibodies and positive and negative control antibodies were added to Raji cells in a gradient, followed by functional cells at a rate of 50,000 cells / well. The mixture was incubated at 37°C in 5% CO2 for 6 hours. A fluorescent reaction substrate was added and the mixture was allowed to react at room temperature in the dark for 10 minutes, after which fluorescence intensity was measured. If the antibody showed CTLA-4 blocking activity, the fluorescence intensity would show an inverse curve with increasing antibody concentration.

[0121] Experimental results showed that humanized anti-human CTLA4 monoclonal antibodies (AH01672, AH01674, AH01679, and AH01695) could specifically relieve the negative immune regulation of CTLA4 and activate T cells to secrete cytokines, with corresponding EC50 values ​​of 7.310 μg / ml, 1.115 μg / ml, 17.10 μg / ml, and 5.464 μg / ml, respectively. Figure 2 (See Table 5). Two humanized antibodies with lower EC50 values, AH01674 (1.115 μg / ml) and AH01695 (5.464 μg / ml), were selected for further studies on drug properties and thermal stability.

[0122] Table 5

[0123] Antibody name Yervoy chimeric antibodies AH1672 AH1674 AH1679 AH1695 EC50 (μg / ml) 0.9334 3.960 7.310 1.115 17.10 5.464

[0124] Example 5: Drugability Evaluation of Humanized Anti-human CTLA4 Monoclonal Antibody

[0125] Three antibodies, AH01674, AH01695, and a chimeric antibody, were expressed in a 200ml system to obtain purified antibody samples of more than 5mg with endotoxin levels controlled at 3EU / mg for subsequent experiments.

[0126] 1. Thermal stability testing

[0127] Thermal stability testing experiment setup

[0128] A. Durability tests were conducted on antibody samples with a concentration >5 mg / ml.

[0129] Antibody samples were treated at 40°C, then centrifuged to remove the precipitate, and the amount of residual antibody was assessed by ELISA. (The samples were tested after 7 days and 14 days of treatment at 40°C, with an untreated sample stored at -80°C serving as a control for each test.)

[0130] B. For each antibody sample with a concentration >5 mg / ml, treat at five different temperature gradients (room temperature, 30°C, 40°C, 50°C, and 60°C) for 20 min each, then centrifuge to remove the precipitate, and finally assess the residual antibody level using ELISA. A control sample stored at -80°C was used simultaneously for each assay.

[0131] A / B treated samples were simultaneously sent for SEC-HPLC analysis.

[0132] As shown in Figures 3-5, the three antibody samples, AH01674, AH01695, and chimeric antibody, did not show large-scale aggregation after heat treatment, and at the same time, they exhibited stable antibody-antigen binding ability.

[0133] 2. Drug-likeness experiment

[0134] Sequence analysis of the CDR region of the anti-human CTLA4 monoclonal antibody (Table 1) revealed predicted hotspots for oxidative modification at W33 and M63 sites in the VH region. The humanized anti-human CTLA4 monoclonal antibody was subjected to...

[0135] A. Oxidative pressure test: Antibody molecules were transferred into 20mM ammonium acetate solution (pH 5.0), and AAPH (2,2'-azobis(2-amidinopropane))(50:1) was added and treated at 40℃ in the dark for 24 hours.

[0136] B. Deamidation and pressurization assay: Antibody molecules were placed in PBS solution (pH 9) for 48 hours at 40°C.

[0137] The effects of oxidative modification / deamide modification on the antigen recognition ability of antibody molecules were determined. Mass spectrometry was used to detect the proportion of corresponding amino acid molecules undergoing chemical changes in the treated antibody samples, Biocore assay was used to determine changes in affinity, and SEC-HPLC was used to determine changes in the degree of polymerization of antibody molecules.

[0138] The results show (Tables 6-8 and 8) Figure 6-7) The mass spectrometry detection coverage reached approximately 95%, yielding reliable results. In the antibody AH01674 control sample, 9.54% oxidation of M@63 was detected, while in the AAPH-24 treated sample, 75.62% oxidation of M@63 was detected. In the antibody AH01695 control sample, 8.91% oxidation of M@63 was detected, while no oxidation of M@250 was detected. In the AAPH-24 treated sample, 70.90% oxidation of M@63 and 26.14% oxidation of M@250 were detected. M250 is located in the constant region of the antibody molecule. Therefore, there is a possibility of oxidation modification at the M63 site in the CDR sequences of AH01674 and AH01695.

[0139] Affinity verification and SEC verification results showed that the oxidative pressure treatment of AH01674 and AH01695 antibody molecules did not affect the affinity between the antibody and the antigen, nor did it affect the uniformity of the antibody molecules.

[0140] Meanwhile, mass spectrometry analysis of the deamidation pressurization experiment showed that no deamidation modification occurred in the CDR sequences of AH01674 and AH01695, which is consistent with the sequence analysis results.

[0141] Table 6. Drugability Detection and Analysis: Mass Spectrometry Detection of Humanized Anti-human CTLA4 Monoclonal Antibody after Oxidative Pressure Test

[0142]

[0143] Table 7. Drugability Analysis: Affinity Detection of Humanized Anti-human CTLA4 Monoclonal Antibody after Oxidative Pressure Test

[0144]

[0145] Table 8. Drugability Detection and Analysis: Mass Spectrometry Detection of Humanized Anti-human CTLA4 Monoclonal Antibody After Deamide Pressure Test

[0146]

[0147]

[0148]

[0149]

Claims

1. A humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is SEQ ID NO:14 and the light chain variable region is SEQ ID NO:

22.

2. The humanized anti-human CTLA4 monoclonal antibody or its functional fragment as described in claim 1, wherein the dissociation constant KD between the antibody and CTLA4 is less than 0.02 nM.

3. An isolated polynucleotide encoding the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof as described in claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A host cell comprising the expression vector as described in claim 4.

6. Use of the humanized anti-human CTLA4 monoclonal antibody or its functional fragment as described in claim 1 or 2, the polynucleotide as described in claim 3, the expression vector as described in claim 4, or the host cell as described in claim 5 in the preparation of a medicament for antitumor purposes, wherein the tumor is selected from multiple myeloma, non-small cell lung cancer, renal cell carcinoma, prostate cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, malignant mesothelioma, gastrointestinal cancer, and liver cancer.

7. The use as described in claim 6, wherein the tumor is colorectal cancer.

8. An antitumor pharmaceutical composition comprising an effective amount of the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

9. A method for preparing the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof as described in claim 1 or 2, comprising transfecting competent cells with the expression vector as described in claim 4 and culturing the competent cells.

10. A method for preparing the humanized anti-human CTLA4 monoclonal antibody or a functional fragment thereof as described in claim 1 or 2, comprising: (1) Humanize the mouse antibody to obtain the variable region coding sequences of the light chain and heavy chain of the humanized anti-human CTLA4 monoclonal antibody or its functional fragment; as well as (2) Use the variable region coding sequence to produce recombinant antibodies to obtain functional humanized anti-human CTLA4 monoclonal antibody or its functional fragment.

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