Humanized anti-cd24 antibodies and uses thereof

Abstract

The application discloses humanized anti-CD24 antibodies and application thereof, relates to the technical field of biological medicine, and specifically provides humanized antibodies capable of specifically recognizing CD24 and application thereof. FR regions and constant regions of a mouse-derived anti-CD24 monoclonal antibody are replaced by human-derived FR regions and constant regions, and CDR of the variable region of the mouse-derived anti-CD24 monoclonal antibody is retained, so that a series of anti-CD24 humanized monoclonal antibodies are obtained. The humanized antibodies obtained by the application have an affinity to CD24 antigens which is basically equivalent to that of a human-mouse chimeric anti-CD24 monoclonal antibody WT, and exhibit strong anti-tumor capacity in vivo.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to humanized antibodies capable of specifically recognizing CD24 and their applications. Background Technology

[0002] CD24, or Cluster of Differentiation 24, is a small, highly glycosylated cell adhesion protein, also known as a heat-stable antigen (HSA). The CD24 molecule is a highly glycosylated sialic acid glycoprotein with an apparent molecular weight between 30 and 70 kDa. CD24 contains a core protein backbone of 33 amino acid residues and has 16 potential O- or N-glycosylation sites, exhibiting high homology with mouse heat-stable antigens. Its mature polypeptide can be anchored to the cell membrane surface via a carboxyl-terminal glycosyl-phosphatidyl-inositol (GPI). Under physiological conditions, CD24 molecules are expressed at low levels only on immature B cells, mature granulocytes, and a few epithelial cells and nerve cells. However, in pathological states, significantly high levels of CD24 expression can be detected on the surface of most malignant tumor cells, and its expression level is closely related to tumor occurrence and development. Recent research indicates that CD24, as a novel immune checkpoint inhibitor, can bind to the inhibitory receptor Siglec-10 expressed on the surface of macrophages, generating a "don't eat me" signal through this interaction. This inhibitory signal suppresses the phagocytosis of cancer cells by macrophages and triggers an immunosuppressive signaling cascade, limiting macrophage phagocytosis and allowing tumor cells to evade macrophage immune surveillance. Furthermore, it has been reported that blocking the CD24-Siglec-10 interaction via antibodies reduces tumor growth via macrophages and prolongs the survival of mice bearing ovarian and breast cancer tumors.

[0003] P-selectin is expressed on the surface of activated platelets and endothelial cells, and CD24 is one of its ligands. High expression of CD24 enhances the adhesion of tumor cells to platelets and endothelial cells, promoting tumor recurrence and metastasis. Of course, as a cell membrane surface signal transduction molecule, CD24 can mediate the proliferation, adhesion, metastasis, and invasion of tumor cells through multiple mechanisms of action.

[0004] In recent years, numerous studies have shown that CD24 is highly expressed in various malignant tumors and cancer stem cells, such as ovarian cancer, breast cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, liver cancer, bladder cancer, and B-cell lymphoma. Research indicates that CD24 is a potential prognostic marker for many types of tumors. For example, in prostate cancer, detecting CD24 expression levels helps determine the patient's recurrence risk level; in colorectal cancer and non-small cell lung cancer, CD24 expression levels can serve as an independent prognostic marker for patient survival; and in bladder cancer, upregulation of CD24 expression is involved in more aggressive cancer development and tumor recurrence. Furthermore, CD24 is considered an important cancer stem cell marker in ovarian cancer, colorectal cancer, pancreatic cancer, and nasopharyngeal carcinoma, and its overexpression can induce the maintenance and activation of cancer stem cells.

[0005] Therefore, although various therapies targeting CD24 have made initial progress, there is still an urgent need in the field to develop novel CD24 monoclonal antibodies as macrophage checkpoint inhibitors to exert anti-tumor immune effects. Summary of the Invention

[0006] The applicant not only successfully obtained a novel anti-human CD24 monoclonal antibody with anti-tumor activity, but more importantly, the applicant humanized the obtained murine anti-human CD24 monoclonal antibody into a humanized monoclonal antibody. Specifically, the FR and constant regions of the murine anti-CD24 monoclonal antibody were replaced with human regions, while the CDR of the variable region of the murine anti-CD24 monoclonal antibody was retained, resulting in a series of humanized anti-CD24 monoclonal antibodies. Furthermore, the applicant discovered that these humanized antibody candidates obtained in this application have an affinity for the CD24 antigen that is substantially equivalent to that of the human-mouse chimeric anti-CD24 monoclonal antibody WT, and exhibit strong in vivo anti-tumor activity.

[0007] Among them, the human-mouse chimeric anti-CD24 monoclonal antibody WT has a light chain variable region VL0 with the amino acid sequence shown in SEQ ID NO.1 and a heavy chain variable region VH0 with the amino acid sequence shown in SEQ ID NO.5.

[0008] This invention provides a humanized antibody or its antigen-binding fragment capable of specifically recognizing CD24, wherein the humanized antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region; wherein:

[0009] The variable region of the light chain includes complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain, and the amino acid sequences are shown in any one of SEQ ID NO.28 to 30, respectively.

[0010] The variable region of the heavy chain includes complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain, with amino acid sequences as shown in any one of SEQ ID NO. 31 to 33.

[0011] Preferably, the complementary determination region CDR1 of the light chain is selected from SEQ ID NO.28, the complementary determination region CDR2 is selected from SEQ ID NO.29, and the complementary determination region CDR3 is selected from SEQ ID NO.30;

[0012] The complementarity determination region CDR1 of the heavy chain is selected from SEQ ID NO.31, the complementarity determination region CDR2 is selected from SEQ ID NO.32, and the complementarity determination region CDR3 is selected from SEQ ID NO.33.

[0013] Preferably, the heavy chain variable region further includes a heavy chain frame region, and the light chain variable region further includes a light chain frame region.

[0014] More preferably, both the heavy chain framework region and the light chain framework region are derived from human IgG antibodies.

[0015] Further preferably, the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO. 2 to 4, and the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO. 6 to 9. In this application, SEQ ID NO. 2 to 4 are referred to as VL1 to VL3, and SEQ ID NO. 6 to 9 are referred to as VH1 to VH4.

[0016] Preferably, the humanized antibody or its antigen-binding fragment further includes a heavy chain constant region and a light chain constant region, wherein the light chain constant region is the light chain constant region of the human Kappa light chain; and the heavy chain constant region is the heavy chain constant region of the human IgG heavy chain. Specifically, the full-length amino acid sequence of the light chain constant region is shown in SEQ ID NO. 10; and the full-length amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 11.

[0017] Specifically, the humanized antibody or its antigen-binding fragment comprises a light chain with an amino acid sequence as shown in any one of SEQ ID NO. 12-14 and a heavy chain with an amino acid sequence as shown in any one of SEQ ID NO. 15-18. In this application, SEQ ID NO. 12-14 are referred to as L1-L3, and SEQ ID NO. 15-18 are referred to as H1-H4. Additionally, the human-mouse chimeric anti-CD24 monoclonal antibody WT has a light chain L0 with the amino acid sequence shown in SEQ ID NO. 19 and a light chain H0 with the amino acid sequence shown in SEQ ID NO. 20.

[0018] In this application, the humanized monoclonal antibody composed of L1 and H1, having IgG1 heavy chain isotype and Kappa light chain isotype, is referred to as hAb-L1H1; the humanized monoclonal antibody composed of L2 and H1, having IgG1 heavy chain isotype and Kappa light chain isotype, is referred to as hAb-L2H1; the humanized monoclonal antibody composed of L3 and H2, having IgG1 heavy chain isotype and Kappa light chain is referred to as hAb-L3H2; and so on.

[0019] According to embodiments of the present invention, the antibody or antigen-binding fragment includes monoclonal antibodies or polyclonal antibodies.

[0020] According to embodiments of the present invention, the monoclonal antibody includes at least one of CDR transplantation antibody, full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody, and minimum recognition unit.

[0021] The present invention also provides a nucleic acid encoding the humanized antibody or its antigen-binding fragment.

[0022] According to embodiments of the present invention, the nucleic acid molecule encodes the aforementioned antibody or its antigen-binding fragment. The antibody or antigen-binding fragment encoded by the nucleic acid molecule according to embodiments of the present invention can specifically target and bind to human CD24 molecules, blocking the binding of Siglec10 and CD24.

[0023] According to embodiments of the present invention, the above-mentioned nucleic acid molecule may further include at least one of the following additional technical features:

[0024] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0025] The nucleic acid includes a nucleic acid encoding a light chain and a nucleic acid encoding a heavy chain; the nucleotide sequence of the nucleic acid encoding the light chain is shown in any one of SEQ ID NO. 21 to 23, and the nucleotide sequence of the nucleic acid encoding the heavy chain is shown in any one of SEQ ID NO. 24 to 27.

[0026] The nucleotide sequences shown in SEQ ID NO. 21–23 encode the light chains L1–L3, respectively, and the nucleotide sequences shown in SEQ ID NO. 24–27 encode the heavy chains H1–H4, respectively. The underlined portions encode the variable regions VL1–VL3 of the light chains and the variable regions VH1–VH4 of the heavy chains, respectively.

[0027] According to an embodiment of the present invention, the nucleotide sequence encoding the antibody light and heavy chain signal peptide is shown in SEQ ID NO.34. The Kozak sequence is ACCACC.

[0028] In a third aspect, the present invention provides an expression vector. According to embodiments of the present invention, the expression vector carries the aforementioned nucleic acid molecule. After the expression vector according to embodiments of the present invention is introduced into suitable recipient cells, under the mediation of a regulatory system, the expression of the aforementioned humanized antibody or its antigen-binding fragment that specifically recognizes CD24 can be effectively achieved, thereby realizing the large-scale in vitro production of the humanized antibody or antigen-binding fragment.

[0029] According to embodiments of the present invention, the expression vector may further include at least one of the following additional technical features:

[0030] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector. This enables the expression of the aforementioned humanized antibody or its antigen-binding fragment that specifically recognizes CD24 in eukaryotic cells, such as HEK293 cells.

[0031] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell carries the aforementioned nucleic acid molecules or expresses the aforementioned humanized antibody or its antigen-binding fragment. The recombinant cell according to embodiments of the invention can be used for the in vitro expression and large-scale acquisition of the aforementioned humanized antibody or its antigen-binding fragment that specifically recognizes CD24.

[0032] According to embodiments of the present invention, the recombinant cells may further include at least one of the following additional technical features: According to embodiments of the present invention, the recombinant cells are obtained by introducing the aforementioned expression vector into a host cell. According to embodiments of the present invention, the recombinant cells are introduced into the host cell by transfection or electrotransduction.

[0033] According to an embodiment of the present invention, the recombinant cells are eukaryotic cells.

[0034] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0035] In a fifth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition contains the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The humanized antibody or expressed humanized antibody contained in the pharmaceutical composition according to embodiments of the invention has an affinity for the CD24 molecule substantially consistent with that of the human-mouse chimeric anti-CD24 monoclonal antibody WT, and exhibits good in vivo activity, effectively inhibiting tumor growth.

[0036] This invention also provides the use of the humanized antibody or its antigen-binding fragment in the preparation of a medicament for treating diseases caused by CD24 overexpression. The diseases include colon cancer, ovarian cancer, prostate cancer, lung cancer, breast cancer, pancreatic cancer, renal cell carcinoma, cervical cancer, endometrial cancer, cholangiocarcinoma, gastric adenocarcinoma, or glioblastoma.

[0037] The beneficial effects of this invention are:

[0038] This invention replaces the FR and constant regions of a murine anti-CD24 monoclonal antibody with human-derived regions while retaining the CDR of the variable region of the murine anti-CD24 monoclonal antibody, resulting in a series of humanized anti-CD24 monoclonal antibodies. The humanized antibodies obtained by this invention possess an affinity for the CD24 antigen that is substantially equivalent to that of the human-mouse chimeric anti-CD24 monoclonal antibody WT, and exhibit robust in vivo anti-tumor activity. Attached Figure Description

[0039] Figure 1 This is a graph showing the results of SDS-PAGE analysis of humanized antibody protein bands according to an embodiment of the present invention;

[0040] Figure 2 This is a graph showing the results of evaluating the monomer purity of monoclonal antibodies using the SEC-HPLC purity detection method according to an embodiment of the present invention.

[0041] Figure 3 A-3C is a graph showing the experimental results of detecting the binding ability of humanized antibodies and human-mouse chimeric antibodies to human CD24 using flow cytometry according to an embodiment of the present invention.

[0042] Figure 4 A-4C is a graph showing the experimental results of evaluating the in vivo antitumor activity of the humanized antibody hAb-L3H4 using a C57BL / 6 mouse model transplanted with MC38-hCD24 tumor cells according to an embodiment of the present invention. Detailed Implementation

[0043] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0044] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. These definitions are merely for the convenience of understanding the invention and should not be construed as limiting the scope of protection of this invention.

[0045] The antibody or antigen-binding fragments described in this invention are typically prepared by biosynthetic methods. Based on the nucleotide sequence described in this invention, those skilled in the art can readily obtain the encoding nucleic acid of this invention using various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc. As one embodiment of this invention, the encoding nucleic acid sequence of this invention can be constructed by segmenting and synthesizing the nucleotide sequence followed by overlap extension PCR. The antibody or antigen fragments are numbered and defined using the Kabat numbering system. The light chain variable region CDR and heavy chain variable region CDR of the antibody or its antibody fragments are jointly determined using both the Kabat and Chothia numbering systems.

[0046] Antibody:

[0047] In this article, the term "antibody" refers to an immunoglobulin molecule capable of binding to a specific antigen. It comprises two lighter chains (H chains) and two heavier chains (L chains), linked by disulfide bonds to form a tetrapeptide chain. The amino-terminal (N-terminus) sequence of the peptide chain varies considerably and is called the variable region (V region), while the carboxyl-terminus (C-terminus) is relatively stable and changes very little, called the constant region (C region). The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system. The V regions of the L and H chains are referred to as VL and VH, respectively.

[0048] Within the variable regions, certain areas exhibit a higher degree of amino acid composition and sequence, termed hypervariable regions (HVRs). These hypervariable regions are the sites where antigens and antibodies bind, and are therefore also called complementary-determining regions (CDRs). They are located within more conserved regions called framework regions (FRs). Each VH and VL can be composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR2, FR2, CDR2, FR3, CDR3, and FR4.

[0049] This invention describes a highly specific, high-affinity anti-CD24 Fab (antigen-binding fragment) antibody fragment. This antibody fragment can specifically bind to the CD24 antigen, thereby enabling targeted therapy for malignant tumors and related diseases.

[0050] In some embodiments, the present invention provides a humanized antibody or antigen-binding fragment having a light chain variable region having an amino acid sequence shown in any one of SEQ ID NO. 2-4 and a heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 6-9. The inventors can obtain the CDR regions of the light chain variable region and the heavy chain variable region using antibody sequence alignment databases (NCBI, IMGT). In other embodiments, the light chain variable region sequence of the antibody or antigen-binding fragment has conserved amino acid substitutions compared to the amino acid sequences shown in SEQ ID NO. 2-4. In other embodiments, the heavy chain variable region sequence of the antibody or antigen-binding fragment has conserved amino acid substitutions compared to the amino acid sequences shown in SEQ ID NO. 6-9. "Conserved amino acid substitution" refers to the substitution of an amino acid by another amino acid with a biologically, chemically, or mechanically similar residue. Of course, these conserved amino acid substitutions do not alter the biological function of the antibody or antigen-binding fragment. In some specific embodiments, these conserved amino acid substitutions can occur on amino acids in the heavy chain variable region and the light chain variable region other than the CDR region. Biological similarity refers to the substitution not disrupting the biological activity of the CD24 antibody or CD24 antigen. Structural similarity refers to amino acids having side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size. Chemical similarity refers to amino acids having the same charge or being either hydrophilic or hydrophobic. For example, hydrophobic amino acid residues such as isoleucine, valine, leucine, or methionine can be substituted for each other. Alternatively, polar amino acids can be substituted for each other, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc.

[0051] The term "antigen-binding fragment of an antibody" refers to a portion of a complete antibody molecule that retains the specific antigen-binding (CD24) ability of the parent antibody, and typically includes at least a portion of the parent antibody's antigen-binding region or variable region (e.g., one or more CDRs). Examples of antigen-binding fragments include, but are not limited to, Fv fragments, disulfide-bonded Fv fragments (dsFv), Fab fragments, (Fab)2, scFv-Fc fusion proteins, scFv-Fv fusion proteins, Fv-Fc fusion proteins, multispecific antibodies formed from antigen-binding fragments, single-domain antibodies, domain antibodies, bivalent domain antibodies, or at least one of the smallest recognition units.

[0052] The term "mouse antibody" usually refers to the fusion of B cells from immunized mice with myeloma cells, followed by screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying antibodies.

[0053] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder originates from a different source or species. A "chimeric antibody" can also be a functional fragment as defined above. "Humanized antibodies" are a subset of "chimeric antibodies."

[0054] The term "humanized antibody" refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to naturally occurring human antibodies. Specifically, a humanized antibody is a molecule having an antigen-binding site substantially derived from a non-human immunoglobulin, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of human immunoglobulins. The antigen-binding site may comprise a complete variable domain fused to a constant domain or only a complementarity-determining region (CDR) grafted to an appropriate frame region within the variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, for example, to make it more similar to human immunoglobulins. Some forms of humanized antibodies retain the entire CDR sequence (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). Other forms have one or more CDRs that have been altered relative to the original antibody.

[0055] In some preferred embodiments, the present invention provides a humanized anti-CD24 antibody having a light chain having an amino acid sequence shown in any one of SEQ ID NO. 12-14 and a heavy chain having an amino acid sequence shown in any one of SEQ ID NO. 15-18.

[0056] In some preferred embodiments, the present invention provides a humanized anti-CD24 single-chain antibody comprising a heavy chain variable region of the amino acid sequence shown in any one of SEQ ID NO. 2-4 and a light chain variable region of the amino acid sequence shown in any one of SEQ ID NO. 6-9, wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the light chain variable region via a linker, or the C-terminus of the light chain variable region is linked to the N-terminus of the heavy chain variable region via a linker.

[0057] Nucleic acid molecules, expression vectors, recombinant cells:

[0058] The present invention also relates to nucleic acid molecules encoding antibodies or portions thereof. The sequences of such nucleic acid molecules include, but are not limited to, the nucleotide sequences shown in any one of SEQ ID NO. 21-23, 24-27.

[0059] In some embodiments, the nucleic acid molecule has at least 90% homology with the nucleotide sequences shown in SEQ ID NO. 21-23 or 24-27, preferably more than 95% homology, and more preferably more than 98% or 99% homology. These sequences, homologous to the nucleotide sequences shown in SEQ ID NO. 21-23 or 24-27, can express amino acid sequences similar to those in SEQ ID NO. 12-14 or SEQ ID NO. 15-18, thereby enabling them to specifically bind to the CD24 antigen and achieve the targeting function of the antibody.

[0060] This invention also provides an expression vector comprising the isolated nucleic acid molecules described above. When ligating the isolated polynucleotides to the vector, the polynucleotides can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translational expression of the polynucleotides. The polynucleotides encoding the antibody heavy and light chains can be independently inserted into different vectors, but are commonly inserted into the same vector. Commonly used vectors include plasmids, bacteriophages, etc., such as pcDNA3.1(+).

[0061] This invention also provides a recombinant cell containing the expression vector. The expression vector can be transfected or introduced into mammalian cells to construct recombinant cells, which can then be used to express the humanized antibody or antigen-binding fragment provided by this invention. The corresponding antibody can be obtained by culturing these recombinant cells. These usable mammalian cells can be, for example, HEK293F cells.

[0062] Pharmaceutical composition:

[0063] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment described above and a pharmaceutically acceptable carrier. The composition may optionally contain one or more other pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of this application may also be administered in combination therapy, for example, with another immunostimulant, anticancer drug, antiviral agent, or vaccine.

[0064] Pharmaceutical compositions may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspenders, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof.

[0065] For example, the antibodies of the present invention can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms, such as liquid, semi-solid, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions. The antibodies can be administered via tail vein infusion or injection, or via intramuscular or subcutaneous injection.

[0066] The pharmaceutical composition of this application has good in vivo application, and according to embodiments of the invention, relates to, for example, the treatment of cancer or immune enhancement for patients with cancer. The antibody can be applied to human subjects, for example, to inhibit tumor growth in vivo. Tumor types include, but are not limited to, ovarian cancer, cervical cancer, endometrial cancer, acute lymphoblastic leukemia, cholangiocarcinoma, pancreatic cancer, bladder cancer, colon cancer, etc. The pharmaceutical composition of this application can be administered in combination with one or more therapies, which can effectively inhibit tumor growth in subjects.

[0067] This application will be further described with reference to the following non-limiting embodiments.

[0068] Example 1: Humanization Strategy for CD24 Monoclonal Antibodies

[0069] 1) The structure of the VH and VL domains of the mouse antibody was predicted using the AlphaFold2 system, and the structure model with the highest score was selected as the basis for subsequent modification.

[0070] 2) By comparing with the IMGT (https: / / www.imgt.org) human antibody light and heavy chain variable region germline gene database, the light and heavy chain variable region germline genes with the highest homology to the murine monoclonal antibodies were selected as templates. Then, the CDRs of the murine monoclonal antibodies were transplanted into the corresponding human templates to form variable region sequences in the order FR1—CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues of the antibody CDRs were determined and annotated by the Kabat numbering system and the Chothia numbering system.

[0071] 3) To maintain the CDR region conformation and minimize the loss of antibody affinity, key amino acid residues in the backbone sequence need to be reverted to the sequences corresponding to the murine antibody. Specifically, based on the structural model of the variable region of the murine anti-CD24 monoclonal antibody predicted by AlphaFold2, Pymol analysis was used to identify residues within 3.5 Å of the CDR region embedded in the protein that interact with the CDR region. Residues that would cause significant steric hindrance before and after modification were reverted to their original form.

[0072] 4) Based on the importance of the reversion mutated residues, three different humanized light chain variable region sequences (VL1–VL3, amino acid sequences as shown in SEQ ID NO. 2–4) and four different heavy chain variable region sequences (VH1–VH4, amino acid sequences as shown in SEQ ID NO. 6–9) were designed. Combining these light and heavy chain variable regions yielded multiple humanized antibody sequences containing different light and heavy chain variable regions. Subsequently, molecular biological cloning was performed to form complete expression plasmids, which were then expressed on a small scale in a mammalian expression system.

[0073] Example 2: Humanization Design Scheme for Anti-CD24 Monoclonal Antibody

[0074] 1) The humanized light chain templates for the murine antibody were IGKV3-11*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV4-4*08 and IGHJ1*01. The CDRs (CDRs 1-3 of the light chain variable region, with amino acid sequences as shown in SEQ ID NO. 28-30; CDRs 1-3 of the heavy chain variable region, with amino acid sequences as shown in SEQ ID NO. 31-33) of the murine antibody were respectively transplanted into their humanized templates to obtain the corresponding humanized sequences. As needed, key amino acids in the FR region sequence of the humanized antibody were reverse-mutated to the corresponding amino acids of the murine antibody to maintain the original affinity. Specific reverse-mutation designs are shown in Table 1.

[0075] Table 1: Humanized Antibody Reverse Mutation Design

[0076]

[0077] Note: Graft indicates that the murine antibody CDR was inserted into the FR region sequence of the human template; I2N indicates that the second I in Graft was mutated to N, and so on. The reversal mutated amino acid is numbered with the kabat number.

[0078] 2) The specific sequence of the variable region of the humanized antibody is as follows:

[0079] The amino acid sequences of VL1-3 are shown in SEQ ID NO.2-4; the amino acid sequences of VH1-4 are shown in SEQ ID NO.6-9.

[0080] 3) From the reverse mutation designs of the variable regions of the light and heavy chains of the above-mentioned humanized antibodies, different light chain (amino acid sequences as shown in SEQ ID NO. 12-14) and heavy chain sequences (amino acid sequences as shown in SEQ ID NO. 15-18) were selected for cross-combination to finally obtain a variety of humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0081] Table 2: Amino acid sequences corresponding to antibody variable regions

[0082] Fv VH1 VH2 VH3 VH4 VL1 hAb-L1H1 hAb-L1H2 hAb-L1H3 hAb-L1H4 VL2 hAb-L2H1 hAb-L2H2 hAb-L2H3 hAb-L2H4 VL3 hAb-L3H1 hAb-L3H2 hAb-L3H3 hAb-L3H4

[0083] Example 3 Expression and purification of anti-CD24 humanized antibody

[0084] 1) Construction of humanized antibody plasmid: The humanized light and heavy chain variable region sequences from Example 2 were integrated with the light and heavy chain constant region sequences of the human IgG1 subtype antibody (the full-length amino acid sequence of the light chain constant region is shown in SEQ ID NO. 10; the full-length amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 11) to form a complete antibody sequence. After codon optimization using a human expression system, nucleotide sequences encoding the light and heavy chains of the humanized antibody were formed. Subsequently, the nucleotide sequence encoding the antibody light and heavy chain signal peptide (sequence shown in SEQ ID NO. 34) was added to the 5' end of the light and heavy chain nucleotide sequences (sequences shown in SEQ ID NO. 21-23 and SEQ ID NO. 24-27), and the Kozak sequence (sequence: ACCACC) was added to the 5' end of the signal peptide sequence. Finally, EcoRI and Not I restriction enzyme sites were added to the 5' and 3' ends of the obtained sequences, respectively. The obtained sequences were synthesized by Nanjing Genscript Biotech Co., Ltd., and cloned into the pcDNA3.1(+) vector.

[0085] 2) The pcDNA3.1(+) vector carrying the humanized anti-human CD24 antibody light and heavy chains was transiently transfected into HEK293F cells to prepare the antibody. One day before transfection, the HEK293F cell density was adjusted to (1–2) × 10⁻⁶ cells / cells. 6 Incubate overnight at 37°C, 5% CO2, and 220 rpm with shaking. On the day of transfection, check cell viability; if it is greater than 95%, transfection can proceed. Transfect the light and heavy chain plasmid at a 1:1 mass ratio into HEK293F cells using PEI (polyethyleneimine hydrochloride, Polysciences) transfection reagent at a PEI:DNA ratio of 4:1 for antibody expression. After culturing for 3–4 days with shaking at 220 rpm, 37°C, and 5% CO2, collect the culture supernatant by centrifugation at 3000 rpm for 20 minutes.

[0086] 3) Antibody purification. Antibodies containing the Fc domain were captured from the expression supernatant using a HiTrap protein A affinity chromatography column (GE17040301). After equilibrating the column with phosphate buffer (pH 7.2), the supernatant was passed through the affinity chromatography column, eluted with elution buffer (100 mM sodium citrate, pH 3.2), and then concentrated and replaced with PBS buffer. Subsequently, protein concentration was determined using a Nanodrop ND-1000. The PBS-replaced antibodies were aliquoted and stored at -80°C. The purity of the purified antibodies was assessed by reducing SDS-PAGE. Figure 1 Two bands were observed, approximately 25 kDa and 50 kDa respectively, consistent with the theoretical band size. The resulting humanized antibody (heavy chain sequence as shown in SEQ ID NO. 12–14, light chain sequence as shown in SEQ ID NO. 15–18) was finally obtained.

[0087] Example 4: Evaluation of monomer purity of humanized antibodies using SEC-HPLC purity detection method

[0088] SEC-HPLC is an analytical method that separates solute molecules based on the relative size of the gel pores and the size of the analyte. Larger molecules in the sample are excluded from the gel pores and pass through the gaps between the porous gel particles, thus being the first to be eluted by the mobile phase. Smaller molecules, on the other hand, can enter the gel pores and experience stronger retention within the column, making them less likely to elute. This process achieves the separation of solute molecules of different sizes.

[0089] Human-mouse chimeric anti-CD24 monoclonal antibody WT: The human-mouse chimeric anti-CD24 monoclonal antibody WT has a light chain variable region VL0 of the amino acid sequence shown in SEQ ID NO.1 and a heavy chain variable region VH0 of the amino acid sequence shown in SEQ ID NO.5; it also has a light chain L0 of the amino acid sequence shown in SEQ ID NO.19 and a light chain H0 of the amino acid sequence shown in SEQ ID NO.20.

[0090] Humanized antibody (hAb-L1H1, hAb-L1H2, hAb-L1H3, hAb-L2H3) samples and chimeric antibody (WT) samples were centrifuged, and approximately 50 μg of the supernatant was injected into HPLC for detection. The percentage of the monomer peak area of ​​the humanized antibody was determined by SEC-HPLC; a higher peak area percentage indicates higher monomer purity. Results are shown below. Figure 2The results in the figure show that the monomer peak retention volumes of humanized antibodies hAb-L1H1, hAb-L1H2, hAb-L1H3, and hAb-L2H3 are all around 12.45 ml, with area percentages of 96.01%, 95.92%, 95.53%, and 98.85%, respectively; the monomer peak area percentage of chimeric antibody H0L0-IgG4 is 91.61%. This indicates that the monomer purity of humanized antibodies hAb-L1H1, hAb-L1H2, hAb-L1H3, hAb-L2H3, and chimeric antibody WT is high.

[0091] Example 5: Humanized Antibody Flow Cytometry Combined with Experiment

[0092] The affinity level of humanized antibodies was evaluated by flow cytometry using a binding assay based on the CD24+HT29 cell line.

[0093] CD24+HT29 cell density was adjusted to 2×10⁻⁶. 6 / mL, take 100μl, wash with pre-cooled PBS, and incubate at 4°C for 30 minutes in 100μl of PBS containing the above-mentioned humanized antibody and chimeric antibody at an initial concentration of 20μg / mL. After incubation, wash the cells twice with cold PBS, and then fluorescently label them with 200μL of FITC-labeled goat anti-human IgG secondary antibody (Beyotime). After washing, resuspend in 200μL PBS, and then detect the mean fluorescence intensity (MFI) on the cell surface using ACEA NovoCyte™ flow cytometry. The shift in MFI indicates the change in antibody affinity level before and after conjugation.

[0094] like Figure 3 As shown in A-3C, the above-mentioned humanized antibody exhibited binding ability to HT29 cells comparable to that of the chimeric antibody.

[0095] Example 6: In vivo antitumor experiment of humanized antibody in a mouse model of colorectal cancer

[0096] MC38-hCD24 cells were cultured at 37°C in a 5% CO2 environment in DMEM containing 10% fetal bovine serum and 2 μg / mL purmycin. MC38-hCD24 tumor cells in the logarithmic growth phase were collected, reselected with pre-chilled PBS, and counted. The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 7 / mL. Using a 1mL syringe, inoculate the cell suspension subcutaneously into the right axillary region of six 6-8 week old C57BL / 6 mice, with each mouse receiving 100μL (approximately 1×10⁶ cells). 6 Several tumor cells. When the average tumor volume reaches approximately 80 mm... 3Animals were randomly divided into two groups of three mice each, based on tumor volume. One group served as a PBS blank control, and the other as an hAb-L3H4 group. The drug concentration was adjusted, and mice in the hAb-L3H4 group were administered 100 μL of hAb-L3H4 via tail vein injection at a dose of 10 mg / kg. Mice in the PBS blank control group were administered 100 μL of PBS solution via tail vein injection. The experimental time was defined as day 0 after tumor inoculation. Administered the drug twice, every two days. Mouse weight and tumor size were measured every 2–3 days. The tumor was considered closed when the average tumor volume in the PBS group reached 1500 mm². 3 The experiment was terminated at that time. The specific formula for calculating tumor volume is: V = (L × W) 2 ) / 2. Where V is the tumor volume (mm). 3 L represents the measured tumor length (mm), and W represents the measured tumor width (mm). At day 27, some mouse tumors exceeded 1500 mm. 3 Therefore, data from D0 to D27 were used for plotting and analysis.

[0097] like Figure 4 As shown in Figure A, hAb-L3H4 demonstrated good tumor growth inhibition in the C57BL / 6 mouse homologous transplantation model of MC38-hCD24 cell tumor. Specifically, the tumor volume in the PBS group mice showed a significant increase, such as... Figure 4 As shown in B; however, at a dose of 10 mg / kg, one of the three mice in the hAb-L3H4 treatment group experienced complete tumor clearance, while the tumors in the other two mice grew slowly, as shown in Figure B. Figure 4 As shown in C.

Claims

1. A humanized antibody capable of specifically recognizing CD24, characterized in that, The humanized antibody includes a light chain variable region and a heavy chain variable region; The amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO.2~4, and the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO.6~9; It also includes a heavy chain constant region and a light chain constant region, wherein the light chain constant region is the light chain constant region of the human Kappa light chain; and the heavy chain constant region is the heavy chain constant region of the human IgG heavy chain. The amino acid sequence of the light chain constant region is shown in SEQ ID NO.10; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.

11.

2. The humanized antibody as described in claim 1, characterized in that, Includes light chains with amino acid sequences as shown in any one of SEQ ID NO. 12-14 and heavy chains with amino acid sequences as shown in any one of SEQ ID NO. 15-18.

3. The nucleic acid encoding the humanized antibody of claim 1 or 2.

4. The nucleic acid as described in claim 3, characterized in that, It includes nucleic acids encoding a light chain and nucleic acids encoding a heavy chain; the nucleotide sequence of the nucleic acid encoding the light chain is shown in any one of SEQ ID NO. 21~23, and the nucleotide sequence of the nucleic acid encoding the heavy chain is shown in any one of SEQ ID NO. 24~27.

5. Application of humanized antibodies in the preparation of drugs for treating diseases caused by CD24 overexpression; The diseases mentioned are colorectal cancer, ovarian cancer, prostate cancer, lung cancer, breast cancer, pancreatic cancer, renal cell carcinoma, cervical cancer, endometrial cancer, bile duct cancer, gastric adenocarcinoma, or glioblastoma. in, The humanized antibody has an amino acid sequence as shown in SEQ ID NO.14 for the light chain and an amino acid sequence as shown in SEQ ID NO.18 for the heavy chain.

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