An anti-CD24 antibody or its antigen-binding fragment and its application

Abstract

The present invention relates to an anti-CD24 antibody or an antigen-binding fragment thereof that can bind to CD24 and / or can exert an anti-tumor effect through an antibody Fc fragment and uses thereof.

Description

Technical Field

[0001] The present invention belongs to the fields of tumor immunotherapy and molecular immunology, and particularly relates to an anti-CD24 antibody or an antigen-binding fragment thereof. Background Art

[0002] As human beings’ understanding of the immune system and the mechanisms of tumor occurrence deepens, tumor immunotherapy has increasingly become a powerful weapon for humans to fight against tumors.

[0003] Tumor-targeted monoclonal antibodies are an important tool in the field of tumor immunotherapy. Macrophage phagocytosis requires the simultaneous activation of two signals: activation of the "eat me" signal on the surface of the targeted cell and inactivation of the "don't eat me" signal on the same cell surface. The absence of either signal alone is insufficient to trigger phagocytosis. Growing evidence indicates that CD47 is a "don't eat me" signal. Tumor cells highly express CD47, which binds to signal regulatory protein α (SIRPα) on the surface of macrophages, releasing the "don't eat me" signal and preventing phagocytosis.

[0004] CD24, also known as a heat-stable antigen, is a highly glycosylated glycosylphosphatidylinositol-anchored surface protein. Studies have found that CD24 signaling often serves as a complementary pathway to CD47 signaling. For example, blood cancers are highly susceptible to CD47 signaling blockade and less responsive to CD24 blockade. However, in other cancers, such as ovarian cancer, the reverse is true, making most cancers susceptible to attack by blocking just one signal. Furthermore, blocking multiple "don't eat me" signals on a tumor may make the cancer more vulnerable. Among the many primary and secondary "don't eat me" signals, CD24 is the most prominent. Studies have shown that CD24, by binding to Siglec-10 on the surface of macrophages, activates the SHP-1 / SHP-2-mediated inhibitory signaling pathway, which cancer cells use to protect themselves. This makes it a promising target for the development of cancer immunotherapy. Clinically, high CD24 expression is negatively correlated with breast cancer prognosis.

[0005] In summary, CD24 is an anti-phagocytic signal that is highly expressed in many cancers. Anti-CD24 antibodies have significant anti-tumor effects and can increase the chemotherapy sensitivity of cancer cells. Blocking CD24 is expected to become a new immunotherapy. Summary of the Invention

[0006] The present invention provides an anti-CD24 antibody or an antigen-binding fragment thereof, which can bind to CD24 and / or can exert an anti-tumor effect through the antibody Fc fragment.

[0007] In an optional embodiment, the anti-CD24 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0008] (a) a heavy chain variable region HCDR1 selected from any one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, or an amino acid sequence that has one or more (preferably two or three) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82;

[0009] (b) a heavy chain variable region HCDR2 selected from any one of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, or an amino acid sequence that has one or more (preferably two or three) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any one of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83;

[0010] (c) a heavy chain variable region HCDR3 selected from any one of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, or 84, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, or 84, or an amino acid sequence that has one or more (preferably two or three) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to any one of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, or 84;

[0011] (d) a light chain variable region LCDR1 selected from any one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, or an amino acid sequence that has one or more (preferably two or three) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86;

[0012] (e) a light chain variable region LCDR2 selected from any one of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, or an amino acid sequence that has one or more (preferably two or three) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any one of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87; and / or

[0013] (f) a LCDR3 of a light chain variable region selected from any one of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, or an amino acid sequence that has one or more (preferably two or three) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any one of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88.

[0014] In an optional embodiment, the HCDR1, HCDR2, HCDR3 of the heavy chain variable region, and the LCDR1, LCDR2, LCDR3 of the light chain variable region are selected from any one of the following amino acid sequences (1)-(11):

[0015] (1) HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 3, HCDR3 shown in SEQ ID NO: 4, LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, LCDR3 shown in SEQ ID NO: 8;

[0016] (2) HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 16;

[0017] (3) HCDR1 shown in SEQ ID NO: 18, HCDR2 shown in SEQ ID NO: 19, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 22, LCDR2 shown in SEQ ID NO: 23, LCDR3 shown in SEQ ID NO: 24;

[0018] (4) HCDR1 shown in SEQ ID NO: 26, HCDR2 shown in SEQ ID NO: 27, HCDR3 shown in SEQ ID NO: 28, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 31, LCDR3 shown in SEQ ID NO: 32;

[0019] (5) HCDR1 shown in SEQ ID NO: 34, HCDR2 shown in SEQ ID NO: 35, HCDR3 shown in SEQ ID NO: 36, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, LCDR3 shown in SEQ ID NO: 40;

[0020] (6) HCDR1 shown in SEQ ID NO: 42, HCDR2 shown in SEQ ID NO: 43, HCDR3 shown in SEQ ID NO: 44, LCDR1 shown in SEQ ID NO: 46, LCDR2 shown in SEQ ID NO: 47, LCDR3 shown in SEQ ID NO: 48;

[0021] (7) HCDR1 shown in SEQ ID NO: 50, HCDR2 shown in SEQ ID NO: 51, HCDR3 shown in SEQ ID NO: 52, LCDR1 shown in SEQ ID NO: 54, LCDR2 shown in SEQ ID NO: 55, LCDR3 shown in SEQ ID NO: 56;

[0022] (8) HCDR1 shown in SEQ ID NO: 58, HCDR2 shown in SEQ ID NO: 59, HCDR3 shown in SEQ ID NO: 60, LCDR1 shown in SEQ ID NO: 62, LCDR2 shown in SEQ ID NO: 63, LCDR3 shown in SEQ ID NO: 64;

[0023] (9) HCDR1 shown in SEQ ID NO: 66, HCDR2 shown in SEQ ID NO: 67, HCDR3 shown in SEQ ID NO: 68, LCDR1 shown in SEQ ID NO: 70, LCDR2 shown in SEQ ID NO: 71, LCDR3 shown in SEQ ID NO: 72;

[0024] (10) HCDR1 shown in SEQ ID NO: 74, HCDR2 shown in SEQ ID NO: 75, HCDR3 shown in SEQ ID NO: 76, LCDR1 shown in SEQ ID NO: 78, LCDR2 shown in SEQ ID NO: 79, LCDR3 shown in SEQ ID NO: 80;

[0025] (11) HCDR1 shown in SEQ ID NO: 82, HCDR2 shown in SEQ ID NO: 83, HCDR3 shown in SEQ ID NO: 84, LCDR1 shown in SEQ ID NO: 86, LCDR2 shown in SEQ ID NO: 87, LCDR3 shown in SEQ ID NO: 88.

[0026] In an alternative embodiment, the heavy chain variable region and the light chain variable region are selected from any one of the following amino acid sequences (1) to (11):

[0027] (1) SEQ ID NO: 1 and SEQ ID NO: 5;

[0028] (2) SEQ ID NO: 9 and SEQ ID NO: 13;

[0029] (3) SEQ ID NO: 17 and SEQ ID NO: 21;

[0030] (4) SEQ ID NO: 25 and SEQ ID NO: 29;

[0031] (5) SEQ ID NO: 33 and SEQ ID NO: 37;

[0032] (6) SEQ ID NO: 41 and SEQ ID NO: 45;

[0033] (7) SEQ ID NO: 49 and SEQ ID NO: 53;

[0034] (8) SEQ ID NO: 57 and SEQ ID NO: 61;

[0035] (9) SEQ ID NO: 65 and SEQ ID NO: 69;

[0036] (10) SEQ ID NO: 73 and SEQ ID NO: 77;

[0037] (11) SEQ ID NO: 81 and SEQ ID NO: 85.

[0038] In alternative embodiments, the antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, and / or a humanized antibody or antigen-binding fragment thereof.

[0039] In alternative embodiments, the antibody is a monoclonal antibody.

[0040] In an optional embodiment, the anti-CD24 antibody or antigen-binding fragment thereof further comprises an Fc region, and the Fc region is selected from mouse IgG1, IgG2a, IgG2b and / or IgG3, or selected from rat IgG1, IgG2a, IgG2b and / or IgG2c.

[0041] In an optional embodiment, the anti-CD24 antibody or antigen-binding fragment thereof further comprises an Fc region, and the Fc region is selected from IgG1, IgG2, IgG3 and / or IgG4.

[0042] The present invention also provides a nucleic acid molecule encoding any of the above-mentioned anti-CD24 antibodies or antigen-binding fragments thereof.

[0043] The present invention also provides a recombinant vector comprising the above nucleic acid molecule.

[0044] The present invention also provides a recombinant cell comprising the aforementioned nucleic acid molecule and / or the aforementioned recombinant vector and capable of expressing the anti-CD24 antibody or antigen-binding fragment thereof.

[0045] The present invention also provides a multifunctional fusion protein comprising any of the above-mentioned anti-CD24 antibodies or antigen-binding fragments thereof.

[0046] In an optional embodiment, the multifunctional fusion protein further comprises one or more second antibodies or antigen-binding portions thereof that specifically bind to other antigens.

[0047] In an alternative embodiment, the antigen bound to the second antibody or antigen-binding portion thereof is selected from a tumor-associated antigen (TAA) or an immune checkpoint.

[0048] The present invention also provides use of any of the above-mentioned anti-CD24 antibodies or antigen-binding fragments thereof, or any of the above-mentioned multifunctional fusion proteins in the preparation of a medicament for treating and / or preventing and / or diagnosing a disease.

[0049] In an optional embodiment, the use is achieved by one or more of tumor immunotherapy, cell therapy and gene therapy.

[0050] The present invention also provides use of any of the above-mentioned anti-CD24 antibodies or antigen-binding fragments thereof, or any of the above-mentioned multifunctional fusion proteins in the preparation of drugs for treating cancer.

[0051] In an alternative embodiment, the cancer is breast cancer, ovarian cancer, lung cancer, liver cancer, melanoma, glioblastoma, head and neck cancer, colorectal cancer, gastric cancer, bladder cancer, pancreatic cancer, stomach cancer, colon cancer, cervical cancer or related tumors.

[0052] The present invention also provides a pharmaceutical composition comprising any of the above-mentioned anti-CD24 antibodies or antigen-binding fragments thereof and an acceptable carrier, diluent or excipient.

[0053] The present invention also provides a pharmaceutical composition comprising any of the multifunctional fusion proteins described above and an acceptable carrier, diluent or excipient.

[0054] Beneficial effects

[0055] The anti-CD24 antibody or antigen-binding fragment thereof provided by the present invention has good binding activity with MCF7 cells, SKOV3 cells and HT55 cells, and can mediate the phagocytosis of MCF7 cells and CD24-SKOV3 cells by macrophages.

[0056] To aid in understanding the invention described herein, the following abbreviations and term definitions are provided.

[0057] The following abbreviations are used in this article:

[0058] CDR: complementarity determining region in the variable region of an antibody

[0059] HCDR: complementarity determining region in the antibody heavy chain variable region

[0060] LCDR: complementarity determining region in the variable region of the antibody light chain

[0061] FR: Antibody framework region, i.e., the amino acid residues in the antibody variable region other than the CDR residues

[0062] VH: antibody heavy chain variable region

[0063] VL: antibody light chain variable region

[0064] IgG: Immunoglobulin G

[0065] mAb: monoclonal antibody

[0066] ELISA: Enzyme-linked immunosorbent assay

[0067] FACS: Fluorescence-activated cell sorting

[0068] ADCP: Antibody-dependent cell-mediated phagocytosis

[0069] In this manual, the term "antibody" refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis. Antibodies can be reconstructed and separated from natural resources such as plasma or serum of the natural antibody, or the culture supernatant of hybridoma cells that produce the antibody, animal immune serum, or phage library screening. Alternatively, they can be synthesized partially or completely using techniques such as genetic recombination. Preferred antibodies include, for example, antibodies of the isotype of immunoglobulin or the subclasses of these isotypes. Known human immunoglobulins include 9 categories (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Among these isotypes, antibodies of the present invention can include IgG1, IgG2, IgG3, and / or IgG4.

[0070] The terms "antibody" and "immunoglobulin" are used interchangeably herein. As used herein, some antibodies are immunoglobulin molecules composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL), or only a light chain constant region (CL). The light chain constant region is composed of a single domain, CL. The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as mediating 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 (C1q) of the classical complement system. The VH and VL regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site.

[0071] The term "antigen-binding fragment" of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical fragmentation of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (e.g., technology from Ablynx), domain antibodies (e.g., technology from Domantis), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to a polypeptide.

[0072] The term "polypeptide" refers to an amino acid chain of any length, regardless of modifications (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. Polypeptides can be "exogenous," meaning they are "heterologous," i.e., foreign to the host cell being utilized, such as human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as sequences of amino acid residues. Those sequences are written from left to right, in the direction of amino terminus to carboxyl terminus. According to standard nomenclature, amino acid residue sequences are designated by three-letter or one-letter codes as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V). The numbering of amino acid positions in the antibodies described herein (e.g., amino acid residues in the Fc region) and target regions, such as CDRs, uses the Kabat system, with the exception that the sequence numbering of SEQ ID NO. 4 and SEQ ID NO. 12 uses the IMGT system.

[0073] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package.

[0074] The term "monoclonal antibody" as used herein refers to a homogeneous antibody directed only against a specific antigenic epitope. Compared to conventional polyclonal antibody preparations that typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous character of the antibody and is not to be construed 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] The term "murine antibody" as used herein refers to a monoclonal antibody prepared according to the knowledge and skill in the art by injecting a test subject with an antigen and then isolating a hybridoma that expresses an antibody with the desired sequence or functional properties.

[0076] The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the mouse hybridoma cells. Furthermore, the constant region genes of the human antibody are cloned as needed. The mouse variable region genes and the human constant region genes are then linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell culture system.

[0077] The term "humanized antibody" refers to an antibody comprising at least one humanized antibody chain (i.e., at least one humanized light chain or heavy chain). The term "humanized antibody chain" (i.e., "humanized immunoglobulin chain") refers to an antibody chain (i.e., a light chain or a heavy chain, respectively) having a variable region comprising a substantial variable framework region and complementarity determination of a human antibody. Regions (CDRs) substantially derived from non-human antibodies (e.g., at least one CDR, two CDRs, or three CDRs). In some embodiments, the humanized antibody chain further comprises a constant region (e.g., in the case of a light chain, one constant region or a portion thereof, in the case of a heavy chain, preferably three constant regions).

[0078] The term "host cell" refers to a cell that has been or is capable of being transformed with a nucleic acid sequence to express a selected gene of interest. The term includes the progeny of a parent cell, regardless of whether the progeny is identical in morphology or genetic makeup to the original parent cell, as long as the gene of interest is present in the progeny. Commonly used host cells include bacteria, yeast, and mammalian cells.

[0079] The term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1a-Figure 1c The binding activity of mouse anti-CD24 antibody to MCF7 cells;

[0081] Figure 2a-2c The binding activity of mouse anti-CD24 antibody to SKOV3 cells;

[0082] Figure 3 is the binding activity of chimeric anti-CD24 antibody to MCF7 cells;

[0083] Figure 4 is the binding activity of the chimeric anti-CD24 antibody to SKOV3 cells;

[0084] Figure 5 is the binding activity of the chimeric anti-CD24 antibody to HT55 cells;

[0085] Figure 6 is the ADCP effect of chimeric anti-CD24 antibody on MCF7 cells;

[0086] Figure 7 The ADCP effect of chimeric anti-CD24 antibody on CD24-SKOV3 cells;

[0087] Figure 8 The anti-tumor effect of chimeric anti-CD24 antibody in SKOV3 subcutaneous xenograft tumor model. DETAILED DESCRIPTION

[0088] The present invention is further described below in conjunction with the accompanying drawings and specific examples, and the protection content of the present invention is not limited to the following examples. It should also be understood that the terms used in the examples of the present invention are for the purpose of describing specific embodiments, rather than for the purpose of limiting the scope of protection of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the scope of protection of the present invention is the attached claims and any equivalents thereof. In the specification and claims of the present invention, unless otherwise expressly indicated in the text, the singular forms "a", "an" and "this" include plural forms. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for those specifically mentioned, are common knowledge and common common sense of those skilled in the art, and the present invention does not particularly limit the content.

[0089] Example 1: Animal Immunization

[0090] Balb / c mice were immunized with recombinant human CD24 protein (prepared by KACTUS). The immunogen was diluted in PBS and added to Freund's complete adjuvant (Sigma; F5881) at a 1:1 volume ratio. Vortex and emulsify until emulsified. Mice were immunized intraperitoneally. A second immunization was performed two weeks after the first immunization, and then again at monthly intervals. Negative serum was collected 3 days before immunization, and 50 μL of blood was collected by tail clipping 6 days after each immunization. Negative and immune serum were diluted in the appropriate ratios (1:0.1K, 1:1K, 1:10K, 1:100K, 1:1000K, and 1:10000K). Serum titers were determined by ELISA using CD24-overexpressing breast cancer MDA-MB-231 cells (MDA-MB-231-CD24). When titers met the required level, with anti-human CD24 antibodies detected at a dilution >1:10K, the spleen and lymph nodes were harvested.

[0091] Example 2: Cell fusion

[0092] Place the spleen and lymph nodes in a cell sieve, then place the cell sieve in a 50 mL centrifuge tube. Pipette DMEM onto the spleen, grind, and prepare a splenocyte suspension. Centrifuge at 1600 rpm for 10 minutes, and discard the supernatant. Resuspend B cells in 2 mL of red blood cell lysis buffer and lyse at room temperature for 2 minutes. Add 30 mL of DMEM, mix thoroughly, and centrifuge at 1600 rpm for 10 minutes. Count the cells.

[0093] Myeloma cells SP2 / 0 (ATCC) were passaged the day before fusion, allowing the cells to be in the logarithmic growth phase at the time of the experiment. Splenocytes and SP2 / 0 were mixed at a ratio of 2:1 and centrifuged at 1600 rpm for 10 minutes. The mixed cells were washed twice with fusion solution and centrifuged at 1600 rpm for 10 minutes. The final cell density was 1×107 Add fusion solution to resuspend the cells. Within 5 minutes, transfer the cell suspension to the fusion chamber of an electrofusion instrument (BTX; ECM 2001) for fusion. After fusion is complete, transfer the cells to complete medium containing HAT and incubate at 37°C for 60 minutes. After incubation, plate the cells into a 96-well plate containing feeder cells and culture at 37°C in 5% CO2.

[0094] Example 3: Preliminary screening of positive clones by ELISA

[0095] After 7 days of culture, the fusion supernatant was screened. MDA-MB-231-CD24 cells were plated in 96 ELISA plates (1.5×10 4 / well), incubate for 36 hours, and then wash twice with PBST. Fix with 4% paraformaldehyde and block with 2% BSA. Pour off the blocking solution and wash three times with a plate washer. Take 100 μL / well of the fusion supernatant and add it to the blocked ELISA plate, incubate at 37°C for 1 hour, and discard the liquid in the well. Wash three times with a plate washer. Dilute the goat anti-mouse secondary antibody-HRP (ABCOM; Ab6789) with 0.5% BSA. 100 μL / well, incubate at 37°C. Wash 6 times with PBST and pat dry on a flat paper. Add 100 μL / well of Solarbio colorimetric solution (Solarbio; PR1200) to the plate wells. Develop at 37°C in the dark. Add 1 M HCl to stop the color reaction, read at 450 nm on a microplate reader, and analyze the data. The cell lines with OD450>1.0 in the supernatant were selected as the candidate positive cell lines for primary screening, the culture supernatant of the positive cell lines was discarded, and new HAT complete medium was added.

[0096] Example 4: Further screening of positive clones by FACS

[0097] Transfer MDA-MB-231-CD24 cells to a centrifuge tube and centrifuge at 1000 rpm for 5 min. 5 Each stably expressing cell was aliquoted into a separate tube and 100 μL of fusion supernatant was added. The cells were incubated at 4°C for 60 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100 μL FACS buffer and goat anti-mouse secondary antibody-FITC (ABCOM; ab6785) was added to the sample, incubated for 30 minutes and washed twice with excess FACS buffer. The cells were fixed in fixation buffer and subsequently analyzed by flow cytometry. Antibodies that specifically bind to MDA-MB-231-CD24 cells were screened by FACS.

[0098] The hybridoma cells were cloned by two rounds of limiting dilution method and detected by ELISA method. 450Monoclonal clones with OD > 1.0 were selected as candidate cell lines for cell culture, and clones without monoclonal antibodies were selected. 450 The clones with the value >1.0 were subjected to the next subcloning.

[0099] Example 5: Antibody Sample Production in Candidate Cell Lines

[0100] Culture hybridoma cells in a T75 medium until the cell confluence is 80-90%. Discard the supernatant from two flasks of cells and add 30 mL of hybridoma-SFM to culture at 37°C in 5% CO2. Observe the cell status and culture medium color for 2-3 days. If the culture medium turns yellow, add 30 mL of fresh hybridoma-SFM. After 6-7 days of culture, centrifuge at low speed and collect the culture supernatant for purification.

[0101] Place the gravity column on an iron stand and rinse the column with 0.5M NaOH. Equilibrate the cleaned gravity column with 10-fold diluted binding buffer (1M Tris-HCl + 1.5M NaCl, pH 8.0). After centrifugation, add an equal volume of 10-fold diluted binding buffer, mix, and load the hybridoma sample. Reequilibrate each gravity column with 10-fold diluted binding buffer. Elute the sample with elution buffer. Finally, adjust the sample pH to approximately 7.0.

[0102] Example 6: FACS detection of binding of candidate antibodies to natural tumor cells

[0103] Using FACS, candidate antibodies were evaluated for binding activity against natural tumor cells (MCF7 (ATCC) and SKOV3 (ATCC)) to confirm the final positive clones. A commercial antibody, SN3 (ABCOM; ab134375), was used as a positive control. MCF7 and SKOV3 cells were transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. 100 μL of MCF7 or SKOV3 cells were plated in a 96U plate, washed twice with PBS, and 100 μL of the fusion supernatant was added. The sample was serially diluted eight times. The cells were incubated at 4°C for 60 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100 μL of FACS buffer, and a secondary goat anti-mouse antibody, FITC (ABCOM; ab6785), was added to the sample, incubated for 30 minutes, and washed twice with excess FACS buffer. The cells were fixed in fixation buffer and analyzed by flow cytometry.

[0104] The binding activities of antibodies MS-01 to MS-10 to MCF7 cells are shown in Figure 1a-Figure 1c The results show that the binding activities of antibodies MS-01 to MS-10 to MCF7 cells are better than or equivalent to the control antibody SN3.

[0105] The binding activities of antibodies MS-01 to MS-10 to SKOV3 cells are shown in Figure 2a-2c The results show that the binding activity of antibodies MS-01 to MS-10 to SKOV3 cells is better than that of the control antibody SN3, or is equivalent to that of the control antibody SN3.

[0106] Example 7: Sequencing of monoclonal antibodies

[0107] Monoclonal antibodies with good binding activity to tumor cells MCF7 and SKOV3 were selected for sequencing; and the amino acid sequences of murine anti-CD24 antibodies MS-01, MS-02, MS-03, MS-04, MS-05, MS-06, MS-07, MS-08, MS-09, MS-10, and MS-11 were finally obtained:

[0108] (1) The amino acid sequence of the MS-01 heavy chain variable region is SEQ ID NO: 1;

[0109] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-01 heavy chain variable region are SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively;

[0110] The amino acid sequence of the light chain variable region of MS-01 is SEQ ID NO: 5;

[0111] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of MS-01 are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0112] (2) The amino acid sequence of the MS-02 heavy chain variable region is SEQ ID NO: 9;

[0113] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-02 heavy chain variable region are SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively;

[0114] The amino acid sequence of the light chain variable region of MS-02 is SEQ ID NO: 13;

[0115] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-02 light chain variable region are SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0116] (3) The amino acid sequence of the MS-03 heavy chain variable region is SEQ ID NO: 17;

[0117] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-03 heavy chain variable region are SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively;

[0118] The amino acid sequence of the light chain variable region of MS-03 is SEQ ID NO: 21;

[0119] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-03 light chain variable region are SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

[0120] (4) the amino acid sequence of the MS-04 heavy chain variable region is SEQ ID NO: 25;

[0121] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-04 heavy chain variable region are SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively;

[0122] The amino acid sequence of the light chain variable region of MS-04 is SEQ ID NO: 29;

[0123] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-04 light chain variable region are SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0124] (5) The amino acid sequence of the MS-05 heavy chain variable region is SEQ ID NO: 33;

[0125] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-05 heavy chain variable region are SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively;

[0126] The amino acid sequence of the light chain variable region of MS-05 is SEQ ID NO: 37;

[0127] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-05 light chain variable region are SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

[0128] (6) The amino acid sequence of the MS-06 heavy chain variable region is SEQ ID NO: 41;

[0129] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-06 heavy chain variable region are SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively;

[0130] The amino acid sequence of the light chain variable region of MS-06 is SEQ ID NO: 45;

[0131] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-06 light chain variable region are SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively.

[0132] (7) The amino acid sequence of the MS-07 heavy chain variable region is SEQ ID NO: 49;

[0133] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-07 heavy chain variable region are SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively;

[0134] The amino acid sequence of the light chain variable region of MS-07 is SEQ ID NO: 53;

[0135] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-07 light chain variable region are SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively.

[0136] (8) The amino acid sequence of the MS-08 heavy chain variable region is SEQ ID NO: 57;

[0137] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-08 heavy chain variable region are SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively;

[0138] The amino acid sequence of the light chain variable region of MS-08 is SEQ ID NO: 61;

[0139] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-08 light chain variable region are SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, respectively.

[0140] (9) The amino acid sequence of the MS-09 heavy chain variable region is SEQ ID NO: 65;

[0141] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-09 heavy chain variable region are SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively;

[0142] The amino acid sequence of the light chain variable region of MS-09 is SEQ ID NO: 69;

[0143] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-09 light chain variable region are SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, respectively.

[0144] (10) The amino acid sequence of the MS-10 heavy chain variable region is SEQ ID NO: 73;

[0145] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-10 heavy chain variable region are SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively;

[0146] The amino acid sequence of the light chain variable region of MS-10 is SEQ ID NO: 77;

[0147] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-10 light chain variable region are SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, respectively.

[0148] (11) The amino acid sequence of the MS-11 heavy chain variable region is SEQ ID NO: 81;

[0149] The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the MS-11 heavy chain variable region are SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively;

[0150] The amino acid sequence of the light chain variable region of MS-11 is SEQ ID NO: 85;

[0151] The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the MS-11 light chain variable region are SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88, respectively.

[0152] Example 8: Construction and expression of chimeric antibodies

[0153] The antibody fragments obtained by sequencing were synthesized and constructed into a human IgG framework. Using molecular cloning techniques, the antibody fragments were then inserted into the PCDNA3.1 vector to construct a mammalian cell expression plasmid. This was then introduced into the host cell line, CHO cells, using lipofectamine transfection. The fermentation supernatant was obtained using a fed-batch method and purified through a series of steps, including affinity chromatography and ion exchange chromatography, to ultimately obtain the constructed antibodies. The amino acid sequences of the heavy and light chain variable regions of the chimeric antibodies CH-01 to CH-11 correspond to those of the murine antibodies MS-01 to MS-11, respectively.

[0154] The amino acid sequence of the heavy chain constant region of CH-01, CH-02, CH-03, CH-04, CH-05, CH-06, CH-07, CH-08, CH-09, CH-10 and CH-11 is SEQ ID NO: 89, and the amino acid sequence of the light chain constant region is SEQ ID NO: 90.

[0155] Example 9: Binding activity of chimeric antibodies to tumor cells

[0156] The binding activity of the chimeric antibody to tumor cells was detected according to the method of Example 6.

[0157] The binding activity of chimeric antibodies CH-01, CH-02, CH-07, CH-08, CH-10, and CH-11 to MCF7 cells is shown in Figure 3 The results show that the binding activity of chimeric antibodies CH-01, CH-02, CH-07, CH-08, CH-10, and CH-11 to MCF7 cells is significantly higher than that of control IgG1.

[0158] The binding activity of chimeric antibodies CH-07, CH-08, CH-10, and CH-11 to SKOV3 cells is shown in Figure 4 The results show that the chimeric antibodies CH-07, CH-08, CH-10, and CH-11 have very good binding activity with SKOV3 cells. 50 All were less than 0.5 μg / mL.

[0159] The binding activity of chimeric antibodies CH-01, CH-07, CH-08, and CH-10 to HT55 cells is shown in Figure 5 The results show that the binding activity of chimeric antibodies CH-01, CH-07, CH-08, and CH-10 to HT55 cells is significantly higher than that of the isotype control IgG1.

[0160] Example 10: ADCP effect of chimeric antibodies

[0161] Dilute the antibody with FACS buffer to create a gradient of different concentrations. Use a 100 μL pipette to add 50 μL of the antibody dilutions to each well of a 96-well round-bottom plate.

[0162] The cells were evenly dispersed and counted, and the cells were washed once with PBS. The cell density of tumor cells (MCF7 cells or SKOV3 cells overexpressing CD24 (named: CD24-SKOV3)) was adjusted to 1×10 6 cells / mL, and CFSE was added to a concentration of 1 μM. The cells were incubated at 37°C for 20 min, centrifuged to remove the supernatant, and resuspended in 5 mL of RPMI-1640 + 10% FBS complete medium. After incubation, the cells were centrifuged and resuspended in RPMI-1640 + 10% FBS complete medium for counting, and the cell density was adjusted to 2 × 10 6 Pour the diluted cells into a sterile sample container and add the cells to the above cell plate using a 300 μL 12-pass pipette, 50 μL per well, and the number of tumor cells per well is 1×10 5 / hole.

[0163] Monocytes were induced into macrophages, and the cells were digested with enzymes. The macrophages were made into a cell suspension, centrifuged, and the supernatant was discarded. RPMI-1640 complete medium was added for resuspending and counting. The cell density was adjusted to 1×10 6 Use a 100 μL pipette to add the antibody dilution solution to a 96-well round-bottom plate, 100 μL per well, mix the two cells and the antibody dilution solution, and incubate at 37°C for 3 h.

[0164] Place the incubated 96-well round-bottom plate in a centrifuge and centrifuge, discarding the supernatant. Dilute the secondary anti-CD14 monoclonal antibody (61D3) APC 1:100 in FACS buffer, add 100 μL / well to each well, and incubate at 4°C for 20 minutes. Wash the plate twice with FACS buffer. Detect and analyze the samples. The results are as follows: Figure 6 and Figure 7 shown.

[0165] Depend on Figure 6 and Figure 7 It can be seen that chimeric antibodies CH-01, CH-07, CH-08, and CH-10 can mediate the phagocytosis of MCF7 cells and CD24-SKOV3 cells by macrophages.

[0166] Example 11: In vivo experiments of chimeric antibodies

[0167] A subcutaneous tumor model of ovarian cancer SKOV3 cells in BALB / C nude mice was established to evaluate the in vivo antitumor efficacy of the chimeric antibody CH-07. SKOV3 cells were revived, cultured, and digested to prepare a cell suspension. When the cells reached the logarithmic growth phase, they were harvested and the tumor cell suspension was injected subcutaneously into BALB / C nude mice. Each mouse was inoculated with 100 μL of cell suspension containing 5×10 6 Observe tumor growth and draw a tumor growth curve.

[0168] When the subcutaneous tumor grows to 500mm 3 When the tumor was about 1 hour old, the subcutaneous tumor was removed and cut into 1 mm pieces in culture medium. 3 The tumor tissue was transplanted into the subcutaneous tissue of BALB / C nude mice. 3 At 4 hr, animals were randomly divided into groups according to tumor volume and treated with anti-CD24 antibody.

[0169] The tumor-bearing mice were intraperitoneally injected with antibody CH-07 and PBS after grouping, twice a week, 10.0 mg / kg each time, for a total of 5 times.

[0170] The tumor inhibition efficacy of the compound was evaluated using the TGI (%). The TGI (%) was calculated as follows: TGI (%) = [1 - (average tumor volume of a treatment group at the end of dosing - average tumor volume of the treatment group at the start of dosing) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0171] The tumor growth curve of the mouse ovarian cancer SKOV3 cell subcutaneous tumor model in which the mouse was given the antibody is shown in the figure. Figure 8 The horizontal axis represents the number of days after the start of treatment, and the vertical axis represents the tumor volume. The tumor inhibition rate TGI (%) is greater than 30%.

[0172] Depend on Figure 8 It can be seen that the chimeric antibody CH-07 has a higher tumor inhibitory effect, and its effect is significantly better than that of the PBS group.

[0173] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. An anti-CD24 antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof can bind to CD24 and / or can exert an anti-tumor effect through the antibody Fc fragment; it comprises a heavy chain variable region and a light chain variable region, wherein the HCDR1, HCDR2, HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, LCDR3 of the light chain variable region have the following amino acid sequences: HCDR1 shown in SEQ ID NO: 50, HCDR2 shown in SEQ ID NO: 51, HCDR3 shown in SEQ ID NO: 52, LCDR1 shown in SEQ ID NO: 54, LCDR2 shown in SEQ ID NO: 55, and LCDR3 shown in SEQ ID NO:

56.

2. The anti-CD24 antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region and the light chain variable region have the following amino acid sequences: SEQ ID NO:49 and SEQ ID NO:

53.

3. The anti-CD24 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

4. The anti-CD24 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof, or a chimeric antibody or antigen-binding fragment thereof.

5. The anti-CD24 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody is a monoclonal antibody.

6. The anti-CD24 antibody or antigen-binding fragment thereof according to claim 1, wherein It further comprises an Fc region selected from mouse IgG1, IgG2a, IgG2b and / or IgG3, or selected from rat IgG1, IgG2a, IgG2b and / or IgG2c.

7. The anti-CD24 antibody or antigen-binding fragment thereof according to claim 1, wherein It further comprises an Fc region selected from IgG1, IgG2, IgG3 and / or IgG4.

8. A nucleic acid molecule encoding the anti-CD24 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. A recombinant vector comprising the nucleic acid molecule of claim 8.

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