Antibodies against CD24

Abstract

The present invention relates to anti-CD24 antibodies that bind CD24 in an O-glycosylation dependent manner. Thereby, these antibodies are specific for tumor-associated CD24. The present invention further provides pharmaceutical compositions containing said anti-CD24 antibodies and their use in the treatment of cancer.

Description

Technical Field

[0001] This invention relates to the field of antibodies. Specifically, it provides an anti-CD24 antibody that exhibits strong antigen binding in a tumor-specific manner. In a specific embodiment, this invention relates to anti-CD24 antibodies for therapeutic and diagnostic purposes. Background Technology

[0002] Antibodies are now widely used agents in the medical and research fields. In medicine, they are applied in many different areas. For example, antibodies are used as therapeutic agents for treating and preventing various diseases such as cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infections. In these therapies, antibodies themselves can possess therapeutic activity, for example, by inhibiting their disease-related functions by blocking receptors or messenger molecules, or by recruiting and activating components of a patient's immune system.

[0003] In cancer treatment, a key characteristic of therapeutic antibodies is their specificity to tumor tissue. This means that antibodies should target epitopes that are present only or primarily on cancer cells, but only in small amounts on cells in normal tissues. Thus, the therapeutic activity of the antibody (e.g., inducing an immune response against the target cells or destroying cells through a cytotoxic payload) specifically acts on the tumor site. Producing corresponding activity in normal tissues (due to antibody binding to non-tumor cells) can lead to serious side effects. Increased specificity to tumor cells thus reduces the risk of adverse reactions and enhances the safety of the considered therapy.

[0004] A potential cancer antigen is CD24. This protein is a highly glycosylated adhesion molecule anchored to glycosylphosphatidylinositol (GPI). It was initially identified as a differentiation marker in mice and described as a heat-stable antigen due to its thermostability. Since then, CD24 has been conferred various functions, including roles in adaptive immunity, inflammation, autoimmunity, and cancer. In cancer, CD24 is thought to be involved in cell migration, invasion, and cell proliferation.

[0005] CD24 overexpression has been shown to be associated with malignant phenotypes and poor prognosis in various cancer types, such as breast cancer, non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), cholangiocarcinoma, urothelial carcinoma, ovarian cancer, and prostate cancer. Therefore, CD24 represents an attractive target for anti-tumor immunotherapy.

[0006] However, under normal physiological conditions, CD24 is expressed throughout a variety of tissues, including lymphocytes, epithelial cells, and inflammatory cells. This can lead to undesirable side effects when using antibodies that cannot distinguish between cancer-associated CD24 and CD24 in normal tissues for cancer treatment.

[0007] Therefore, there is a need in the art to provide tumor-specific anti-CD24 antibodies that can distinguish between CD24 on cancer cells and CD24 on normal tissues. Invention Overview

[0009] The inventors have developed anti-CD24 antibodies with enhanced tumor-binding specificity. These antibodies bind to tumor-associated CD24 in an O-glycosylation-dependent manner. O-glycosylation in cancer cells comprises a large number of short-chain structures, particularly the Thomsen-Friedenreich antigen (TF; Galβ1-3GalNAcα1-). In contrast, O-glycosylation in normal cells encompasses much longer oligosaccharide chains. Because the developed antibodies specifically bind to CD24 carrying short, cancer-derived TF structures, they distinguish between tumor-associated CD24 (i.e., CD24 present on cancer cells) and CD24 on cells of normal tissue.

[0010] Given that while tumor-associated short-chain structures are almost entirely absent in most normal tissues, the O-glycosylation of certain blood cells (such as B cells and granulocytes) contains sialylated Thomsen-Friedenreich antigen (sTF) structures, the developed antibody is particularly significant for its high specificity against CD24 cells carrying TF structures. The developed antibody is able to distinguish between closely related TF and sTF structures on CD24, thereby preventing targeting of healthy blood cells expressing sTF-carrying CD24 cells.

[0011] Therefore, the antibody according to the present invention preferentially binds to CD24 on tumor cells, thus exhibiting excellent cancer specificity and reduced binding to normal tissues, resulting in a lower risk of adverse reactions in cancer immunotherapy across a broad patient population. Consequently, the developed antibody is particularly suitable for cancer immunotherapy.

[0012] In view of the foregoing, in a first aspect, the present invention relates to an antibody capable of binding to human CD24 glycosylated at one or more serine residues and / or threonine residues by Galβ1-3GalNAcα1.

[0013] In a second aspect, the present invention relates to an antibody capable of binding to human CD24 and comprising:

[0014] (i) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 5, and CDR-H3 having the amino acid sequence of SEQ ID NO: 10, and

[0015] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 20; or

[0016] (ii) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 6, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and

[0017] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 17, and CDR-L3 having the amino acid sequence of SEQ ID NO: 21; or

[0018] (iii) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and

[0019] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; or

[0020] (iv) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 3, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and

[0021] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or

[0022] (v) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 4, CDR-H2 having the amino acid sequence of SEQ ID NO: 9, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13, and

[0023] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24; or

[0024] (vi) The heavy chain variable region and the light chain variable region according to any one of (i) to (v) above contain a total of 1, 2 or 3 amino acid substitutions in the six CDR sequences.

[0025] In some implementations, the antibody based on the first aspect is based on the antibody based on the second aspect.

[0026] In a third aspect, the present invention provides a conjugate comprising an antibody conjugated with a further reagent according to the first or second aspect of the present invention.

[0027] In a fourth aspect, the present invention provides a nucleic acid encoding an antibody or conjugate according to the first or second aspect of the invention, or according to the third aspect, wherein the further formulation is a polypeptide or protein fused with the antibody. Furthermore, in a fifth aspect, an expression cassette or vector is provided comprising a nucleic acid according to the invention and a promoter operatively linked to the nucleic acid; and in a sixth aspect, a host cell is provided comprising the nucleic acid or expression cassette or vector according to the invention.

[0028] In a seventh aspect, the present invention relates to compositions comprising antibodies according to the first or second aspect of the invention, conjugates according to the third aspect of the invention, nucleic acids according to the fourth aspect of the invention, expression cassettes or vectors according to the fifth aspect of the invention, or host cells according to the sixth aspect of the invention. These compositions are particularly pharmaceutical compositions.

[0029] According to the eighth aspect, the present invention provides antibodies, conjugates, nucleic acids, expression cassettes or vectors, host cells or compositions according to the invention for use in medicine, particularly for the treatment of cancer, bacterial or viral infections, or vascular diseases.

[0030] Other objects, features, advantages, and aspects of the invention will become apparent to those skilled in the art from the following description and appended claims. However, it should be understood that the specific embodiments of the preferred embodiments indicated in the following description, appended claims, and examples are given by way of illustration only. Various changes and modifications will become apparent to those skilled in the art upon reading the following, and these changes and modifications are within the spirit and scope of the disclosed invention.

[0031] definition

[0032] The following expressions used herein are generally preferred to have the meanings described below, unless the context in which they are used indicates otherwise.

[0033] As used herein, the expression “comprising” includes, in addition to its literal meaning, meaning “substantially constitutes” and “consisting of”. Therefore, the expression “comprising” means an implementation in which the subject matter “comprising” specifically listed elements does not contain further elements, and an implementation in which the subject matter “comprising” specifically listed elements may and / or does cover further elements. Similarly, the expression “having” should be understood to mean that the expression “comprising” also includes and specifically refers to meaning “substantially constitutes” and “consisting of”. Where possible, the term “substantially constitutes” specifically refers to an implementation in which the subject matter contains 20% or less, particularly 15% or less, 10% or less, or especially 5% or less of further elements besides the specifically listed elements in which the subject matter substantially constitutes.

[0034] The term "antibody" specifically refers to a protein comprising at least two heavy chains and two light chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The heavy chain constant region contains three heavy chain constant domains or (in the case of IgM or IgE antibodies) four heavy chain constant domains (CH1, CH2, CH3, and CH4), wherein the first constant domain CH1 is adjacent to the variable region and can be linked to the second constant domain CH2 via a hinge region. The light chain constant region consists of only one constant domain. The variable region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are alternated with more conserved regions called framework regions (FRs), wherein each variable region contains three CDRs and four FRs. The amino acid residues of the CDRs are determined, in particular, based on CDR localization according to the IMGT system.

[0035] The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the heavy chain can be of any type, such as γ-, δ-, α-, μ-, or ε-type heavy chains. Preferably, the heavy chain of the antibody is a γ-chain. Furthermore, the constant region of the light chain can also be of any type, such as a κ- or λ-type light chain. Preferably, the light chain of the antibody is a κ-chain. The terms “γ-(δ-, α-, μ-, or ε-) type heavy chain” and “κ-(λ-) type light chain” respectively refer to antibody heavy chains or antibody light chains having a constant region amino acid sequence derived from the amino acid sequence of a naturally occurring heavy chain constant region or light chain constant region (particularly the amino acid sequence of a human heavy chain constant region or light chain constant region). Specifically, the amino acid sequence of the constant domain of the γ-type (particularly γ1-type) heavy chain is at least 95%, particularly at least 98%, identical to the amino acid sequence of the constant domain of the human γ (particularly an allotype of human γ1) antibody heavy chain. Furthermore, the amino acid sequence of the constant domain of the κ-type light chain is at least 95%, and particularly at least 98%, identical to the amino acid sequence of the constant domain of an allotype of the human κ antibody light chain. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system. The antibody can be, for example, a humanized antibody, a human antibody, or a chimeric antibody.

[0036] The antigen-binding portion of an antibody typically refers to a full-length antibody or one or more fragments of an antibody that retain the ability to specifically bind antigens. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody-binding fragments include Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by disulfide bonds in their hinge regions, each binding the same antigen; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; and dAb fragments consisting of VH domains.

[0037] The term "Fab moiety" of an antibody specifically refers to the antibody moiety comprising the first domain of the heavy chain variable region and the light chain variable region (VH and VL), as well as the first domain of the heavy chain constant region and the light chain constant region (CH1 and CL). In cases where the antibody does not contain all of these regions, the term "Fab moiety" refers only to those regions of VH, VL, CH1, and CL present in the antibody. Preferably, "Fab moiety" refers to the antibody moiety containing the antigen-binding activity corresponding to the fragment obtained by digesting a natural antibody with papain. Specifically, the Fab moiety of an antibody encompasses its antigen-binding site or antigen-binding capacity. Preferably, the Fab moiety comprises at least the first domain of the heavy chain variable region and the first domain of the light chain constant region (CH1 and CL) of the antibody. H area.

[0038] The term "Fc portion" of an antibody specifically refers to a portion of the antibody containing heavy chain constant regions 2, 3, and (where applicable) 4 (CH2, CH3, and CH4). Specifically, the Fc portion contains two of each of these regions. If the antibody does not contain all of these regions, the term "Fc portion" refers only to those regions of CH2, CH3, and CH4 present in the antibody. Preferably, the Fc portion contains at least the CH2 region of the antibody. Preferably, "Fc portion" refers to an antibody portion that does not contain antigen-binding activity corresponding to a fragment obtained by digesting a natural antibody with papain. Specifically, the Fc portion of the antibody is capable of binding to an Fc receptor and therefore, for example, contains an Fc receptor binding site or Fc receptor binding capacity.

[0039] According to the present invention, the term "chimeric antibody" specifically refers to an antibody in which the constant region is derived from a human antibody or a human antibody common sequence, and in which at least one variable region, preferably two variable regions, is derived from a non-human antibody (e.g., derived from rodent antibodies such as mouse antibodies).

[0040] As used herein, the term "antibody" may refer in some embodiments to a group of antibodies of the same type. Specifically, all antibodies in the antibody group exhibit the characteristics used to define that antibody. In some embodiments, all antibodies in the antibody group have the same amino acid sequence. References to specific types of antibodies, such as anti-CD24 antibodies, specifically refer to a group of antibodies of that type.

[0041] As used herein, the term "antibody" includes full-length antibodies as well as fragments and derivatives of said antibodies. A "fragment or derivative" of an antibody is specifically a protein or glycoprotein derived from said antibody and capable of binding the same antigen, particularly the same epitope as that antibody. Therefore, as used herein, a fragment or derivative of an antibody generally refers to a functional fragment or derivative. In a particularly preferred embodiment, the fragment or derivative of the antibody comprises a heavy chain variable region. It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody or a derivative thereof. Examples of antibody fragments include: (i) Fab fragments, monovalent fragments consisting of variable regions and a first constant domain in each heavy and light chain; (ii) F(ab)2 fragments, divalent fragments comprising two Fab fragments linked by disulfide bonds through hinge regions; (iii) Fd fragments consisting of variable regions in the heavy chain and the first constant domain CH1; (iv) Fv fragments consisting of variable regions in the heavy and light chains of a single arm of the antibody; (v) scFv fragments, Fv fragments consisting of a single polypeptide chain; (vi) (Fv)2 fragments consisting of two covalently linked Fv fragments; (vii) heavy chain variable domains; and (viii) multibody fragments consisting of heavy chain variable regions and light chain variable regions covalently linked together in a manner that allows only intermolecular, not intramolecular, association. Antibody derivatives particularly include antibodies that bind to the same antigen as the parent antibody but have a different amino acid sequence than the parent antibody from which they are derived. These antibody fragments and derivatives are obtained using conventional techniques known to those skilled in the art.

[0042] If a target amino acid sequence has at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity with the corresponding portion of a reference amino acid sequence over its entire length, then the target amino acid sequence is “derived from” or “corresponds to” the reference amino acid sequence. “Corresponding portion” means, for example, that frame region 1 (FRH1) of the heavy chain variable region of the target antibody corresponds to frame region 1 of the heavy chain variable region of the reference antibody. In a specific embodiment, a target amino acid sequence “derived from” or “corresponds to” the reference amino acid sequence is 100% homologous, or particularly 100% identical, to the corresponding portion of the reference amino acid sequence over its entire length. The “homology” or “identity” of an amino acid sequence or nucleotide sequence is preferably determined according to the invention over the entire length of the reference sequence or over the entire length of a corresponding portion of the reference sequence, which corresponds to the sequence defining its homology or identity. Antibodies derived from parent antibodies, defined by one or more amino acid sequences (such as specific CDR sequences or specific variable region sequences), particularly antibodies having such amino acid sequences (such as CDR sequences or variable region sequences) that are at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homologous to or identical, particularly identical to, the corresponding amino acid sequence of the parent antibody. In some embodiments, antibodies derived from parent antibodies (i.e., derivatives of parent antibodies) contain the same CDR sequence as the parent antibody, but differ in the remaining sequences of the variable region.

[0043] The term "antibody" as used in this article also refers to multivalent antibodies and multispecific antibodies, namely antibody constructs having more than two binding sites that each bind to the same epitope, and antibody constructs having one or more binding sites that bind to a first epitope and one or more binding sites that bind to a second epitope, and optionally or even further binding sites that bind to other epitopes.

[0044] "Specific binding" preferably refers to a reagent (such as an antibody) binding more strongly to its specific target (such as an epitope) than to another target. If the dissociation constant (K) of a reagent binding to the first target is... d If the dissociation constant of the reagent with the first target is lower than that with the second target, then the binding of the reagent to the first target is stronger than that to the second target. Preferably, the dissociation constant of the target to which the reagent specifically binds is 10, 30, 100, or more than 500 times lower than that of the target to which the reagent non-specifically binds. Furthermore, the term "specific binding" specifically refers to the binding affinity between binding partners, where the affinity constant K is... a At least 10 5 M -1 Preferably at least 10 6 M-1 More preferably at least 10 7 M -1 For example, at least 10 8 M -1 Antibodies that specifically target a particular antigen, especially those capable of producing antibodies with at least 10... 5 M -1 Preferably at least 10 6 M -1 More preferably at least 10 7 M -1 K a Antibodies that bind to the antigen with an affinity of at least 10. For example, the term "anti-CD24 antibody" refers to an antibody that specifically binds to CD24, and preferably has an affinity of at least 10. 5 M -1 Preferably at least 10 6 M -1 More preferably at least 10 7 M -1 K a It binds to CD24 with its affinity.

[0045] As used in this article, the term "epitope" refers to the amino acid residues and glycan structures on the antigen of an antibody that are directly contacted by the amino acids of the antibody (especially the amino acids of the antibody's CDR), or to those amino acid residues and glycan structures in their immediate vicinity that affect the binding of the antibody to its antigen.

[0046] According to the present invention, the term "CD24" specifically refers to the human CD24 protein, particularly the mature human CD24 protein, including all its variants. As used herein, CD24 specifically refers to the human CD24 protein corresponding to UniProt entry P25063. CD24 specifically comprises the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence that is at least 90%, particularly at least 95%, identical in length to SEQ ID NO: 58. In some embodiments, "CD24" refers to the most relevant isoform 1 of human CD24, which consists of the amino acid sequence of SEQ ID NO: 58. CD24 is particularly post-translational modified and may carry O-glycosylation and / or N-glycosylation. For example, CD24 may carry O-glycosylation at one or more positions corresponding to Thr4, Thr5, Ser8, Ser9, Ser11, Ser12, Ser14, Thr15, Ser16, Ser18, Thr25, Thr28, and Thr29 of SEQ ID NO: 58.

[0047] The term “GalNAcα1-” (also known as “Tn”, “Tn antigen”, or “Thomsen nouvelle antigen”) refers to a monosaccharide structure consisting of N-acetylgalactosamine residues linked by α-glycosidic bonds to a supporting structure (especially serine or threonine residues of proteins or peptides).

[0048] The term "sialylated GalNAcα1-" (also known as "sTn", "sialylated Tn antigen", or "sialylated Thomsen nouvelle antigen") refers to a disaccharide structure consisting of N-acetylgalactosamine residues linked by α-glycosidic bonds to a supporting structure (especially serine or threonine residues of proteins or peptides). In this structure, sialic acid residues are linked to N-acetylgalactosamine residues via α2-6 bonds, forming the disaccharide structure Siaα2-6GalNAcα1-.

[0049] The term “Galβ1-3GalNAcα1-” (also known as “TF”, “TF antigen”, “T antigen”, or “Thomsen-Friedenreich antigen”) refers to a disaccharide structure consisting of a galactose residue linked to an N-acetylgalactosamine residue via a β1-3 bond, which in turn is linked to a supporting structure (especially a serine or threonine residue of a protein or peptide) via an α1-glycosidic bond.

[0050] The term “sialylated Galβ1-3GalNAcα1-” (also known as “sTF”, “sTF antigen”, “sialylated TF antigen”, “sialylated T antigen”, or “sialylated Thomsen Friedenreich antigen”) refers to a trisaccharide or tetrasaccharide structure consisting of a galactose residue linked to an N-acetylgalactosamine residue via a β1-3 bond, which in turn is linked to a supporting structure (especially a serine or threonine residue of a protein or peptide) via an α1-glycosidic bond. In this structure, sialic acid residues are linked to galactose residues via α2-3 bonds, and / or sialic acid residues are linked to N-acetylgalactosamine residues via α2-6 bonds, forming trisaccharide structures Siaα2-3Galβ1-3GalNAcα1- and Galβ1-3(Siaα2-6)GalNAcα1- or tetrasaccharide structures Siaα2-6(Siaα2-3Galβ1-3)GalNAcα1-.

[0051] In the above structure, Gal represents a galactose residue, and GalNAc represents an N-acetylgalactosamine residue. "β1-3", "α2-3", and "α2-6" represent the connecting bonds between two adjacent monosaccharide residues, especially between carbon atom C1 or C2 of the monosaccharide on the left and carbon atom C3 or C6 of the monosaccharide on the right. This connecting bond can be at the α or β position (as shown in the schematic diagram of glucose below).

[0052]

[0053] The term "GalNAcα1-" indicates that the GalNAc residue at the reducing end of the oligosaccharide is attached to the supporting structure in an α configuration via its carbon atom C1.

[0054] The term "sialic acid" specifically refers to any N- or O-substituted derivative of neuraminic acid. It can refer to both 5-N-acetylneuraminic acid (NeuNAc) and 5-N-hydroxyacetylneuraminic acid (NeuGc), but preferably only 5-N-acetylneuraminic acid.

[0055] The terms “polysaccharide,” “polysaccharide structure,” “sugar,” “sugar chain,” and “sugar structure” are generally used synonymously in this document.

[0056] In a "conjugate," two or more compounds are linked together. In some embodiments, the conjugate retains at least a portion of the properties from each compound. The linking can be achieved by covalent or non-covalent bonds. Preferably, the compounds of the conjugate are linked by covalent bonds. The different compounds of the conjugate can be directly linked to each other by one or more covalent bonds between the atoms of the compounds. Alternatively, the compounds can be linked to each other by chemical parts (such as linking molecules), where the linker is covalently attached to the atoms of the compounds. If the conjugate consists of more than two compounds, these compounds can be linked, for example, in a chain conformation, with one compound linking to the next, or several compounds can each be linked to a central compound.

[0057] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids, as well as ribonucleic acid and deoxyribonucleic acid. It can contain naturally occurring nucleotides and synthetic nucleotides, and can be naturally or synthetically modified (e.g., through methylation, 5' capping, and / or 3' capping).

[0058] The term "expression cassette" specifically refers to a nucleic acid construct capable of expressing and regulating the expression of a coding nucleic acid sequence introduced therein. Expression cassettes may contain promoters, ribosome binding sites, enhancers, and other regulatory elements that regulate gene transcription or mRNA translation. The exact structure of an expression cassette can vary depending on species or cell type, but typically includes 5' non-transcriptional sequences involved in transcription and translation initiation, as well as 5' and 3' untranslated regions, such as TATA boxes, capped sequences, and CAAT sequences. More specifically, the 5' non-transcriptional expression regulatory sequence contains a promoter region containing a promoter sequence for the operational linker nucleic acid used in transcriptional regulation. Expression cassettes may also contain enhancer sequences or upstream activator sequences.

[0059] According to the present invention, the term "promoter" refers to a nucleic acid sequence located upstream (5') of the nucleic acid sequence to be expressed and regulating the expression of that sequence by providing a recognition and binding site for RNA polymerase. A "promoter" may also contain additional recognition and binding sites for other factors involved in the regulation of gene transcription. Promoters can regulate the transcription of prokaryotic or eukaryotic genes. Furthermore, promoters can be "inducible," i.e., initiating transcription in response to an inducer, or they can be "constitutive," in which case transcription is not regulated by an inducer. Genes regulated by inducible promoters are not expressed or are expressed only in small amounts in the absence of an inducer. In the presence of an inducer, the gene is activated or the transcriptional level increases. This is typically mediated by the binding of specific transcription factors.

[0060] The term "vector" as used herein is used in its most general sense and includes any intermediate medium for nucleic acids that enables the nucleic acids to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. Such vectors preferably replicate and / or are expressed in cells. Vectors include plasmids, phage particles, bacteriophages, or viral genomes. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material (typically a circular DNA double strand) that can replicate independently of chromosomal DNA.

[0061] According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. According to the present invention, the term "host cell" includes prokaryotic cells (e.g., *Escherichia coli*). E. coli Nucleic acid may be a single or multiple copy in the host cell, and in one embodiment, the nucleic acid is expressed in the host cell. The cell may be derived from a variety of tissue types and includes primary cells and cell lines.

[0062] According to the present invention, the term "patient" refers to a human, a non-human primate or other animal, particularly a mammal such as a cow, horse, pig, sheep, goat, dog, cat, or rodent such as a mouse and rat. In a particularly preferred embodiment, the patient is a human.

[0063] According to the present invention, the term "cancer" specifically includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, bowel cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, oral cancer, bladder cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer and their metastases. According to the present invention, the term cancer also includes cancer metastases. The term cancer further refers to and / or includes cancer stem cells, especially cancer stem cells of the specific types of cancer listed above.

[0064] A tumor is a group of cells or tissues that form due to disordered cell proliferation. Tumors can exhibit partial or complete loss of the structural organization and functional coordination of normal tissues, and usually form a distinct tissue mass, which can be benign or malignant.

[0065] Metastasis refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process and typically involves cancer cells detaching from the primary tumor, entering the systemic circulation, and settling and growing in normal tissues in other parts of the body. When tumor cells metastasize, the new tumor is called a secondary tumor or metastatic tumor, and its cells are usually similar to those in the original tumor. For example, this means that if breast cancer metastasizes to the lungs, the secondary tumor is composed of abnormal breast cells, not abnormal lung cells. The tumor in the lungs is called metastatic breast cancer, not lung cancer.

[0066] The term "pharmaceutical composition" specifically refers to a composition suitable for administration to humans or animals, i.e., a composition containing pharmaceutically acceptable components. Preferably, the pharmaceutical composition contains an active compound or a salt or prodrug thereof, as well as a carrier, diluent, or pharmaceutical excipient, such as a buffer, preservative, and tension modifier. Invention Details

[0068] This invention is based on the development of anti-CD24 antibodies that specifically bind to tumor-associated CD24. These antibodies are generated using CD24 or CD24 fragments carrying O-glycan structures produced by cancer cells and by screening for antibodies that bind CD24 in an O-glycosylation-dependent manner. O-glycosylation in normal cells differs significantly from that produced by cancer cells. Most normal cells produce large O-glycan structures, while tumor cells contain numerous short-chain structures, especially monosaccharides and disaccharides, such as Thomsen-Friedenreich antigen (TF; Galβ1-3GalNAcα1-), Thomsen nouvelle antigen (Tn; GalNAcα1-), and sialylated Thomsen nouvelle antigen (sTn). The developed antibodies specifically bind to CD24 carrying short-chain, cancer cell-derived TF structures, thus distinguishing tumor-associated CD24 (i.e., CD24 present on cancer cells) from CD24 on normal tissue cells. The developed antibody is particularly significant because it specifically binds to CD24 with the glycosylation structure TF, but not to CD24 with the glycosylation structure sialylated Thomsen-Friedenreich antigen (sTF), which is present in the glycosylation pattern of CD24 expressed by certain blood cells, such as B cells and granulocytes. Therefore, the antibody according to the invention preferentially binds to CD24 on tumor cells, thus exhibiting excellent cancer specificity and reduced binding to normal tissues, resulting in a lower risk of adverse reactions in cancer immunotherapy. Therefore, the developed antibody is particularly suitable for cancer immunotherapy in a broad patient population.

[0069] 1. Anti-CD24 antibody

[0070] In view of these findings, the present invention provides an antibody in a first aspect that is capable of binding to human CD24 glycosylated by Galβ1-3GalNAcα1 at one or more serine residues and / or threonine residues.

[0071] In some embodiments, the antibody is capable of specifically binding to human CD24 glycosylated at one or more serine and / or threonine residues by at least one oligosaccharide structure selected from the group consisting of GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated GalNAcα1-. In some embodiments, the antibody is capable of specifically binding to human CD24 glycosylated at one or more serine and / or threonine residues by Galβ1-3GalNAcα1-.

[0072] In a specific embodiment, the antibody is capable of binding to human CD24 glycosylated by any one of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, or a mixture thereof. Glycosylation as described herein generally refers to a glycan structure linked to serine and / or threonine residues on CD24. These serine and / or threonine residues are particularly selected from the group comprising Thr4, Thr5, Ser8, Ser9, Ser11, Ser12, Ser14, Thr15, Ser16, Ser18, Thr25, Thr28, and Thr29 of SEQ ID NO: 58.

[0073] Antibodies capable of binding to glycosylated human CD24 exhibit a higher binding affinity to glycosylated human CD24 than to non-glycosylated human CD24. In some embodiments, the binding affinity of antibodies capable of binding to glycosylated human CD24 is higher than that of unrelated proteins carrying the same glycan structure, and / or higher than that of unrelated carrier molecules carrying the same glycan structure. For example, the carrier molecule may be polyacrylamide (PAA) or a random peptide.

[0074] In some embodiments, antibodies capable of binding to human CD24 glycosylated at one or more serine and / or threonine residues (Galβ1-3GalNAcα1-glycosylated) exhibit a higher binding affinity to glycosylated human CD24 than to human CD24 glycosylated with O-glycans from healthy human cells. In some embodiments, antibodies capable of binding to human CD24 glycosylated at one or more serine and / or threonine residues (Galβ1-3GalNAcα1-glycosylated) exhibit a higher binding affinity to glycosylated human CD24 than to human CD24 glycosylated with sialylated Galβ1-3GalNAcα1-glycosylated. O-glycosylation of normal human cells from healthy tissues specifically refers to glycans containing 5, 6, 7 or more monosaccharide units, particularly selected from the group consisting of: extended core-1 structures, such as Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-3GalNAcα-; extended core-2 structures, such as NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-3)GalNAcα- and Neu5Acα2-3Galβ1-3(Galβ1-4(Fucα1-3)GlcNAc(β1-6)G alNAcα-; and extended core-3 structures, such as Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3GalNAcα-. As used herein, the term "higher than" means that the binding affinity of the reagent to the first target is higher than that to the second target. If the dissociation constant (Kd) of the reagent with the first target is lower than that with the second target, then the binding affinity of the reagent to the first target is higher than that to the second target. As used herein, "higher than" specifically means a difference in dissociation constant of at least 10-fold, particularly at least 25-fold, and especially at least 100-fold.

[0075] In some embodiments, the antibody is capable of binding to human CD24 comprising more than one oligosaccharide structure, such as 2, 3, or 4 oligosaccharide structures linked to serine and / or threonine residues of CD24. One or more of these other oligosaccharide structures may also be selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, and Galβ1-3GalNAcα1-, especially Galβ1-3GalNAcα1-. In a specific embodiment, the antibody is capable of binding to human CD24 comprising at least two oligosaccharide structures, each selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, and Galβ1-3GalNAcα1-, and each structure linked to a serine or threonine residue. In particular, the antibody is able to bind to human CD24 containing at least three oligosaccharide structures, each selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, and Galβ1-3GalNAcα1-, and each structure is linked to a serine or threonine residue of CD24.

[0076] In some embodiments, the antibody is capable of binding to human CD24 containing more than one Galβ1-3GalNAcα1- structure, such as 2, 3, or 4 Galβ1-3GalNAcα1- structures, which are linked to serine and / or threonine residues of CD24. In specific embodiments, the antibody is capable of binding to human CD24 containing at least two Galβ1-3GalNAcα1- structures, each linked to a serine or threonine residue of CD24. Specifically, the antibody is capable of binding to human CD24 containing at least three Galβ1-3GalNAcα1- structures, each linked to a serine or threonine residue of CD24.

[0077] This antibody can bind to a peptide-glycoside epitope of human CD24. This epitope contains one or more amino acids of SEQ ID NO: 58, particularly three or more amino acids of SEQ ID NO: 58, particularly five or more amino acids of SEQ ID NO: 58. In a specific embodiment, at least 75%, particularly at least 85%, particularly at least 90% of the amino acids in the epitope are present in SEQ ID NO: 58.

[0078] In this regard, the term "capable of binding to a peptide-glycosylated epitope of human CD24" means that the antibody either interacts directly with the amino acids and one or more glycan structures of CD24, or interacts only directly with the amino acids of CD24. In some aspects, if one or more Galβ1-3GalNAcα1- structures are attached to the amino acids of CD24, the antibody has a higher binding affinity for CD24 than for non-glycosylated CD24. In embodiments where the antibody interacts only directly with the amino acids of CD24 rather than the glycan chain, the higher affinity for O-glycosylated CD24 is based on conformational changes in the polypeptide chain induced by the oligosaccharides attached to the amino acids of CD24.

[0079] In some embodiments, CD24 is present on the cell surface. Therefore, in some embodiments, the antibody is capable of binding to human CD24 glycosylated with Galβ1-3GalNAcα1 at one or more serine and / or threonine residues as described herein, wherein the human CD24 is present on the cell surface. In some embodiments, the antibody specifically binds to tumor-associated CD24. In some embodiments, the antibody is capable of binding to tumor-associated CD24 with a higher binding affinity than to CD24 expressed in normal tissue cells.

[0080] In a specific embodiment, the antibody is capable of binding to human CD24 glycosylated with Galβ1-3GalNAcα1-. Therefore, the antibody binds to Galβ1-3GalNAcα1-glycosylated CD24. In these embodiments, CD24 may also carry other glycan structures, provided that Galβ1-3GalNAcα1- is also present. Specifically, these antibodies specifically bind to human CD24 glycosylated with Galβ1-3GalNAcα1-.

[0081] In a second aspect, the present invention provides an antibody capable of binding to human CD24 and comprising:

[0082] (i) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 5, and CDR-H3 having the amino acid sequence of SEQ ID NO: 10, and

[0083] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 20; or

[0084] (ii) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 6, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and

[0085] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 17, and CDR-L3 having the amino acid sequence of SEQ ID NO: 21; or

[0086] (iii) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and

[0087] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; or

[0088] (iv) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 3, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and

[0089] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or

[0090] (v) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 4, CDR-H2 having the amino acid sequence of SEQ ID NO: 9, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13, and

[0091] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24; or

[0092] (vi) The heavy chain variable region and the light chain variable region according to any one of (i) to (v) above contain a total of 1, 2 or 3 amino acid substitutions in the six CDR sequences.

[0093] In specific embodiments, the anti-CD24 antibody may have a total of 1, 2, or 3 amino acid substitutions in the six CDR sequences, particularly 1 or 2, especially 1 amino acid substitution. In these embodiments, the anti-CD24 antibody retains the antigen specificity of an antibody without said amino acid substitution. As used herein, "amino acid substitution" also includes amino acid addition and amino acid deletion. In some embodiments, the amino acid substitution is a conserved amino acid substitution.

[0094] The "antibody" according to the second aspect of the invention specifically exhibits one or more binding activities against the antibody defined in the first aspect. Specifically, the antibody according to the second aspect is the antibody according to the first aspect.

[0095] In a specific embodiment, the antibody according to the invention comprises a heavy chain variable region containing a complementarity-determining region (CDR): CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain variable region containing a complementarity-determining region (CDR): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22.

[0096] In some embodiments, the antibody according to the invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

[0097] (i) The heavy chain variable region, which is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 25, and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 5, and CDR-H3 having the amino acid sequence of SEQ ID NO: 10; and

[0098] The light chain variable region is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 38 and includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 20; or

[0099] (ii) A heavy chain variable region that is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 27 and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 6, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and

[0100] The light chain variable region is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 44 and includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 17, and CDR-L3 having the amino acid sequence of SEQ ID NO: 21; or

[0101] (iii) A heavy chain variable region that is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 30 and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and

[0102] The light chain variable region is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 47 and includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; or

[0103] (iv) A heavy chain variable region that is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 35 and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 3, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 12; and

[0104] The light chain variable region is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 55 and includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or

[0105] (v) A heavy chain variable region that is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 36 and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 4, CDR-H2 having the amino acid sequence of SEQ ID NO: 9, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13; and

[0106] The light chain variable region is at least 60% identical in length to the amino acid sequence of SEQ ID NO: 56 and contains the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24.

[0107] In the above embodiments, sequence identity can be, in particular, at least 70%, preferably at least 80%, and more preferably at least 90%. The above embodiments are particularly humanized versions of the respective antibodies, wherein the changes in the amino acid sequence are substitutions of amino acid residues of the relevant human antibody sequence.

[0108] In some embodiments, the anti-CD24 antibody may additionally have a total of 1, 2, or 3 amino acid substitutions in the six CDR sequences, particularly 1 or 2, especially 1 amino acid substitution. In these embodiments, the anti-CD24 antibody retains the antigen specificity of the antibody without the aforementioned amino acid substitutions.

[0109] In a further embodiment, the antibody according to the invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

[0110] (i) The heavy chain variable region, which is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 25, and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 5, and CDR-H3 having the amino acid sequence of SEQ ID NO: 10; and

[0111] The light chain variable region is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 38 and includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 20; or

[0112] (ii) A heavy chain variable region that is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 27 and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 6, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and

[0113] The light chain variable region is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 44 and includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 17, and CDR-L3 having the amino acid sequence of SEQ ID NO: 21; or

[0114] (iii) A heavy chain variable region that is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 30 and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and

[0115] The light chain variable region is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 47 and includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; or

[0116] (iv) A heavy chain variable region that is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 35 and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 3, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 12; and

[0117] The light chain variable region is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 55 and includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or

[0118] (v) A heavy chain variable region that is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 36 and comprises the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 4, CDR-H2 having the amino acid sequence of SEQ ID NO: 9, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13; and

[0119] The light chain variable region is at least 90% identical in length to the amino acid sequence of SEQ ID NO: 56 and contains the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24.

[0120] In the above embodiments, sequence identity can be, in particular, at least 95%. In some embodiments, the anti-CD24 antibody may additionally have a total of 1, 2, or 3 amino acid substitutions in the six CDR sequences, particularly 1 or 2, especially 1 amino acid substitution. In these embodiments, the anti-CD24 antibody retains the antigen specificity of the antibody without the aforementioned amino acid substitutions.

[0121] In a specific embodiment, the antibody according to the invention comprises a heavy chain variable region that is at least 80%, particularly 90%, identical in length to the amino acid sequence of SEQ ID NO: 30, and comprises complementarity-determining regions (CDRs) having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and comprises a light chain variable region that is at least 80%, particularly 90%, identical in length to the amino acid sequence of SEQ ID NO: 47, and comprises complementarity-determining regions (CDRs) having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22.

[0122] Specifically, the antibody according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein...

[0123] (i) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 25, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 38; or

[0124] (ii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 44; or

[0125] (iii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 30, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 47; or

[0126] (iv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 35, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 55; or

[0127] (v) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 56; or

[0128] (vi) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 37, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 56; or

[0129] (vii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 37, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 57; or

[0130] (viii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 57.

[0131] In some embodiments, the antibody is a humanized version of any one of the antibodies described in (i) to (viii). This humanized version, in particular, has at least 60%, especially at least 70%, preferably at least 80%, and more preferably at least 90% amino acid sequence identity with the corresponding antibody over the entire original sequence length.

[0132] In a specific embodiment, the antibody according to the present invention is a humanized antibody, comprising a heavy chain variable region and a light chain variable region, wherein...

[0133] (i) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 39; or

[0134] (ii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 40; or

[0135] (iii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 41; or

[0136] (iv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 42; or

[0137] (v) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 43; or

[0138] (vi) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 28, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 45; or

[0139] (vii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 29, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 46; or

[0140] (viii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 48; or

[0141] (ix) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 32, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 48; or

[0142] (x) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 49; or

[0143] (xi) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 50; or

[0144] (xii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 51; or

[0145] (xiii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 52; or

[0146] (xiv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 33, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 53; or

[0147] (xv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 34, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 54.

[0148] In some embodiments, the antibody according to the invention is a humanized antibody that has at least 90%, especially at least 95%, more preferably at least 97% amino acid sequence identity with any of the aforementioned humanized antibodies over the entire length of the original heavy chain variable region sequence and the entire original light chain variable region sequence.

[0149] In embodiments where the heavy chain variable region and / or light chain variable region contains an amino acid sequence having a certain identity with any of the amino acid sequences in SEQ ID NO: 25 to 57, any sequence deviation from said amino acid sequence is located, in particular, in the frame region and not in the CDR. Therefore, in these embodiments, the heavy chain variable region and light chain variable region contain the corresponding CDR sequence as defined herein.

[0150] In a specific embodiment, the antibody according to the present invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 31 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48.

[0151] In a specific embodiment, the antibody according to the present invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 32 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48.

[0152] In a specific embodiment, the antibody according to the present invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 31 and a light chain variable region having the amino acid sequence of SEQ ID NO: 49.

[0153] In a specific embodiment, the antibody according to the present invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 31 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50.

[0154] In a specific embodiment, the antibody according to the present invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 31 and a light chain variable region having the amino acid sequence of SEQ ID NO: 51.

[0155] In a specific embodiment, the antibody according to the present invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 31 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52.

[0156] In a specific embodiment, the antibody according to the present invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 33 and a light chain variable region having the amino acid sequence of SEQ ID NO: 53.

[0157] In a specific embodiment, the antibody according to the present invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 34 and a light chain variable region having the amino acid sequence of SEQ ID NO: 54.

[0158] In a specific implementation, the antibody is capable of binding to human CD24 glycosylated by any one of Galβ1-3GalNAcα1- and / or GalNAcα1-, and comprises a light chain variable region and a heavy chain variable region selected from the group consisting of:

[0159] (i) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 3, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and

[0160] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or

[0161] (ii) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 4, CDR-H2 having the amino acid sequence of SEQ ID NO: 9, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13, and

[0162] The light chain variable region includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24.

[0163] In a specific embodiment, the antibody is capable of binding to human CD24 glycosylated by Galβ1-3GalNAcα1, particularly specifically binding to human CD24 glycosylated by Galβ1-3GalNAcα1, and comprises a light chain variable region and a heavy chain variable region selected from the group consisting of:

[0164] (i) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 5, and CDR-H3 having the amino acid sequence of SEQ ID NO: 10, and

[0165] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 20; or

[0166] (ii) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 6, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and

[0167] Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 17, and CDR-L3 having the amino acid sequence of SEQ ID NO: 21; or

[0168] (iii) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and

[0169] The light chain variable region includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22.

[0170] In the above embodiments, sequence identity can be, in particular, at least 70%, preferably at least 80%, and more preferably at least 90%. In the above embodiments involving sequence identity, the antibody is, in particular, a humanized version of each corresponding antibody, wherein the change in the amino acid sequence is the substitution of amino acid residues with those of the relevant human antibody sequence.

[0171] In some embodiments, the anti-CD24 antibody may additionally have a total of 1, 2, or 3 amino acid substitutions in the six CDR sequences, particularly 1 or 2, especially 1 amino acid substitution. In these embodiments, the anti-CD24 antibody retains the antigen specificity of the antibody without the aforementioned amino acid substitutions.

[0172] In some embodiments, the antibody comprises an Fc region. The antibody can be, in particular, an intact antibody. Specifically, the antibody may comprise two heavy chains and two light chains. The antibody can be any isotype, and is particularly an IgG antibody, especially IgG1, IgG2, or IgG4. In a specific embodiment, the antibody is an IgG1 antibody. The antibody is particularly capable of binding to one or more human Fc receptors, especially human Fcγ receptors such as Fcγ receptor IIIa. In some embodiments, the anti-CD24 antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the binding of the antibody to human Fc receptors is reduced or eliminated. For example, this can be achieved by mutating and deleting the glycosylation site in the CH2 domain of the antibody (e.g., by replacing Asn297 with, for example, Gln). In another embodiment, the antibody contains an L234A / L235A double mutation in the heavy chain.

[0173] In a further embodiment, the anti-CD24 antibody is an antibody fragment. Specifically, the fragment is selected from the group consisting of: (i) a Fab fragment; (ii) an F(ab)2 fragment; (iii) an Fd fragment; (iv) an Fv fragment; (v) a scFv fragment; and (vi) an (Fv)2 fragment. In some embodiments, the anti-CD24 antibody does not contain an Fc region.

[0174] 2. Production of anti-CD24 antibodies

[0175] Anti-CD24 antibodies are preferably produced recombinantly in host cells. Therefore, this antibody is particularly a monoclonal antibody. The host cell used to produce this antibody can be any host cell suitable for antibody production. Suitable host cells are particularly eukaryotic host cells, especially mammalian host cells. Exemplary host cells include yeast cells such as Pichia pastoris (…). Pichia pastoris Cell lines, insect cells such as SF9 and SF21, plant cells, avian cells such as EB66 duck cells, rodent cells such as CHO, NS0, SP2 / 0 and YB2 / 0, and human cells such as HEK293, PER.C6, CAP, CAP-T, AGE1.HN, Mutz-3 and KG1.

[0176] In some embodiments, anti-CD24 antibodies are recombinantly generated in human cell lines (particularly in human myeloid leukemia cell lines). Preferred human cell lines for generating anti-CD24 antibodies and suitable generation procedures are described in WO 2008 / 028686 A2. In a specific embodiment, anti-CD24 antibodies are obtained by expression in human myeloid leukemia cell lines selected from the group consisting of NM-H9D8, NM-H9D8-E6, and NM-H9D8-E6Q12. In accordance with the requirements of the Budapest Treaty, these cell lines are deposited with accessions DSM ACC2806 (NM-H9D8; deposited September 15, 2006), DSM ACC2807 (NM-H9D8-E6; deposited October 5, 2006), and DSM ACC2856 (NM-H9D8-E6Q12; deposited August 8, 2007) at the German Center for Microbial and Cell Culture Collection (DSMZ), address: Inhoffenstraße 7B, 38124 Braunschweig (DE), depositary Glycotope GmbH, Robert-Rössle-Str. 10, 13125 Berlin (Germany). NM-H9D8 cells exhibit a glycosylation pattern characterized by high sialylation, high glucocorticization of GlcNAc, high galactosylation, and high fucosylation. NM-H9D8-E6 and NM-H9D8-E6Q12 cells provide a similar glycosylation pattern to NM-H9D8 cells, except that the degree of fucosylation is very low. Other suitable cell lines include K562 (a human myeloid leukemia cell line present at the American Center for Type Culture Collection (ATCC CCL-243)), CHO cells, and cell lines derived from the above cell lines. In a specific embodiment, anti-CD24 antibodies are recombinantly generated in CHO cells (particularly in CHO dhfr- cells).

[0177] 3. Conjugates of anti-CD24 antibodies

[0178] In specific embodiments, the anti-CD24 antibody is provided as a conjugate comprising an antibody conjugated to other reagents, such as detectable markers or therapeutically active substances. The antibody may be conjugated to one or more other reagents. If more than one other reagent is present in the conjugate, these other reagents may be the same or different, particularly all identical. The conjugation of other reagents to the antibody can be achieved by any method known in the art. Other reagents may be covalently (particularly by fusion or chemical coupling) or non-covalently linked to the antibody. In some embodiments, other reagents are covalently (particularly by a linker portion) linked to the antibody. The linker portion may be any chemical entity suitable for linking other reagents to the antibody.

[0179] Other reagents are preferably intended for the treatment, diagnosis, prognosis, and / or monitoring of diseases, particularly cancer. For example, other reagents may be selected from the group consisting of: radionuclides, chemotherapeutic agents, antibodies, bispecific antibodies or antibody fragments (especially those of different species and / or different specificities than anti-CD24 antibodies), enzymes, interacting domains, detectable tags, toxins, cytolysin components, immunomodulators, immune effectors, cytokines, chemokines, MHC class I antigens or MHC class II antigens, and liposomes.

[0180] In some embodiments, the other reagent is a peptide or protein. This peptide or protein is particularly fused to the peptide chain of the anti-CD24 antibody. In some embodiments, the other reagent, as a peptide or protein, is fused to the C-terminus of the antibody light chain of the anti-CD24 antibody. In embodiments where the anti-CD24 antibody comprises two antibody light chains, the other reagent, as a peptide or protein, may be fused to the C-terminus of each of the two antibody light chains. In further embodiments, the other reagent, as a peptide or protein, is fused to the C-terminus of the antibody heavy chain of the anti-CD24 antibody. In embodiments where the antibody comprises two antibody heavy chains, the other reagent, as a peptide or protein, may be fused to the C-terminus of each of the two antibody heavy chains. The other reagent may be the same or different, particularly having the same amino acid sequence. In embodiments where the antibody does not comprise one or more light chains and one or more heavy chains, for example, in the case where the antibody is an antibody fragment, the other reagent, as a peptide or protein, may be fused to the C-terminus or N-terminus of the antibody's peptide chain. Suitable examples of such other reagents as peptides or proteins are selected from the group consisting of: cytokines, chemokines, antibodies, antigen-binding fragments, enzymes, and interacting domains.

[0181] In some embodiments, other agents as peptides or proteins are checkpoint antibodies that block and / or trigger activation signals. In further embodiments, other agents as peptides or proteins are anticancer antibodies against tumor-associated antigens. In further embodiments, other agents as peptides or proteins are immunomodulatory compounds (such as chemokines, cytokines, or growth factors).

[0182] Conjugates comprising anti-CD24 antibodies conjugated with other reagents are, in particular, chimeric antigen receptors (CARs). Such conjugates are also referred to herein as anti-CD24 CARs. In these embodiments, the other reagents are antigen receptors, such as T-cell receptors, T-cell co-receptors, or activating NK-cell receptors, or portions thereof and / or chimeras. Specifically, the anti-CD24 antibody is fused with a transmembrane domain and an intracellular T-cell or NK-cell signaling domain to form a chimeric antigen receptor (CAR). The intracellular domain is particularly derived from one or more T-cell or NK-cell receptors or co-receptors. Optionally, the CAR also includes a hinge region between the antibody and the transmembrane domain.

[0183] In these embodiments, anti-CD24 antibodies are particularly single-chain antibody fragments (especially scFv fragments) containing both heavy chain and light chain variable regions within a single polypeptide chain. Hinge regions may be based, for example, on hinge regions or proximal membrane regions of immunoglobulin superfamily members. Exemplary hinge regions include those derived from IgG, CD8, and CD28. Transmembrane domains may be hydrophobic α-helices spanning the cell membrane. These may be derived, for example, from CD28 or NKG2D. Intracellular T cell signaling domains are particularly cytoplasmic domains containing the ζ chain of the T cell receptor. Furthermore, intracellular T cell signaling domains may contain other domains of T cell co-stimulatory proteins. Exemplary other domains include signaling domains derived from CD28, CD27, CD134 (OX40), CD137 (4-1BB), 2B4, KIR2DS4, DNAM1, DAP10, and DAP12.

[0184] An exemplary CAR may comprise, from the N-terminus to the C-terminus: (i) an anti-CD24 antibody in the form of an scFv fragment, (ii) an extracellular hinge region derived from CD8, (iii) a transmembrane domain derived from CD28, (iv) a cytoplasmic signal transduction domain derived from CD28, and (v) a signal transduction domain derived from the T cell receptor ζ chain.

[0185] Alternatively, anti-CD24 antibodies (especially single-chain forms such as scFv) can be fused at the N-terminus to the CD3 chain (especially the CD3ε chain) of the T-cell receptor complex to form a chimeric antigen receptor. Alternatively, anti-CD24 antibodies (especially single-chain forms such as scFv) can be fused to a binding domain of a naturally occurring or engineered receptor on T cells or NK cells that can specifically bind to it.

[0186] In some implementations, the other agents are cytotoxic agents or chemotherapeutic agents (especially cytotoxins). Specific examples of chemotherapeutic agents that can be conjugated as other agents include alkylating agents (such as cisplatin), antimetabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes (such as paclitaxel), topoisomerase inhibitors (such as irinotecan and topotecan), antitumor drugs (such as doxorubicin), or microtubule inhibitors (such as olipattin and metansine / metansine).

[0187] 4. Nucleic acid encoding anti-CD24 antibody

[0188] In another aspect, the present invention provides a nucleic acid encoding an anti-CD24 antibody. In some aspects, the present invention provides a nucleic acid encoding an antibody according to a first or second aspect, or a conjugate according to a third aspect, wherein other reagents are polypeptides or proteins fused to the antibody. The nucleic acid sequence of said nucleic acid may have any nucleotide sequence suitable for encoding the antibody. However, preferably, the nucleic acid sequence is at least partially adapted to the specific codon usage of the host cell or organism to which the nucleic acid is to be expressed, particularly human codon usage. The nucleic acid may be double-stranded or single-stranded DNA or RNA, preferably double-stranded DNA such as cDNA or single-stranded RNA such as mRNA. The nucleic acid may be a single continuous nucleic acid molecule, or it may consist of several nucleic acid molecules, each encoding a different portion of the antibody.

[0189] If the anti-CD24 antibody consists of more than one distinct amino acid chain (such as a light chain and a heavy chain), the nucleic acid can be, for example, a single nucleic acid molecule containing several coding regions, each coding region encoding one amino acid chain of the antibody, preferably separated by regulatory elements such as IRES elements to generate independent amino acid chains; or the nucleic acid can consist of several nucleic acid molecules, each containing one or more coding regions, each coding region encoding one amino acid chain of the antibody. Alternatively, the nucleic acid can be a single nucleic acid molecule containing a coding region encoding both a heavy chain and a light chain, separated by a self-cleaving peptide such as a 2A peptide and / or a linker peptide containing a protease recognition site (such as a furin protease recognition site). In addition to the coding region encoding the antibody, the nucleic acid may contain other nucleic acid sequences or other modifications, such as encoding other proteins, affecting transcription and / or translation of the coding region, affecting the stability or other physical or chemical properties of the nucleic acid, or may be completely non-functional.

[0190] In some embodiments, the nucleic acid is a viral vector that can be used to infect human cells. These viral vectors can be used, for example, for human treatment, such as by directing viral infection and / or replication to disease cells such as tumor cells, or, in embodiments where the anti-CD24 antibody is present in the form of a chimeric antigen receptor, to modify T cells or NK cells to obtain CAR cells or CAR NK cells.

[0191] In another aspect, the present invention provides an expression cassette or vector comprising a nucleic acid according to the invention and a promoter operatively linked to said nucleic acid. Furthermore, the expression cassette or vector may include other elements, particularly those capable of influencing and / or regulating the transcription and / or translation of the nucleic acid, the amplification and / or replication of the expression cassette or vector, the integration of the expression cassette or vector into the genome of a host cell, and / or the copy number of the expression cassette or vector in the host cell. Suitable expression cassettes and vectors comprising corresponding expression cassettes for expressing antibodies are well known in the art and therefore require no further description herein.

[0192] 5. Host cells

[0193] Furthermore, the present invention provides a host cell containing nucleic acids according to the invention or expression cassettes or vectors according to the invention. The host cell can be any host cell. It can be an isolated cell or a cell contained in a tissue. Preferably, the host cell is a cultured cell, particularly a primary cell or a cell line with an established structure, preferably a cell derived from tumor cells. Suitable host cells are particularly eukaryotic host cells, especially mammalian host cells. Exemplary host cells include yeast cells such as Pichia pastoris cell lines, insect cells such as SF9 and SF21 cell lines, plant cells, avian cells such as the EB66 duck cell line, rodent cells such as CHO, NSO, SP2 / 0, and YB2 / 0 cell lines, and human cells such as HEK293, PER.C6, CAP, CAP-T, AGE1.HN, Mutz-3, and KG1 cell lines.

[0194] In a preferred embodiment of the invention, the host cell is a CHO cell or a cell derived from human myeloid leukemia cells. Preferably, it is selected from cells or cell lines including K562, KG1, MUTZ-3, CHO, or cells or cell lines derived therefrom. The host cell is preferably selected from the group consisting of CHO, NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12, and cells or cell lines derived from any of the aforementioned host cells. These cell lines and their properties are described in detail in PCT application WO2008 / 028686 A2. In some embodiments, the host cell is optimized for expressing glycoproteins, particularly antibodies, with a specific glycosylation pattern. Preferably, the codon usage and / or promoter in the nucleic acid coding region according to the invention, as well as other elements of the expression cassette or vector, are compatible with the type of host cell used; more preferably, they are optimized for the type of host cell used. Preferably, the anti-CD24 antibody is produced from the host cell or host cell line as described above.

[0195] The present invention further provides a host cell carrying an anti-CD24 CAR. Such host cells are also referred to herein as CAR cells. CAR cells specifically contain a nucleic acid according to the invention encoding an anti-CD24 CAR or an expression cassette or vector according to the invention. In some embodiments, CAR cells are engineered to express an anti-CD24 CAR, for example by introducing a vector containing an anti-CD24 CAR expression cassette.

[0196] In specific implementations, CAR cells are leukocytes, particularly lymphocytes such as T cells, NK cells, and NKT cells, or monocytes such as macrophages. CAR cells can be primary leukocytes or cells from immune cell lines. In some implementations, CAR cells are selected from the group consisting of: primary T cells, primary NK cells, primary NKT cells, and primary macrophages, especially primary T cells.

[0197] 6. Pharmaceutical Compositions and Therapeutic Uses

[0198] In another aspect, the present invention provides a composition comprising an anti-CD24 antibody, a nucleic acid, an expression cassette or vector, a host cell, or a conjugate according to the invention. The composition may also comprise more than one of these components. Furthermore, the composition may comprise one or more other components selected from the group consisting of solvents, diluents, and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, all components of the composition are preferably pharmaceutically acceptable. The composition may be a solid or liquid composition, particularly (preferably) a solution, emulsion, or suspension, or a lyophilized powder.

[0199] Anti-CD24 antibodies or their conjugates, or CAR cells, are particularly useful in medicine, especially in the treatment, diagnosis, prognosis, and / or monitoring of diseases, particularly those described herein, such as cancer and infections, preferably cancer. Therefore, in another aspect, the present invention provides anti-CD24 antibodies, nucleic acids, expression cassettes or vectors, host cells, conjugates, or compositions for medical use. Preferably, the medical use is for the treatment, prognosis, diagnosis, and / or monitoring of diseases, particularly those related to CD24. Exemplary diseases include those associated with abnormal cell growth such as cancer, bacterial and viral infections, and vascular diseases.

[0200] In a preferred embodiment, the disease is cancer. Preferably, the cancer is selected from the group consisting of: endometrial cancer, breast cancer, ovarian cancer, skin cancer, thyroid cancer, prostate cancer, head and neck cancer, oral cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, stomach cancer, lung cancer, esophageal cancer, and pancreatic cancer. The cancer may be, for example, breast cancer, endometrial cancer, or ovarian cancer. The cancer is CD24 positive and particularly comprises cancer cells that carry CD24 on their cell surface.

[0201] In a specific implementation, the anti-CD24 antibody is used in combination with another anticancer therapeutic agent. The other therapeutic agent can be any known anticancer drug, and in particular, can be an antibody against a cancer antigen, including bispecific antibodies, especially immune cell-recruiting bispecific antibodies.

[0202] Treatment with anti-CD24 antibodies can be further combined with immunostimulants, cytokines, chemokines, radiotherapy, therapeutic cells such as innate immune cells or engineered immune cells (CAR-T cells, TCR-T cells, CAR-NK cells, CAR-monocytes, CAR-NKT cells, CAR-macrophages, CAR-neutrophils, etc.), vaccines such as protein vaccines, peptide vaccines or RNA vaccines, B-Raf inhibitors such as vemurafenib and dexamethasone, protease inhibitors such as bortezomib, and lenalidomide.

[0203] When used in cancer therapy involving cell expression of CD24, the antibody can be conjugated to other agents as described above, preferably cytotoxic agents such as radionuclides or cytotoxins. Cytotoxic agents also include precursor compounds that produce cytotoxic activity only upon activation (e.g., by light irradiation or enzymatic reactions in vivo). Furthermore, the antibody can be engineered to enhance its ability to activate the patient's immune response, particularly its ability to activate ADCC (antibody-dependent cell-mediated cytotoxicity) and / or CDC (complement-dependent cytotoxicity). This can be achieved, for example, by optimizing the antibody, particularly the amino acid sequence and / or glycosylation pattern of its constant regions.

[0204] When used as a diagnostic reagent for disease diagnosis, prognosis, and / or monitoring, the antibody is preferably conjugated to a labeled reagent capable of generating a detectable signal. Specifically, the labeled reagent may be a radionuclide, a fluorophore, or an enzyme. Attached Figure Description

[0205] Figure 1 The process of antibody generation is visualized.

[0206] Figure 2 The biosynthetic pathways and structures of Tn and TF carbohydrate antigens are shown. In this structure, squares represent GalNAc, circles represent Gal, and rhombuses represent sialic acid.

[0207] Figure 3The binding of anti-CD24 clones to different glycosylated CD24 variants in antigen ELISA was demonstrated. The binding of 250 ng / ml (A + B + C) or 5 µg / ml (D) anti-CD24 mAb to an equimolar amount of antigen (35 nM) was evaluated in antigen ELISA. Anti-CD24 (SWA-11) and anti-glycan antibody (data not shown) were used as controls. sTF-, TF-, and Tn-carrying proteins were recombinantly expressed and purified from NM-H9D8, NM-F9, and NM F9-GalKO cells, and subjected to enzymatic desialylation (siaA) or deO-glycosylation to remove terminal sialic acid or non-sialylated TF-glycans and Tn-glycans. High OD signal indicates strong antibody binding to the antigen.

[0208] Figure 4 This study demonstrates the titration of anti-CD24 clones on their corresponding primarily on-target protein, CD24-TF. Clones 1E9(A), 1E1(B), 2C9(CH), and their humanized variants were titrated on a fixed amount of CD24-TF protein (35 nM). EC50 of the dose-response curves were calculated using nonlinear regression with ELISA analysis in conjunction with GraphPad Prism 5 software. 50 value.

[0209] Figure 5 This study demonstrates the binding of anti-CD24 clones 1E1(A), 1E9(A), 2C9 (A+B+C) and several humanized variants to different glycosylated CD24 expressed in cell lines: CD24-F9 (NM-F9 cells expressing high levels of CD24 and TF glycans), CD24-HEK non-glyc. (expressing high levels of CD24 but without O-glycans), and F9 (NM-F9 cells expressing low levels of CD24 and high levels of TF). Cells were stained with 10 µg / ml anti-CD24 clones, protein-specific glycosylation-independent control anti-CD24 mAb SWA-11, and anti-glycan control antibodies (data not shown). The signal-to-noise ratio of stained cells is shown as the median fluorescence intensity (MFI) of stained cells divided by the MFI of isotype stained cells.

[0210] Figure 6This study demonstrates the binding of anti-CD24 clone 1E1 and its humanized variants to CD24-expressing tumor cell lines (including those treated with neuraminidase (+NA) and those not treated with neuraminidase (wt)). Binding data for tumor cell lines MCF7 (A) and SKOV-3 (B+C) are shown. Cancer cells, treated with neuraminidase (5 mU / ml, 30 min) without or with neuraminidase, were stained with a 10 µg / ml anti-CD24 clone and fluorophore-conjugated anti-human IgG secondary reagent after washing. The expression of CD24, TF, and Tn was confirmed using control anti-CD24 (SWA-11), anti-TF mAb, and anti-Tn mAb (data not shown). The signal-to-noise ratio of stained cells is shown as the MFI of stained cells divided by the MFI of isotype stained cells.

[0211] Figure 7 This study demonstrates the binding of anti-CD24 clone 1E9 and its humanized variants to CD24-expressing tumor cell lines (including those treated with neuraminidase (+NA) and those not treated with neuraminidase (wt)). Binding data for tumor cell lines MCF7 (A), SKOV-3 (B), and MDA-MB-468 (C) are shown. Cancer cells, treated with neuraminidase (5 mU / ml, 30 min) without or with neuraminidase, were stained with a 10 µg / ml anti-CD24 clone and fluorophore-conjugated anti-human IgG secondary reagent after washing. The expression of CD24, TF, and Tn was confirmed using control anti-CD24 (SWA-11), anti-TF mAb, and anti-Tn mAb (data not shown). The signal-to-noise ratio of stained cells is shown: MFI of stained cells divided by MFI of isotype stained cells.

[0212] Figure 8 This study demonstrates the binding of anti-CD24 clone 2C9 and its humanized variants to CD24-expressing tumor cell lines, including those treated with neuraminidase (+NA) and those not treated with neuraminidase (wt). Binding data for tumor cell lines MCF7 (A+D), SKOV-3 (B), and MDA-MB468 (C+D) are shown. Cancer cells, treated with neuraminidase (5 mU / ml, 30 min) without or with neuraminidase, were stained with a 10 µg / ml anti-CD24 clone and fluorophore-conjugated anti-human IgG secondary reagent after washing. The expression of CD24, TF, and Tn was confirmed using control anti-CD24 (SWA-11), anti-TF mAb, and anti-Tn mAb (data not shown). The signal-to-noise ratio of stained cells is shown as the MFI of stained cells divided by the MFI of isotype stained cells.

[0213] Figure 9The results of quantitative flow cytometry for two clones, 1E9 and 2C9, on different cell lines are presented, including exemplary humanized variants of each clone and the glycan-independent anti-CD24 antibody SWA-11. For quantitative analysis of antibody binding capacity (ABC), Quantum Simply Cellular anti-human IgG microspheres (Bangs Laboratories) were stained with the same secondary reagents and measured on the same day, using the same instrument and settings as cells labeled with a saturated amount (100 µg / ml) of the anti-CD24 clone. Cell ABC values ​​were calculated from a standard curve of the Quantum microspheres, subtracting the isotype control. The detection limit was determined using a blank microsphere population.

[0214] Figure 10 This study demonstrates the titration of anti-CD24 clone 2C9 and exemplary humanized 2C9 variants on MCF7 cells expressing TF and CD24. Cells were stained with different concentrations of the anti-CD24 clone and detected using a fluorophore-conjugated anti-human IgG secondary reagent. MFI of live cells is shown.

[0215] Figure 11 This study demonstrates the binding of (A) anti-TF mAb to healthy blood cells treated with and untreated with neuraminidase, and (B) binding of the glycan-independent anti-CD24 clone SWA-11 and an exemplary anti-CD24 clone to blood cells from two healthy donors. Granulocytes and B cells from healthy donors were stained with 10 µg / ml anti-TF mAb and fluorophore-conjugated anti-IgG secondary reagent, or CD24-positive granulocytes and B cells were stained with 1 µg / ml DIG-conjugated anti-CD24 clone and fluorophore-conjugated anti-DIG secondary reagent. The expression of CD24, as well as O-glycan TF and O-glycan Tn, was confirmed using control anti-CD24 (SWA-11) and anti-glycan antibodies (data not shown). Binding is shown as median fluorescence intensity in live cells.

[0216] Figure 12This study demonstrates the ability of anti-CD24 clones 1E9 and 2C9, as well as humanized variants 1E9-2A04 and 2C9-3A12, to be internalized by TF-positive and CD24-positive MCF7 cells (A+B), CD24 F9 (TF) cells (C+D), and CD24+ granulocytes (E+F) and B cells purified from healthy donors (G+H). The glycan-independent anti-CD24 antibody SWA-11 was used as a control. Internalization was analyzed using an Incucyte S3 instrument. The mAb was labeled with a pH-sensitive fluorophore conjugated to an anti-human Fab fragment and incubated with cells at 37°C for 24 h. Upon internalization and entry into acidic lysosomes, red fluorescence was induced and quantified using an Incucyte S3 (Sartorius). The percentage of lysosomal transport is expressed as the percentage of cells with high red intensity.

[0217] Figure 13 This study exemplifies the additional mode of action of clone 2C9 as a naked mAb, demonstrating its ADCC (antibody-dependent cell-mediated cytotoxicity) activity. MCF-7 tumor cells loaded with europium were used as target cells. The human NK cell line KHYG-CD16aV was used as effector cells, with an effector-to-target cell (E:T) ratio of 10:1. Titrations of 2C9 antibody, SWA-11, or hIgG1 were added as controls to the co-culture system. Europium release was quantified by fluorescence readings, and specific lysis was analyzed after 4 hours.

[0218] Figure 14 This study demonstrated the ability of anti-CD24 clones to inhibit the proliferation of CD24-expressing NM F9 cells. Cells were incubated with different concentrations of anti-CD24 clones conjugated with MMAE via protein G. Proliferation was measured after 4 days. Proliferation is shown as a percentage of luminescent signal relative to an antibody-free medium control.

[0219] Example

[0220] Example 1: Antigen generation

[0221] To optimize the screening of antibodies that specifically bind to human CD24 with tumor-associated glycosylation patterns, different O-glycosylated variants of human CD24 were generated. The CD24 sequence was obtained from Uniprot accession number P25063.

[0222] The most relevant human CD24 isoform 1 was selected as the target structure (“CD24”; SEQ ID NO: 58) for antibody generation in the antibody generation method described in Example 2, which has the high potential to generate glycosylation-dependent anti-CD24 antibodies.

[0223] Cells of cancer-derived cancer cell lines NM F9 and NM-H9D8 were transfected with DNA encoding CD24. Soluble protein constructs were recombinantly expressed as fusion proteins, for example, fused with a tandem version of Strep-tag® (Twin-Strep-tag®, IBA, Germany) and maltose-binding lectin (“MBL”), for purification from cell supernatant by affinity chromatography and detection of the protein by a fusion moiety independent of CD24 (StrMBL-CD24).

[0224] The NM F9 cell line was used to express human CD24 protein with a tumor-associated O-glycosylation pattern, primarily producing CD24 carrying TF and a smaller amount of Tn. Glycopeptide analysis showed that >99% of the CD24 peptides were glycosylated, with 87% of the glycan structure being TF and the remaining 12% being Tn. Soluble CD24 protein with only Tn-glycosylation or no O-glycosylation was generated by enzymatic digestion of protein purified from NM-F9 transfectants with appropriate glycosidases, or by expression in NM-F9-GalKO cells for Tn-glycosylation, where the glycosylation pathway responsible for linking galactose to Tn to convert it to TF was knocked out. Soluble CD24 protein with a major sTF structure and a small amount of TF structure was primarily produced in NM-H9D8 cells.

[0225] To screen antibody clones and perform binding analysis, other constructs (CD24-mFc) were developed by fusing CD24 with the N-terminus of the Fc portion of a mouse antibody.

[0226] As an off-target control for ELISA binding studies, the extracellular domains of irrelevant glycoproteins were expressed in NM-F9 or NM-F9-de-O-glyc, fused with Strep-tag® and MBL, or fused to the N-terminus of the Fc portion of a mouse antibody. As a control anti-CD24 antibody, the commercially available mouse anti-human CD24 IgG1 antibody SWA-11 was used.

[0227] Example 2: Generation of anti-CD24 antibody

[0228] Monoclonal antibodies that specifically recognize hCD24 with tumor-associated glycosylation patterns were generated through animal immunization.

[0229] CD24-specific antibodies were isolated from chicken and rabbit spleen cells immunized with CD24-enhanced from CD24 purified from transfected NM-F9 cells using HybriFree technology. Following final immunization, spleen cells were isolated from animals that demonstrated an antigen-specific antibody response in serum or egg yolk preparations, as assessed by flow cytometry and / or ELISA. Spleen B cells exhibiting antigen specificity for O-glycosylated CD24 were enriched by removing cells showing unwanted protein binding. The removed cells included those bound to non-glycosylated CD24 and / or those bound to irrelevant glycoproteins generated in a similar manner to O-glycosylated CD24 (and these irrelevant glycoproteins thereby containing the same protein fusion chaperones and similar glycan structures linked to non-homologous protein sequences). In this negative selection step, spleen cells were first incubated with off-target proteins immobilized on microplates and / or present in a panning solution. Unbound cells were then transferred to plates coated with O-glycosylated CD24 protein purified from NM-F9 cells to capture target-specific spleen B cells.

[0230] cDNA containing the variable domain of antibodies was amplified from captured cells and cloned into plasmids containing separate expression cassettes of IgG heavy and light chains to construct a combined mouse IgG1 coding library in a mammalian expression vector. The plasmid DNA from the resulting antibody library pool was transfected into CHO cells to transiently generate chimeric antibodies. Target-specific binding of the antibody pool cell culture supernatant was detected by ELISA using multiple target and off-target proteins. Monoclonal antibodies were generated from the pool showing specific binding to O-glycosylated CD24. The VH and VL cDNAs of the antigen-specific monoclonal antibodies were sequenced, and antibodies with unique sequences were expressed in CHO cells. The production of antigen-specific antibodies in the monoclonal supernatant was confirmed by ELISA and flow cytometry prior to antibody purification.

[0231] The following glycosylation-dependent anti-CD24 antibodies were obtained through immunization of chickens or rabbits:

[0232] Table 1

[0233]

[0234] In addition, a cysteine-modified version of antibody 10B5 was generated, wherein serine 49 of the heavy chain was replaced with cysteine ​​(SEQ ID NO: 37), and / or cysteine ​​80 of the light chain was replaced with serine (SEQ ID NO: 57). All selected antibodies were scaled up for expression as chimeric IgG1 with a human backbone and subsequently purified from mammalian cell culture supernatant by protein A affinity chromatography. The purity and integrity of the mAbs were confirmed by SDS-PAGE and analytical SEC.

[0235] Example 3: Humanization of the heavy chain variable region and light chain variable region of anti-CD24 antibody

[0236] Humanization of chicken-derived anti-CD24 antibodies was performed via CDR transplantation. Different VH and VL variants of each lead clone were combined in a Fab phage library, and the desired glycosylation-dependent CD24 binding modalities were selected and screened by ELISA. The optimal variants were expressed as hIgG1 in CHO cells, and their fine specificity and tumor cell binding ability were screened. The following variants were further evaluated:

[0237] Table 2

[0238]

[0239] Example 4: Antigen ELISA containing on-target and off-target controls

[0240] In antigen ELISA assays, the specific binding of antibodies to CD24 carrying tumor-associated glycans was analyzed.

[0241] In short, different glycosylated and non-glycosylated protein antigens are coated onto a 96-well plate and left overnight to block non-specific binding, and then detection antibody samples are added.

[0242] The glycosylation-dependent binding of generated anti-CD24 antibodies, parental clones, and humanized variants to mature human CD24 was evaluated in antigen ELISA (chimeric clones are shown in Table 3). Figure 3 (Simultaneous comparison of chimeric clones and all humanized variants of these clones). All antibodies showed significant binding to O-glycosylated CD24 protein, confirming that the epitopes recognized by these antibodies are present in the amino acid sequence of CD24. Deletion of O-glycosylation on the detected CD24 protein eliminated binding of anti-CD24 antibodies. This clearly demonstrates that O-glycosylated CD24 epitopes are essential for antibody binding, thus confirming the glycosylation-dependent nature of CD24 binding.

[0243] The results also showed that the anti-CD24 antibody clones exhibited diverse and fine specificity in terms of the type of CD24 O-glycosylation. This was evident from the varying degrees of recognition of CD24 primarily carrying Tn-, TF-, or sTF-glycosylation. Specifically, antibody clones 1E1, 1E9, and 2C9 showed strong recognition of CD24 carrying the TF structure, weak recognition of CD24 carrying Tn and sTF, and no binding to de-O-glycosylated CD24 at all. Clones 10B5 and 10F9 also showed strong recognition of CD24 carrying both TF and Tn structures, little recognition of CD24 carrying sTF, and little or no recognition of de-O-glycosylated CD24 (Table 3).

[0244] Table 3

[0245]

[0246] TF: Thomsen-Friedenreich antigen; sTF: sialylated Thomsen-Friedenreich antigen; Tn: Thomsen nouvelle antigen; de-O: de-glycosylation; sTF+NA: sifted Thomsen-Friedenreich antigen treated with neuraminidase to produce TF.

[0247] Furthermore, no off-target binding to irrelevant proteins (off-target proteins) or individual glycan structures was observed (Table 4).

[0248] Table 4

[0249]

[0250] Example 5: Titration of anti-CD24 clone on CD24 protein

[0251] like Figure 4 As shown, anti-CD24 clones 1E9 (A), 1E1 (B), and 2C9 (CH) and their humanized variants were titrated on a fixed amount of CD24-TF protein, with the variants consistently compared to the parental and chimeric clones. For all clones, the parental and humanized sequences exhibited highly similar performance, with their EC50 values ​​falling within a very narrow range, demonstrating successful humanization of the parental and chicken-derived sequences.

[0252] Example 6: Binding with O-glycosylated and de-glycosylated CD24 expressed in cell lines

[0253] The binding of anti-CD24 clones to NM-F9, NM-F9 GalKO and HEK cell lines with different CD24 glycosylation states was investigated.

[0254] NM-F9 cells transfected with CD24 expressing CD24, which mainly carries TF and a small amount of Tn (CD24-F9), and HEK-O-glycKO cells transfected with CD24 expressing non-O-glycosylated CD24 (CD24-HEK-non-glyc.) were stained using an anti-CD24 clone and detected by a fluorophore-conjugated anti-human IgG secondary reagent. Untransfected NM-F9 (TF) cells expressing only low levels of CD24, as well as anti-TF antibody and anti-CD24 antibody SWA-11, served as controls.

[0255] The results showed that the parental anti-CD24 clone and the humanized anti-CD24 clone bound to CD24 in different cell lines in an O-glycosylation-dependent manner. Figure 5 Specifically, all clones showed strong binding to CD24 carrying the TF structure, while none of the clones bound to non-glycosylated CD24 expressed in CD24-HEK cells.

[0256] In addition, the binding of clones 1E1, 1E9, 2C9, 10B5, and 10F9 to Tn-glycosylated F9 GalKO cells was examined (Table 5). F9 GalKO cells and CD24-F9 GalKO cells (expressing high levels of Tn-glycosylated CD24) were not bound.

[0257] Table 5

[0258]

[0259] Example 7: Binding of anti-CD24 clones to tumor cell lines

[0260] To investigate the binding of anti-CD24 clones to tumor cell lines, different cell lines with varying endogenous expression levels of CD24, TF, sTF, sTn, and Tn were selected: MCF7 cells expressing CD24 and exhibiting a high TF but low sTF, Tn, or sTn glycosylation pattern; and MDA-MB-468 cells and SKOV-3 cells exhibiting high sTF but low TF and Tn expression. Treatment of these cell lines (especially SKOV-3 and MDA-MB-468) with neuraminidase removed sialic acid from the cell surface and generated major TF glycosylation.

[0261] Tumor cell lines were stained with anti-CD24 clones, and binding was detected using a fluorophore-conjugated anti-human IgG secondary reagent. The expression of CD24, TF, and Tn was confirmed using a control anti-CD24 antibody (SWA-11), as well as anti-TF and anti-Tn antibodies (data not shown).

[0262] like Figure 6 , 7As shown in Figure 8, due to the low sTF expression in MCF7 cells, all anti-CD24 clones exhibited strong binding to MCF7 cells and were unaffected by neuraminidase treatment. Conversely, after neuraminidase treatment, tumor cells released additional TF-glycosylated CD24 epitopes, resulting in enhanced binding of anti-CD24 clones to SKOV-3 and MDA-MB-468 cells. This demonstrates that the anti-CD24 clones are specific for TF-glycosylated CD24. CD24-negative tumor cell lines were not identified (data not shown). Quantum analysis of exemplary clones ( Figure 9 This data correlates with the number of binding sites per cell and confirms that the number of binding sites is within a similar range to that of the glycosylation-independent SWA-11 control antibody.

[0263] Example 8: Titration of anti-CD24 clones on tumor cells

[0264] To compare the binding strength of parental anti-CD24 clones and humanized anti-CD24 clones, the MCF7 tumor cell line expressing TF-glycosylated CD24 was stained with different concentrations of anti-CD24 clones. All anti-CD24 clones showed dose-dependent binding to CD24 on cells, with clone 2C9 and its variants exhibiting the strongest binding. Figure 10 Titration of 2C9 and humanized 2C9 variants 2C9-3A12, 2C9-3A12-VL1VH, 2C9-3A12-VL5VH, 2C9-3A12-VL6VH, and 2C9-3A12-VL7VH on MCF7 cells was demonstrated. Antibody binding was detected by flow cytometry using a fluorophore-conjugated anti-human IgG secondary reagent. The EC50 values ​​of all humanized variants (gray symbols) were similar to those of the parental clone 2C9 (black dots) and the glycosylation-independent control antibody SWA-11 (black triangles).

[0265] Example 9: Anti-CD24 clone does not bind to CD24 expressed on human blood cells

[0266] To demonstrate that the anti-CD24 clone does not bind to the CD24 glycoform primarily found in normal human blood cells, human B cells and human granulocytes expressing CD24 from healthy donors were treated with an excess of hIgG (Octagam) to block Fc receptor-mediated binding. The anti-CD24 clone was then stained with DIG-conjugated anti-CD24 and detected by a fluorophore-conjugated anti-DIG secondary reagent. CD24 expression was confirmed using a glycan-independent control anti-CD24 mAb SWA-11. Figure 11 A). Use DAPI to distinguish between live and dead cells.

[0267] In addition, the glycosylation patterns of normal human blood cells from healthy donors were analyzed. Figure 11(B) The results showed that human granulocytes and human B cells carry a large number of sTF structures, which was confirmed by the fact that anti-TF antibodies only bind to these blood cells after treatment with neuraminidase. Therefore, the fine specificity of the anti-CD24 antibody clone to CD24 carrying TF rather than CD24 carrying sTF enables it to specifically target tumor cells without off-target effects on normal blood cells.

[0268] As a result, none of the anti-CD24 clones showed binding to CD24 expressed on human CD24+ B cells, and the binding to highly CD24-positive granulocytes was only significantly reduced, while the commercially available glycosylation-independent anti-CD24 antibody SWA-11 strongly bound to both blood cell populations.

[0269] Example 10: Internalization of anti-CD24 antibody

[0270] Anti-CD24 clones 1E9, 1E9-2A04, 2C9, and 2C9-3A12 were incubated with different cell lines, and the percentage of cells showing antibody uptake was determined by fluorescence measurement to analyze the internalization of these antibodies. Figure 12 CD24-F9 cells (NM-F9 cells expressing TF-glycosylated CD24) and MCF7 cells were used as target cells to determine internalization. Additionally, purified granulocytes and B cells were analyzed to investigate their potential internalization in a population of CD24-positive hematologic cells from healthy donors. Internalization analysis was performed using an Incucyte S3 instrument. The mAbs used for detection were labeled with anti-human Fab fragments conjugated to a pH-sensitive fluorophore. After internalization and entry into acidic lysosomes, red fluorescence was induced and quantifiable. Briefly, cells were harvested and seeded at 5 × 10³ cells per well in 96-well flat-bottom plates. The corresponding antibody was incubated with a 12-fold molar excess of anti-human Fabfluor-pH antibody-labeled dye at RT for 15 min before being added to the cells. Phase contrast and red fluorescence were monitored over time (up to 24 hours) at 37°C in an Incucyte S3. The degree of internalization was expressed as the percentage of cells with high red intensity.

[0271] The results showed that in the tested CD24-F9 cells, anti-CD24 clones 1E9 and 2C9, as well as humanized clones 1E9-2A04 and 2C9-3A12, underwent strong internalization. All clones were also strongly internalized into MCF7 cells expressing TF and CD24, but unlike the control antibody SWA-11, which binds to CD24 in a glycosylation-independent manner, no internalization was detected in CD24-positive granulocytes and B cells from healthy donors.

[0272] Example 11: Anti-CD24 clone-mediated ADCC

[0273] In europium release assays, the potential of clonal 2C9 to initiate antibody-dependent cytotoxicity was investigated. NK cell line KHYG-CD16aV mediated specific cleavage of the target tumor cell line MCF-7, dependent on 2C9 concentration. Figure 13 The results showed that 2C9-mediated cleavage was concentration-dependent and very similar in range to the specific cleavage mediated by the glycan-independent anti-CD24 antibody SWA-11.

[0274] Example 12: Inhibition of proliferation using protein G-drug conjugated anti-CD24 antibody

[0275] To investigate the potential of anti-CD24 clones to deliver cytotoxic drugs to target cells, a protein G-drug conjugate assay was performed. Five × 10³ CD24-F9 cells (NM-F9 cells primarily expressing TF-glycosylated CD24) were seeded per well in 96-well flat-bottom plates, with the antibody dilution shown and a constant concentration of protein G preloaded with the toxin MMAE (protein G-MMAE, Levena Biopharma). After 4 days of incubation, cell viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega) according to the manufacturer's instructions and analyzed on a TECAN Infinite F200 microplate reader (Tecan). The percentage of proliferation relative to the antibody-free medium control was calculated based on the luminescence signal.

[0276] The results showed that all anti-CD24 clones conjugated to protein G-MMAE inhibited CD24-F9 proliferation in a dose-dependent manner, indicating effective internalization of the anti-CD24 clones with low IC50 values. Figure 14 Anti-CD24 antibody SWA-11 and unrelated human IgG1 antibody (hIgG1 Iso) were used as controls.

[0277] Example 13: Tissue binding of anti-CD24 antibody

[0278] Formalin-fixed paraffin-embedded tissue sections were stained using an anti-CD24 clone and detected with a secondary antibody, or stained using a DIG-conjugated anti-CD24 clone. The commercial anti-CD24 antibody SWA-11 was included to detect CD24 protein levels in the corresponding tissues.

[0279] Staining of cancer tissue sections showed that the anti-CD24 clone had a strong binding to, for example, breast cancer. Importantly, compared with glycosylation-independent control anti-CD24 antibodies, the anti-CD24 clone showed no or significantly reduced responsiveness to healthy tissues.

[0280] Strong binding of anti-CD24 clones was detected in several tumor tissues, including breast cancer, esophageal cancer, head and neck cancer, kidney cancer, oral cancer, ovarian cancer, bladder cancer, colorectal cancer, skin cancer, endometrial cancer, thyroid cancer, prostate cancer, and cervical cancer (data not shown).

[0281] sequence list

[0282]

[0283]

[0284]

[0285] Identification of Preserved Biological Materials

[0286] Cell line DSM ACC 2606 was deposited on the dates shown in the table below at the DSMZ (German Center for Microbiology and Cell Culture), address: Mascheroder Weg 1b, 38124 Braunschweig (Germany), and the depositary is Nemod Biotherapeutics GmbH & Co. KG, address: Robert-Rössle-Str. 10, 13125 Berlin (Germany). Glycotope has the right to use this biological material because it has been transferred from Nemod Biotherapeutics GmbH & Co. KG to Glycotope GmbH.

[0287] Cell lines DSM ACC 2806, DSM ACC 2807, and DSM ACC 2856 are deposited on the dates shown in the table below at DSMZ (German Center for Microbial and Cell Culture Collection), address: Inhoffenstraße 7B, 38124 Braunschweig (Germany), with Glycotope GmbH at Robert-Rössle-Str. 10, 13125 Berlin (Germany).

[0288]

Claims

1. An antibody capable of binding to human CD24 glycosylated by Galβ1-3GalNAcα1 at one or more serine and / or threonine residues.

2. The antibody according to claim 1, which is capable of specifically binding to human CD24 glycosylated by Galβ1-3GalNAcα1 at one or more serine residues and / or threonine residues.

3. An antibody capable of binding to human CD24 and comprising: (i) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 5, and CDR-H3 having the amino acid sequence of SEQ ID NO: 10, and Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 20; or (ii) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 6, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 17, and CDR-L3 having the amino acid sequence of SEQ ID NO: 21; or (iii) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; or (iv) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 3, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or (v) Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 4, CDR-H2 having the amino acid sequence of SEQ ID NO: 9, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13, and Light chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24; or (vi) The heavy chain variable region and the light chain variable region according to any one of (i) to (v) above contain a total of 1, 2 or 3 amino acid substitutions in the six CDR sequences.

4. The antibody according to claim 3, comprising: Heavy chain variable regions comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and The light chain variable region includes the following complementarity-determining regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO:

22.

5. The antibody according to claim 3, wherein: (i) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 25, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 38; or (ii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 44; or (iii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 30, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 47; or (iv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 35, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 55; or (v) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 56; or (vi) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 37, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 56; or (vii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 37, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 57; or (viii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 57; or (ix) The heavy chain variable region and the light chain variable region each contain an amino acid sequence that is at least 60% identical in length to the heavy chain variable region sequence and the light chain variable region sequence of any one of (i) to (viii) above, wherein the antibody is preferably a humanized version of the antibody of any one of (i) to (viii) above.

6. The antibody according to claim 3, wherein it is a humanized antibody, wherein: (i) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 39; or (ii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 40; or (iii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 41; or (iv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 42; or (v) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 43; or (vi) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 28, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 45; or (vii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 29, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 46; or (viii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 48; or (ix) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 32, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 48; or (x) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 49; or (xi) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 50; or (xii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 51; or (xiii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 52; or (xiv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 33, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 53; or (xv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 34, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

54.

7. The antibody according to any one of claims 3 to 6, wherein the antibody exhibits one or more binding properties as defined in claims 1 and 2.

8. The antibody according to any one of claims 1 to 7, wherein it is an IgG1, IgG2, IgG3 or IgG4 antibody, particularly an IgG1 antibody.

9. A conjugate comprising an antibody conjugated with other reagents according to any one of claims 1 to 8, wherein the other reagents are optionally cytotoxic agents, tumor-specific antibodies, or immune checkpoint blocking antibodies or immune checkpoint activating antibodies, or wherein the conjugate is optionally a chimeric antigen receptor.

10. A nucleic acid encoding an antibody according to any one of claims 1 to 8 or a conjugate according to claim 9, wherein the other reagent is a polypeptide or protein fused with the antibody.

11. An expression cassette or vector comprising the nucleic acid of claim 10 and a promoter operatively linked to the nucleic acid.

12. A host cell comprising the nucleic acid of claim 10 or the expression cassette or vector of claim 11.

13. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 8, a conjugate according to claim 9, a nucleic acid according to claim 10, an expression cassette or vector according to claim 11, or a host cell according to claim 12, wherein the pharmaceutical composition optionally further comprises one or more components selected from the group consisting of solvents, diluents, and excipients.

14. The antibody according to any one of claims 1 to 8, the conjugate according to claim 9, the host cell according to claim 12, or the composition according to claim 13, for use in medicine.

15. The use of the antibody, conjugate, host cell, or composition according to claim 14 in medicine for the treatment of cancer, bacterial or viral infections, or vascular diseases.

16. The antibody, conjugate, host cell, or composition according to claim 15, for treating cancer, wherein the cancer is selected from the group consisting of: endometrial cancer, breast cancer, ovarian cancer, skin cancer, thyroid cancer, prostate cancer, head and neck cancer, oral cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, stomach cancer, lung cancer, esophageal cancer, pancreatic cancer, and particularly wherein the cancer is breast cancer, endometrial cancer, or ovarian cancer.

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