Antibodies against CD24

Abstract

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

Description

[0001] ..Antibodies against CD24“

[0002] FIELD OF THE INVENTION

[0003] The present invention pertains to the field of antibodies. In particular, anti-CD24 antibodies showing strong antigen binding in a tumor-specific manner are provided. In specific embodiments, the present invention is directed to anti-CD24 antibodies for therapeutic and diagnostic use.

[0004] BACKGROUND OF THE INVENTION

[0005] Today, antibodies are widely used agents in the field of medicine and research. In medicine, they find application in many different fields. For example, antibodies are used as therapeutic agents in the treatment and prophylaxis of a variety of diseases such as cancer, cardiovascular diseases, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infections. In these therapies, the antibody may possess therapeutic activity on its own, for example by blocking receptors or messenger molecules, thereby inhibiting their disease-relevant functions, or by recruiting and activating components of the patient's immune system.

[0006] In cancer treatment, a key characteristic of therapeutic antibodies is their specificity for tumor tissue. This means that the antibodies should target an epitope which is exclusively or predominantly found on cancer cells, but only to a low extent on cells of normal tissue. Thereby, the therapeutic activity of the antibody, e.g., inducing an immune response against the targeted cells or destroying the cells by a cytotoxic payload, specifically acts at the tumor site. Respective activity at normal tissue - due to binding of the antibody to non-tumor cells - may result in severe side effects. Increasing the specificity for tumor cells hence reduces the risk of adverse effects and enhances the safety of the envisioned therapy.

[0007] A possible cancer antigen is CD24. This protein is a glycosylphosphatidylinositol (GPI)- anchored, highly glycosylated adhesion molecule. It was first identified as a murine differentiation marker described as a heat stable antigen due to its heat resistance. Since then, CD24 has been attributed various functions including roles in the adaptive immunity, inflammation autoimmunity and cancer. In cancer, CD24 has been suggested to be involved in cell migration, invasiveness and cell proliferation.

[0008] Overexpression of CD24 has been shown to correlate with a malignant phenotype, being associated with a poor prognosis in a variety of cancer types such as breast cancer, nonsmall cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), cholangiocarcinoma, urothelial carcinoma, ovarian cancer and prostate carcinomas. Therefore, CD24 represents an interesting target for anti-tumor immunotherapy.

[0009] However, under normal physiologic conditions CD24 is expressed throughout various tissues including lymphocytes, epithelial cells and inflammatory cells. This may cause unwanted side effects in a cancer therapy with an antibody which cannot discriminate between cancer-associated CD24 and CD24 on normal tissue.

[0010] Therefore, there is a need in the art to provide tumor-specific anti-CD24 antibodies which can distinguish between CD24 on cancer cells and normal tissue.

[0011] SUMMARY OF THE INVENTION

[0012] The present 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 of cancer cells comprises high amounts of short chain structures, especially the Thomsen-Friedenreich antigen (TF; Gaipi-3GalNAca1-). In contrast, O-glycosylation in normal cells encompasses much longer oligosaccharide chains. Since the developed antibodies specifically bind to CD24 carrying the short, cancer cell-derived TF structures, they discriminate between tumor- associated CD24 (i.e. CD24 present on cancer cells) and CD24 on cells of normal tissue.

[0013] The high specificity of the developed antibodies for CD24 carrying TF structures is of particular relevance in view of the fact that, while the tumor-associated short chain structures are virtually absent from most normal tissue, O-glycosylation of certain blood cells such as e.g. B cells and granulocytes comprises sialylated Thomsen-Friedenreich antigen (sTF) structures. The developed antibodies are able to discriminate between the closely related TF and sTF structures on CD24 and thereby prevent targeting of healthy blood cells expressing CD24 carrying sTF.

[0014] Thereby, the antibodies according to the present invention preferentially bind to CD24 on tumor cells and hence, have a superior cancer specificity and reduced binding to normal tissue, resulting in lower risk for adverse effects in cancer immunotherapy for a broad patient population. Therefore, the developed antibodies are particularly suitable for use in cancer immunotherapy.

[0015] In view of the above, in a first aspect, the present invention is directed to an antibody which is capable of binding to human CD24 glycosylated at one or more serine and / or threonine residues with Gaipi-3GalNAca1-.

[0016] In a second aspect, the present invention is directed to an antibody which is capable of binding to human CD24 and which comprises

[0017] (i) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0018] (ii) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0019] (iii) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0021] (v) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0022] (vi) a heavy chain variable region and a light chain variable region according to any one of items (i) to (v), above, comprising 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences.

[0023] In certain embodiments, the antibody according to the first aspect is an antibody according to the second aspect.

[0024] In a third aspect, the present invention provides a conjugate comprising the antibody according to the first or second aspect of the invention conjugated to a further agent.

[0025] In a fourth aspect, the present invention provides a nucleic acid encoding the antibody according to the first or second aspect of the invention or the conjugate according to the third aspect, wherein the further agent is a polypeptide or protein fused to the antibody. Furthermore, in a fifth aspect an expression cassette or vector comprising the nucleic acid according to the invention and a promoter operatively connected with said nucleic acid and, in a sixth aspect, a host cell comprising the nucleic acid or the expression cassette or vector according to the invention are provided.

[0026] In a seventh aspect, the present invention is directed to a composition comprising the antibody according to the first or second aspect of the invention, the conjugate according to third aspect of the invention, the nucleic acid according to the fourth aspect of the invention, the expression cassette or vector according to the fifth aspect of the invention, or the host cell according to the sixth aspect of the invention. The composition in particular is a pharmaceutical composition.

[0027] According to an eighth aspect, the invention provides the antibody, the conjugate, the nucleic acid, the expression cassette or vector, the host cell, or the composition according to the invention for use in medicine, in particular in the treatment of cancer, bacterial or viral infections, or vascular diseases.

[0028] Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, which indicate preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

[0029] DEFINITIONS

[0030] As used herein, the following expressions are generally intended to preferably have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0031] The expression "comprise", as used herein, besides its literal meaning also includes and specifically refers to the expressions "consist essentially of" and "consist of". Thus, the expression "comprise" refers to embodiments wherein the subject-matter which "comprises" specifically listed elements does not comprise further elements as well as embodiments wherein the subject-matter which "comprises" specifically listed elements may and / or indeed does encompass further elements. Likewise, the expression "have" is to be understood as the expression "comprise", also including and specifically referring to the expressions "consist essentially of' and "consist of". The term "consist essentially of", where possible, in particular refers to embodiments wherein the subject-matter comprises 20% or less, in particular 15% or less, 10% or less or especially 5% or less further elements in addition to the specifically listed elements of which the subject-matter consists essentially of.

[0032] The term "antibody" in particular refers to a protein comprising at least two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The heavy chain-constant region comprises three or - in the case of antibodies of the IgM- or IgE-type - four heavy chain-constant domains (CH1 , CH2, CH3 and CH4) wherein the first constant domain CH1 is adjacent to the variable region and may be connected to the second constant domain CH2 by a hinge region. The light chainconstant region consists only of one constant domain. The variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR), wherein each variable region comprises three CDRs and four FRs. The amino acid residues of the CDRs are in particular determined based on the CDR localization according to the IMGT system.

[0033] The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The heavy chain constant regions may be of any type such as y-, 5-, a-, p- or e-type heavy chains. Preferably, the heavy chain of the antibody is a y-chain. Furthermore, the light chain constant region may also be of any type such as K- or A-type light chains. Preferably, the light chain of the antibody is a K-chain. The terms "y- (5-, a- , p- or £-) type heavy chain" and "K- (A-) type light chain" refer to antibody heavy chains or antibody light chains, respectively, which have constant region amino acid sequences derived from naturally occurring heavy or light chain constant region amino acid sequences, especially human heavy or light chain constant region amino acid sequences. In particular, the amino acid sequence of the constant domains of a y-type (especially y1-type) heavy chain is at least 95%, especially at least 98%, identical to the amino acid sequence of the constant domains of a human y (especially one of the allotypes of the human y1) antibody heavy chain. Furthermore, the amino acid sequence of the constant domain of a K-type light chain is in particular at least 95%, especially at least 98%, identical to the amino acid sequence of the constant domain of one of the allotypes of the human K antibody light chain. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibody can be, e.g., a humanized, human or chimeric antibody.

[0034] The antigen-binding portion of an antibody usually refers to full length or one or more fragments of an antibody that retains the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2fragment, a bivalent fragment comprising two Fab fragments, each of which binds to the same antigen, linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and a dAb fragment, which consists of a VH domain.

[0035] The "Fab part" of an antibody in particular refers to a part of the antibody comprising the heavy and light chain variable regions (VH and VL) and the first domains of the heavy and light chain constant regions (CH1 and CL). In cases where the antibody does not comprise all of these regions, then the term "Fab part" only refers to those of the regions VH, VL, CH1 and CL which are present in the antibody. Preferably, "Fab part" refers to that part of an antibody corresponding to the fragment obtained by digesting a natural antibody with papain which contains the antigen binding activity of the antibody. In particular, the Fab part of an antibody encompasses the antigen binding site or antigen binding ability thereof. Preferably, the Fab part comprises at least the VH region of the antibody.

[0036] The "Fc part" of an antibody in particular refers to a part of the antibody comprising the heavy chain constant regions 2, 3 and - where applicable - 4 (CH2, CH3 and CH4). In particular, the Fc part comprises two of each of these regions. In cases where the antibody does not comprise all of these regions, then the term "Fc part" only refers to those of the regions CH2, CH3 and CH4 which are present in the antibody. Preferably, the Fc part comprises at least the CH2 region of the antibody. Preferably, "Fc part" refers to that part of an antibody corresponding to the fragment obtained by digesting a natural antibody with papain which does not contain the antigen binding activity of the antibody. In particular, the Fc part of an antibody is capable of binding to the Fc receptor and thus, e.g. comprises an Fc receptor binding site or an Fc receptor binding ability.

[0037] According to the present invention, the term "chimeric antibody" in particular refers to an antibody wherein the constant regions are derived from a human antibody or a human antibody consensus sequence, and wherein at least one and preferably both variable regions are derived from a non-human antibody, e.g. from a rodent antibody such as a mouse antibody.

[0038] The term "antibody", as used herein, refers in certain embodiments to a population of antibodies of the same kind. In particular, all antibodies of the population of the antibody exhibit the features used for defining the antibody. In certain embodiments, all antibodies in the population of the antibody have the same amino acid sequence. Reference to a specific kind of antibody, such as an anti-CD24 antibody, in particular refers to a population of this kind of antibody.

[0039] The term "antibody" as used herein includes the full-length antibody as well as fragments and derivatives of said antibody. A "fragment or derivative" of an antibody in particular is a protein or glycoprotein which is derived from said antibody and is capable of binding to the same antigen, in particular to the same epitope as the antibody. Thus, a fragment or derivative of an antibody herein generally refers to a functional fragment or derivative. In particularly preferred embodiments, the fragment or derivative of an antibody comprises a heavy chain variable region. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody or derivatives thereof. Examples of fragments of an antibody include (i) Fab fragments, monovalent fragments consisting of the variable region and the first constant domain of each the heavy and the light chain; (ii) F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the variable region and the first constant domain CH 1 of the heavy chain; (iv) Fv fragments consisting of the heavy chain and light chain variable region of a single arm of an antibody; (v) scFv fragments, Fv fragments consisting of a single polypeptide chain; (vi) (Fv)2 fragments consisting of two Fv fragments covalently linked together; (vii) a heavy chain variable domain; and (viii) multibodies consisting of a heavy chain variable region and a light chain variable region covalently linked together in such a manner that association of the heavy chain and light chain variable regions can only occur intermolecular but not intramolecular. Derivatives of an antibody in particular include antibodies which bind to the same antigen as the parent antibody, but which have a different amino acid sequence than the parent antibody from which it is derived. These antibody fragments and derivatives are obtained using conventional techniques known to those with skill in the art.

[0040] A target amino acid sequence is "derived" from or "corresponds" to a reference amino acid sequence if the target amino acid sequence shares a homology or identity over its entire length with a corresponding part of the reference amino acid sequence of 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%. The "corresponding part" means that, for example, framework region 1 of a heavy chain variable region (FRH1) of a target antibody corresponds to framework region 1 of the heavy chain variable region of the reference antibody. In particular embodiments, a target amino acid sequence which is "derived" from or "corresponds" to a reference amino acid sequence is 100% homologous, or in particular 100% identical, over its entire length with a corresponding part of the reference amino acid sequence. A "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 the corresponding part of the reference sequence which corresponds to the sequence which homology or identity is defined. An antibody derived from a parent antibody which is defined by one or more amino acid sequences, such as specific CDR sequences or specific variable region sequences, in particular is an antibody having amino acid sequences, such as CDR sequences or variable region sequences, which 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 or identical, especially identical, to the respective amino acid sequences of the parent antibody. In certain embodiments, the antibody derived from (i.e. derivative of) a parent antibody comprises the same CDR sequences as the parent antibody, but differs in the remaining sequences of the variable regions.

[0041] The term "antibody" as used herein also refers to multivalent and multispecific antibodies, i.e. antibody constructs which have more than two binding sites each binding to the same epitope and antibody constructs which have one or more binding sites binding to a first epitope and one or more binding sites binding to a second epitope, and optionally even further binding sites binding to further epitopes.

[0042] "Specific binding" preferably means that an agent such as an antibody binds stronger to a target such as an epitope for which it is specific compared to the binding to another target. An agent binds stronger to a first target compared to a second target if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. Preferably the dissociation constant for the target to which the agent binds specifically is more than 10-fold, 30-fold, 100-fold or more than 500-fold lower than the dissociation constant for the target to which the agent does not bind specifically. Furthermore, the term "specific binding" in particular indicates a binding affinity between the binding partners with an affinity constant Kaof at least 105M’1, preferably at least 106M-1, more preferably at least 107M-1, for example at least 108M-1. An antibody specific for a certain antigen in particular refers to an antibody which is capable of binding to said antigen with an affinity having a Kaof at least 105M’1, preferably at least 106M-1, more preferably at least 107M-1. For example, the term "anti- CD24 antibody" refers to an antibody specifically binding CD24 and preferably is capable of binding to CD24 with an affinity having a Kaof at least 105M-1, preferably at least 106M’1, more preferably at least 107M-1.

[0043] The term "epitope" as used herein refers to the amino acid residues and glycan structures on the antigen of an antibody which are either directly contacted by the amino acids of the antibody, in particular the amino acids of the CDRs of the antibody, or which are in direct vicinity thereof and influence the binding of the antibody to its antigen.

[0044] The term "CD24" according to the present invention in particular refers to the human CD24 protein, especially the mature human CD24 protein, including all its variants. CD24 as used herein especially refers to human CD24 protein according to the UniProt entry P25063. CD24 in particular comprises the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence which is at least 90%, especially at least 95% identical to SEQ ID NO: 58 over the entire length. In certain 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 in particular is post-translationally modified and may carry O- glycosylation and / or N-glycosylation. For example, CD24 may carry O-glycosylation structures 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.

[0045] The term "GalNAcal-", also called "Tn", "Tn antigen" or "Thomsen nouvelle antigen" refers to a monosaccharide structure consisting of an N-acetyl galactosaminyl residue attached via an a-glycosidic bond to a supporting structure, especially to a serine or threonine residue of a protein or peptide.

[0046] The term "sialylated GalNAcal-", also called "sTn", "sialylated Tn antigen", or "sialylated Thomsen nouvelle antigen" refers to a disaccharide structure consisting of an N-acetyl galactosaminyl residue attached via an a-glycosidic bond to a supporting structure, especially to a serine or threonine residue of a protein or peptide. To this structure, a sialic acid residue is attached via a2-6 bond to the N-acetyl galactosaminyl residue resulting in the disaccharide structure Siaa2-6GalNAca1-.

[0047] The term "Gaipi-3GalNAca1-", also called "TF", "TF antigen", "T antigen" or "Thomsen Friedenreich antigen" refers to a disaccharide structure consisting of a galactosyl residue attached via a (31-3 bond to an N-acetyl galactosaminyl residue attached via an a1- glycosidic bond to a supporting structure, especially to a serine or threonine residue of a protein or peptide.

[0048] The term "sialylated Gaipi-3GalNAca1-", also called "sTF", "sTF antigen", "sialylated TF antigen", "sialylated T antigen" or "sialylated Thomsen Friedenreich antigen" refers to trior tetrasaccharide structures consisting of a galactosyl residue attached via a pi -3 bond to an N-acetyl galactosaminyl residue attached via an a1-glycosidic bond to a supporting structure, especially to a serine or threonine residue of a protein or peptide. To this structure, a sialic acid residue is attached via an a2-3 bond to the galactosyl residue and / or a sialic acid residue is attached via an a2-6 bond to the N-acetyl galactosaminyl residue resulting in the trisaccharide structures Siaa2-3Gaipi-3GalNAca1- and Gaipi- 3(Siaa2-6)GalNAca1- or the tetrasaccharide structure Siaa2-6(Siaa2-3Gaipi- 3)GalNAca1-.

[0049] In the above indicated structures, Gal represents a galactose residue and GalNAc represents an N-acetyl galactosamine residue. "P1-3", "a2-3" and "a2-6" indicate the linkage of the two adjacent monosaccharide residues, especially between carbon atom C1 or C2, respectively, of the left monosaccharide and carbon atom C3 or 06, respectively, of the right monosaccharide, wherein the linkage can be in a- or p-position (shown in the following scheme for glucose):

[0050] The term "GalNAcal-" indicates that the GalNAc residue at the reducing end of the oligosaccharide is linked via its carbon atom C1 in a-configuration to the support structure.

[0051] The term "sialic acid" in particular refers to any N- or O-substituted derivatives of neuraminic acid. It may refer to both 5-N-acetylneuraminic acid (NeuNAc) and 5-N- glycolylneuraminic acid (NeuGc), but preferably only refers to 5-N-acetylneuraminic acid.

[0052] The terms "glycan", "glycan structure", "carbohydrate", "carbohydrate chain" and "carbohydrate structure" are generally used synonymously herein.

[0053] In a "conjugate" two or more compounds are linked together. In certain embodiments, at least some of the properties from each compound are retained in the conjugate. Linking may be achieved by a covalent or non-covalent bond. Preferably, the compounds of the conjugate are linked via a covalent bond. The different compounds of a conjugate may be directly bound to each other via one or more covalent bonds between atoms of the compounds. Alternatively, the compounds may be bound to each other via a chemical moiety such as a linker molecule wherein the linker is covalently attached to atoms of the compounds. If the conjugate is composed of more than two compounds, then these compounds may, for example, be linked in a chain conformation, one compound attached to the next compound, or several compounds each may be attached to one central compound.

[0054] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids and ribonucleic acids as well as deoxyribonucleic acids. It may comprise naturally occurring as well as synthetic nucleotides and can be naturally or synthetically modified, for example by methylation, 5'- and / or 3'-capping.

[0055] The term "expression cassette" in particular refers to a nucleic acid construct which is capable of enabling and regulating the expression of a coding nucleic acid sequence introduced therein. An expression cassette may comprise promoters, ribosome binding sites, enhancers and other control elements which regulate transcription of a gene or translation of an mRNA. The exact structure of expression cassette may vary as a function of the species or cell type, but generally comprises 5'-untranscribed and 5'- and 3'-untranslated sequences which are involved in initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, and the like. More specifically, 5'-untranscribed expression control sequences comprise a promoter region which includes a promoter sequence for transcriptional control of the operatively connected nucleic acid. Expression cassettes may also comprise enhancer sequences or upstream activator sequences.

[0056] According to the invention, the term "promoter" refers to a nucleic acid sequence which is located upstream (5') of the nucleic acid sequence which is to be expressed and controls expression of the sequence by providing a recognition and binding site for RNA- polymerases. The "promoter" may include further recognition and binding sites for further factors which are involved in the regulation of transcription of a gene. A promoter may control the transcription of a prokaryotic or eukaryotic gene. Furthermore, a promoter may be "inducible", i.e. initiate transcription in response to an inducing agent, or may be "constitutive" if transcription is not controlled by an inducing agent. A gene which is under the control of an inducible promoter is not expressed or only expressed to a small extent if an inducing agent is absent. In the presence of the inducing agent the gene is switched on or the level of transcription is increased. This is mediated, in general, by binding of a specific transcription factor.

[0057] The term "vector" is used here in its most general meaning and comprises any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and / or eukaryotic cells and, where appropriate, to be integrated into a genome. Vectors of this kind are preferably replicated and / or expressed in the cells. Vectors comprise plasmids, phagemids, bacteriophages or viral genomes. The term "plasmid" as used herein generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0058] According to the invention, the term "host cell" relates to any cell which can be transformed or transfected with an exogenous nucleic acid. The term "host cells" comprises according to the invention prokaryotic (e.g. E. coli) or eukaryotic cells (e.g. mammalian cells, in particular human or hamster cells, yeast cells and insect cells). Particular preference is given to mammalian cells such as cells from humans, mice, hamsters, pigs, goats, or primates. The cells may be derived from a multiplicity of tissue types and comprise primary cells and cell lines. A nucleic acid may be present in the host cell in the form of a single copy or of two or more copies and, in one embodiment, is expressed in the host cell.

[0059] The term "patient" means according to the invention a human being, a nonhuman primate or another animal, in particular a mammal such as a cow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse and rat. In a particularly preferred embodiment, the patient is a human being.

[0060] The term "cancer" according to the invention in particular comprises leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, oral cancer, bladder cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof. The term cancer according to the invention also comprises cancer metastases. The term cancer further also refers to and / or includes cancer stem cells, especially the cancer stem cells of the specific types of cancer listed above.

[0061] By "tumor" is meant a group of cells or tissue that is formed by misregulated cellular proliferation. Tumors may show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign or malignant.

[0062] By "metastasis" is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and normally involves detachment of cancer cells from a primary tumor, entering the body circulation and settling down to grow within normal tissues elsewhere in the body. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells normally resemble those in the original tumor. This means, for example, that, if breast cancer metastasizes to the lungs, the secondary tumor is made up of abnormal breast cells, not of abnormal lung cells. The tumor in the lung is then called metastatic breast cancer, not lung cancer.

[0063] The term "pharmaceutical composition" particularly refers to a composition suitable for administering to a human or animal, i.e., a composition containing components which are pharmaceutically acceptable. Preferably, a pharmaceutical composition comprises an active compound or a salt or prodrug thereof together with a carrier, diluent or pharmaceutical excipient such as buffer, preservative and tonicity modifier.

[0064] DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention is based on the development of anti-CD24 antibodies which specifically bind tumor-associated CD24. These antibodies were generated using CD24 or CD24 fragments carrying O-glycan structures produced by cancer cells and selecting those antibodies which bind to CD24 in an O-glycosylation-dependent manner. O- glycosylation in normal cells differs significantly from O-glycosylation produced by cancer cells. While most normal cells produce large O-glycan structures, tumor cells comprise high amounts of short chain structures, especially mono- and disaccharides such as the Thomsen-Friedenreich antigen (TF; Gaipi-3GalNAca1-), the Thomsen nouvelle antigen (Tn; GalNAcal-) and sialylated Thomsen nouvelle antigen (sTn). The developed antibodies specifically bind to CD24 carrying the short, cancer cell-derived TF structures, therefore discriminating between tumor-associated CD24 (i.e. CD24 present on cancer cells) and CD24 on cells of normal tissue. Since the developed antibodies are capable of specifically binding to CD24 glycosylated with the glycosylation structure TF but not to CD24 glycosylated with sialylated Thomsen-Friedenreich antigen (sTF), they are of particular relevance in view of the fact that sTF is present in the glycosylation pattern of CD24 expressed on certain blood cells such as B cells and granulocytes. Thereby, the antibodies according to the present invention preferentially bind to CD24 on tumor cells and hence, have a superior cancer specificity and reduced binding to normal tissue, resulting in lower risk for adverse effects in cancer immunotherapy. Therefore, the developed antibodies are particularly suitable for use in cancer immunotherapy for a broad patient population.

[0066] 1. Anti-CD24 antibodies

[0067] In view of these findings, the present invention provides in a first aspect an antibody which is capable of binding to human CD24 glycosylated at one or more serine and / or threonine residues with Gaipi-3GalNAca1-.

[0068] In certain embodiments, the antibody is capable of specifically binding to human CD24 glycosylated at one or more serine and / or threonine residues with at least one oligosaccharide structure selected from the group consisting of GalNAcal-, Gaipi- 3GalNAca1-, and sialylated GalNAcal-. In certain embodiments, the antibody is capable of specifically binding to human CD24 glycosylated at one or more serine and / or threonine residues with Gaipi-3GalNAca1-.

[0069] In a specific embodiment, the antibody is capable of binding to human CD24, glycosylated with any one of GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, and mixtures thereof. Glycosylation as referred to herein generally refers to glycan structures attached to serin and / or threonine residues of CD24. These serine and / or threonine residues in particular are selected from the group comprising Thr4, Thr5, Ser8, Ser9, Ser11 , Ser12, Ser14, Thr15, Ser16, Ser18, Thr25, Thr28 and Thr29 of SEQ ID NO: 58.

[0070] An antibody capable of binding to glycosylated human CD24 binds to glycosylated human CD24 with a higher binding affinity compared to non-glycosylated human CD24. In certain embodiments, an antibody capable of binding to glycosylated human CD24 binds to glycosylated human CD24 with a higher binding affinity compared to an unrelated protein carrying the same glycan structures and / or compared to a carrier molecule unrelated to CD24 carrying the same glycan structures. For example, the carrier molecule may be polyacrylamide (PAA) or a random peptide.

[0071] In certain embodiments, an antibody capable of binding to human CD24 glycosylated at one or more serine and / or threonine residues with Gaipi-3GalNAca1- binds to glycosylated human CD24 with a higher binding affinity compared to human CD24 glycosylated with O-glycans of normal human cells from healthy tissue. In certain embodiments, an antibody capable of binding to human CD24 glycosylated at one or more serine and / or threonine residues with Gaipi-3GalNAca1- binds to glycosylated human CD24 with a higher binding affinity compared to human CD24 glycosylated with sialylated Gaipi-3GalNAca1-. Glycosylation with O-glycans of normal human cells from healthy tissue in particular refers to glycans comprising 5, 6, 7 or more monosaccharide units, especially to glycans selected from the group consisting of extended core-1 structures such as Neu5Aca2-3Gaipi-4GlcNAcpi-3Gaipi-3GalNAca-, extended core-2 structures such as NeuAca2-3Gaipi-4GlcNAcpi-6(NeuAca2-3Gaipi-3)GalNAca- and Neu5Aca2-3Gaipi-3(Gaipi-4(Fuca1-3)GlcNAc(pi-6)GalNAca-, and extended core-3 structures such as Gaipi-4GlcNAcpi-3Gaipi-4GlcNAcpi-3GalNAca-. The term "a higher binding affinity" as used herein refers to an agent binding a first target with a higher binding affinity compared to a second target. An agent binds a first target with a higher binding affinity compared to a second target if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. The term "a higher binding affinity" as used herein in particular refers to a difference in the dissociation constant of at least 10-fold, especially at least 25-fold, in particular at least 100-fold.

[0072] In certain embodiments, the antibody is capable of binding to human CD24 comprising more than one oligosaccharide structure, for example 2, 3 or 4 oligosaccharide structures, which are attached to serine and / or threonine residues of CD24. One or more of these further oligosaccharide structures may also be selected from the group consisting of GalNAcal-, sialylated GalNAcal-, and Gaipi-3GalNAca1-, especially Gaipi-3GalNAca1-. In specific embodiments, the antibody is capable of binding to human CD24 comprising at least two oligosaccharide structures, each selected from the group consisting of GalNAcal-, sialylated GalNAcal-, and Gaipi-3GalNAca1-, and each being attached to a serine or threonine residue. Especially, the antibody is capable of binding to human CD24 comprising at least three oligosaccharide structures, each selected from the group consisting of GalNAcal-, sialylated GalNAcal-, and Gaipi- 3GalNAca1-, and each being attached to a serine or threonine residue of CD24. In certain embodiments, the antibody is capable of binding to human CD24 comprising more than one Gaipi-3GalNAca1- structure, for example 2, 3 or 4 Gaipi-3GalNAca1- structures, which are attached to serine and / or threonine residues of CD24. In specific embodiments, the antibody is capable of binding to human CD24 comprising at least two Gaipi-3GalNAca1- structures, each being attached to a serine or threonine residue. Especially, the antibody is capable of binding to human CD24 comprising at least three Gaipi-3GalNAca1- structures, each being attached to a serine or threonine residue of CD24.

[0073] The antibody is capable of binding to human CD24 at a combined peptide and carbohydrate epitope. The epitope comprises one or more amino acids of SEQ ID NO: 58, in particular 3 or more or especially 5 or more amino acids of SEQ ID NO: 58. In specific embodiments, at least 75%, especially at least 85%, in particular at least 90% of the amino acids of the epitope are present in SEQ ID NO: 58.

[0074] The term "capable of binding to human CD24 at a combined peptide and carbohydrate epitope" in this respect means that the antibody either directly interacts with amino acids and one or more glycan structures of CD24, or directly interacts only with amino acids of CD24. In certain aspects, the binding affinity of the antibody to CD24 is higher if one or more Gaipi-3GalNAca1- are attached to the amino acids of CD24 compared to the nonglycosylated CD24. In embodiments where the antibody directly interacts only with amino acids of CD24, but not with carbohydrate chains, the higher affinity for the O-glycosylated CD24 is based on conformational changes of the polypeptide chain induced by the oligosaccharides attached to the amino acids of CD24.

[0075] In certain embodiments, the CD24 is present on the surface of a cell. Hence, in certain embodiments, the antibody is capable of binding to human CD24 glycosylated at one or more serine and / or threonine residues with Gaipi-3GalNAca1-, as described herein, wherein the human CD24 is present on the surface of a cell. In certain embodiments, the antibody specifically binds to tumor-associated CD24. In certain embodiments, the antibody is capable of binding tumor-associated CD24 with a higher binding affinity than CD24 expressed by cells of normal tissue.

[0076] In specific embodiments, the antibody is capable of binding to human CD24 glycosylated with Gaipi-3GalNAca1-. Thus, the antibody binds to CD24 glycosylated with Gaipi- 3GalNAca1-. In these embodiments, CD24 may also carry other glycan structures as long as Gaipi-3GalNAca1- is also present. In particular, these antibodies specifically bind to human CD24 glycosylated with Gaipi-3GalNAca1-.

[0077] In a second aspect, the present invention provides an antibody which is capable of binding to human CD24 and which comprises (i) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0078] (ii) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0079] (iii) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0080] (iv) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0081] (v) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0082] (vi) a heavy chain variable region and a light chain variable region according to any one of items (i) to (v), above, comprising 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences.

[0083] In specific embodiments, the anti-CD24 antibody may have 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences, in particular 1 or 2, especially 1 amino acid substitution. In these embodiments, the anti-CD24 antibody retains the antigen specificity of the antibody without said amino acid substitutions. An “amino acid substitution” as used herein also includes an amino acid addition and an amino acid deletion. In certain embodiments, an amino acid substitution is a conservative amino acid substitution.

[0084] The “antibody” according to the second aspect of the present invention in particular exhibits one or more of the binding activities defined for the antibody according to the first aspect. In particular, the antibody according to the second aspect is an antibody according to the first aspect.

[0085] In specific embodiments, the antibody according to the invention comprises a heavy chain variable region comprising the 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 a light chain variable region comprising the 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.

[0086] In certain 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

[0087] (i) a heavy chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 25 over its entire length and comprises the 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 a light chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 38 over its entire length and comprises the 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

[0088] (ii) a heavy chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 27 over its entire length and comprises the 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 a light chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 44 over its entire length and comprises the 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

[0089] (iii) a heavy chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 30 over its entire length and comprises the 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 a light chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 47 over its entire length and comprises the 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

[0090] (iv) a heavy chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 35 over its entire length and comprises the 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 a light chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 55 over its entire length and comprises the 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) a heavy chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 36 over its entire length and comprises the 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 a light chain variable region which is at least 60% identical to the amino acid sequence of SEQ ID NO: 56 over its entire length and comprises the 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.

[0091] In the above embodiments the sequence identity may in particular be at least 70%, preferably at least 80%, and more preferably at least 90%. The above embodiments in particular are humanized versions of the respective antibodies wherein the changes in the amino acid sequence are substitutions to amino acid residues of a related human antibody sequence.

[0092] In certain embodiments, the anti-CD24 antibody may additionally have 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences, in particular 1 or 2, especially 1 amino acid substitution. In these embodiments, the anti-CD24 antibody retains the antigen specificity of the antibody without said amino acid substitutions.

[0093] In further 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

[0094] (i) a heavy chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 25 over its entire length and comprises the 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 a light chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 38 over its entire length and comprises the 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

[0095] (ii) a heavy chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 27 over its entire length and comprises the 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 a light chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 44 over its entire length and comprises the 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

[0096] (iii) a heavy chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 30 over its entire length and comprises the 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 a light chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 47 over its entire length and comprises the 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

[0097] (iv) a heavy chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 35 over its entire length and comprises the 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 a light chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 55 over its entire length and comprises the 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

[0098] (v) a heavy chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 36 over its entire length and comprises the 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 a light chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 56 over its entire length and comprises the 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.

[0099] In the above embodiments the sequence identity may in particular be at least 95%. In certain embodiments, the anti-CD24 antibody may additionally have 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences, in particular 1 or 2, especially 1 amino acid substitution. In these embodiments, the anti-CD24 antibody retains the antigen specificity of the antibody without said amino acid substitutions.

[0100] In specific embodiments, the antibody according to the invention comprises a heavy chain variable region which is at least 80%, especially 90%, identical to the amino acid sequence of SEQ ID NO: 30 over its entire length and comprises the 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 a light chain variable region which is at least 80% especially 90%, identical to the amino acid sequence of SEQ ID NO: 47 over its entire length and comprises the 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.

[0101] In particular, the antibody according to the invention comprises a heavy chain variable region and a light chain variable region, wherein

[0102] (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 38; or

[0103] (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 44; or

[0104] (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 47; or

[0105] (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55; or

[0106] (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 36, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 56; or (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 56; or

[0107] (vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 57; or

[0108] (viii)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 36, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 57.

[0109] In certain embodiments, the antibody is a humanized version of any one of the antibodies according to items (i) to (viii), above. The humanized version in particular has an amino acid sequence identity with the respective above antibody of at least 60%, especially at least 70%, preferably at least 80% and more preferably at least 90% over the entire length of the original sequence.

[0110] In specific embodiments, the antibody according to the invention is a humanized antibody comprising a heavy chain variable region and a light chain variable region, wherein

[0111] (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39; or

[0112] (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40; or

[0113] (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 41 ; or

[0114] (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42; or

[0115] (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 43; or (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 45; or

[0116] (vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 46; or

[0117] (viii)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48; or

[0118] (ix) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48; or

[0119] (x) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49; or

[0120] (xi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 50; or

[0121] (xii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51 ; or

[0122] (xiii)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 52; or

[0123] (xiv)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 53; or

[0124] (xv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54.

[0125] In certain embodiments, the antibody according to the invention is a humanized antibody which has an amino acid sequence identity with any one of above humanized antibodies of at least 90%, especially at least 95% and more preferably at least 97% over the entire length of the original heavy chain variable region sequence and the original light chain variable region sequence.

[0126] In embodiments wherein the heavy and / or light chain variable region comprises an amino acid sequence which has a certain identity to an amino acid sequence of SEQ ID NOs: 25 to 57, any sequence deviations to said amino acid sequence are in particular located in the framework regions, but not in the CDRs. Hence, in these embodiments the heavy and light chain variable regions comprise the respective CDR sequences as defined herein.

[0127] In a specific embodiment, the antibody according to the 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.

[0128] In a specific embodiment, the antibody according to the 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.

[0129] In a specific embodiment, the antibody according to the 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.

[0130] In a specific embodiment, the antibody according to the 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.

[0131] In a specific embodiment, the antibody according to the 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.

[0132] In a specific embodiment, the antibody according to the 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.

[0133] In a specific embodiment, the antibody according to the 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.

[0134] In a specific embodiment, the antibody according to the 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. In specific embodiments, the antibody is capable of binding to human CD24 glycosylated with any one or both of Gaipi-3GalNAca1- and GalNAcal-, and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of

[0135] (i) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0136] (ii) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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.

[0137] In specific embodiments, the antibody is capable of binding to human CD24 glycosylated with Gaipi-3GalNAca1-, in particular capable of specifically binding to human CD24 glycosylated with Gaipi-3GalNAca1-, and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of

[0138] (i) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0139] (ii) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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

[0140] (iii) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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.

[0141] In the above embodiments the sequence identity may in particular be at least 70%, preferably at least 80%, and more preferably at least 90%. In the above embodiments which refer to a sequence identity, the antibody in particular is a humanized version of the respective antibody wherein the changes in the amino acid sequence are substitutions to amino acid residues of a related human antibody sequence.

[0142] In certain embodiments, the anti-CD24 antibody may additionally have 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences, in particular 1 or 2, especially 1 amino acid substitution. In these embodiments, the anti-CD24 antibody retains the antigen specificity of the antibody without said amino acid substitutions.

[0143] In certain embodiments, the antibody comprises an Fc region. The antibody may especially be a whole antibody. In particular, the antibody may comprise two heavy chains and two light chains. The antibody may be of any isotype, and in particular is an IgG-type antibody, especially lgG1 , lgG2 or lgG4. In specific embodiments, the antibody is an lgG1-type antibody. The antibody in particular is capable of binding to one or more human Fc receptors, especially human Fey receptors such as Fey receptor Illa. In certain embodiments, the anti-CD24 antibody is a chimeric, humanized or human antibody. In certain embodiments, the antibody has a reduced or eliminated binding to human Fc receptors. For example, the glycosylation site in the CH2 domain of the antibody may be deleted by mutation, for example by substitution of Asn297, for example to Gin. In another embodiment, the antibody comprises the L234A / L235A double mutation in the heavy chain.

[0144] In further embodiments, the anti-CD24 antibody is a fragment of an antibody. Especially the fragment is selected from the group consisting of (i) Fab fragments; (ii) F(ab)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) scFv fragments; and (vi) (Fv)2 fragments. In certain embodiments, the anti-CD24 antibody does not comprise an Fc region.

[0145] 2. Production of the anti-CD24 antibodies

[0146] The anti-CD24 antibody is preferably recombinantly produced in a host cell. Hence, the antibody in particular is a monoclonal antibody. The host cell used for the production of the antibody may be any host cells which can be used for antibody production. Suitable host cells are in particular 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, bird cells such as EB66 duck cell lines, 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.

[0147] In certain embodiments, the anti-CD24 antibody is produced recombinantly in a human cell line, in particular in a human myeloid leukemia cell line. Preferred human cell lines which can be used for production of the anti-CD24 antibody as well as suitable production procedures are described in WO 2008 / 028686 A2. In a specific embodiment, the anti-CD24 antibody is obtained by expression in a human myeloid leukemia cell line selected from the group consisting of NM-H9D8, NM-H9D8-E6 and NM-H9D8-E6Q12. These cell lines were deposited under the accession numbers DSM ACC2806 (NM- H9D8; deposited on September 15, 2006), DSM ACC2807 (NM-H9D8-E6; deposited on October 5, 2006) and DSM ACC2856 (NM-H9D8-E6Q12; deposited on August 8, 2007) according to the requirements of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), InhoffenstraBe 7B, 38124 Braunschweig (DE) by Glycotope GmbH, Robert- Rbssle-Str. 10, 13125 Berlin (DE). NM-H9D8 cells provide a glycosylation pattern with a high degree of sialylation, a high degree of bisecting GIcNAc, a high degree of galactosylation and a high degree of fucosylation. NM-H9D8-E6 and NM-H9D8-E6Q12 cells provide a glycosylation pattern similar to that of 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 in the American Type Culture Collection (ATCC CCL-243), CHO cells, as well as cell lines derived from the aforementioned. In specific embodiments, the anti-CD24 antibody is produced recombinantly in CHO cells, especially in CHO dhfr cells.

[0148] 3. Conjugates of the anti-CD24 antibodies

[0149] In specific embodiments, the anti-CD24 antibody is provided as conjugate comprising the antibody conjugated to a further agent such as a detectable marker or a therapeutically active substance. The antibody can be conjugated to one or more further agents. If more than one further agent is present in the conjugate, these further agents may be identical or different, and in particular are all identical. Conjugation of the further agent to the antibody can be achieved using any methods known in the art. The further agent may be covalently, in particular by fusion or chemical coupling, or non-covalently attached to the antibody. In certain embodiments, the further agent is covalently attached to the antibody, especially via a linker moiety. The linker moiety may be any chemical entity suitable for attaching the further agent to the antibody.

[0150] The further agent preferably is useful in therapy, diagnosis, prognosis and / or monitoring of a disease, in particular cancer. For example, the further agent may be selected from the group consisting of radionuclides, chemotherapeutic agents, antibodies, bispecific antibodies or antibody fragments, in particular those of different species and / or different specificity than the anti-CD24 antibody, enzymes, interaction domains, detectable labels, toxins, cytolytic components, immunomodulators, immunoeffectors, cytokines, chemokines, MHC class I or class II antigens, and liposomes.

[0151] In certain embodiments, the further agent is a polypeptide or protein. This polypeptide or protein may in particular be fused to a polypeptide chain of the anti-CD24 antibody. In certain embodiments, the further agent being a polypeptide or protein is fused to the C terminus of an antibody light chain of the anti-CD24 antibody. In embodiments wherein the anti-CD24 antibody comprises two antibody light chains, a further agent being a polypeptide or protein may be fused to the C terminus of each of the two antibody light chains. In further embodiments, the further agent being a polypeptide or protein is fused to the C terminus of an antibody heavy chain of the anti-CD24 antibody. In embodiments wherein the antibody comprises two antibody heavy chains, a further agent being a polypeptide or protein may be fused to the C terminus of each of the two antibody heavy chains. The further agents may be identical or different and in particular have the same amino acid sequence. In embodiments wherein the antibody does not comprise one or more light chains and one or more heavy chains, for example in cases where the antibody is an antibody fragment, a further agent being a polypeptide or protein may be fused to the C terminus or the N terminus of a polypeptide chain of the antibody. Suitable examples of such further agents being a polypeptide or protein may be selected from the group consisting of cytokines, chemokines, antibodies, antigen binding fragments, enzymes, and interaction domains.

[0152] In certain embodiments, the further agent being a polypeptide or protein is a checkpoint antibody which blocks and / or triggers activating signals. In further embodiment the further agent being a polypeptide or protein is an anti-cancer antibody directed to a tumor-associated antigen. In further embodiments, the further agent being a polypeptide or protein is an immunomodulatory compound such as a chemokine, cytokine or growth factor.

[0153] The conjugate comprising the anti-CD24 antibody conjugated to a further agent in particular is a chimeric antigen receptor (CAR). Such conjugates are also referred herein as anti-CD24 CARs. In these embodiments, the further agent is an antigen receptor, such as a T cell receptor, a T cell co-receptor or an activating NK cell receptor, or a part and / or chimera thereof. In particular, the anti-CD24 antibody is fused to a transmembrane domain and an intracellular T cell or NK cell signaling domain, forming a chimeric antigen receptor (CAR). The intracellular domain is in particular derived from one or more T cell or NK cell receptors or co-receptors. Optionally, the CAR further comprises a hinge region between the antibody and the transmembrane domain.

[0154] In these embodiments, the anti-CD24 antibody in particular is a single chain antibody fragment which comprises the heavy chain variable region and the light chain variable region in one polypeptide chain, especially a scFv fragment. The hinge region may for example be based on a hinge region or membrane-proximal region of a member of the immunoglobulin superfamily. Exemplary hinge regions include those derived from IgG, CD8 and CD28. The transmembrane domain may be a hydrophobic alpha helix that spans the cell membrane. It is for example derived from CD28 or NKG2D. The intracellular T cell signaling domain in particular comprises the cytoplasmic domain of the chain of the T cell receptor. In addition, the intracellular T cell signaling domain may comprise further domains of co-stimulatory proteins of T cells. Exemplary further domains include signaling domains from CD28, CD27, CD134 (0X40), CD137 (4-1 BB), 2B4, KIR2DS4, DNAM1 , DAP10, and DAP12.

[0155] An exemplary CAR may comprise, from N terminus to C terminus, (i) the anti-CD24 antibody in the form of a scFv fragment, (ii) an extracellular hinge region derived from CD8, (iii) a transmembrane domain derived from CD28, (iv) a cytoplasmic signaling domain derived from CD28, and (v) a signaling domain derived from the T cell receptor -chain.

[0156] Alternatively, the anti-CD24 antibody, especially in single chain format such as scFv, may be fused N terminally to a CD3 chain of the T cell receptor complex, especially the CD3E chain, to form a chimeric antigen receptor. Or the anti-CD24 antibody, especially in single chain format such as scFv, may be fused to a binding domain which is capable of specifically binding to naturally occurring or engineered receptors on T cells or NK cells.

[0157] In certain embodiments, the further agent is a cytotoxic or chemotherapeutic agent, especially a cytotoxin. Specific examples of chemotherapeutic agents that can be conjugated as further agent include alkylating agents such as cisplatin, anti-metabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes such as taxol, topoisomerase inhibitors such as irinotecan and topotecan, antineoplastics such as doxorubicin or microtubule inhibitors such as auristatins and maytansin / maytansinoids. 4. Nucleic acids encoding the anti-CD24 antibodies

[0158] In a further aspect, the present invention provides a nucleic acid encoding the anti-CD24 antibody. In some aspects, the present invention provides a nucleic acid encoding the antibody according to the first or the second aspect, or the conjugate according to the third aspect, wherein the further agent is a polypeptide or protein 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 in which the nucleic acid is to be expressed, in particular the human codon usage. The nucleic acid may be double-stranded or single-stranded DNA or RNA, preferably doublestranded DNA such as cDNA or single-stranded RNA such as mRNA. It may be one consecutive nucleic acid molecule or it may be composed of several nucleic acid molecules, each coding for a different part of the antibody.

[0159] If the anti-CD24 antibody is composed of more than one different amino acid chain, such as a light chain and a heavy chain, the nucleic acid may, for example, be a single nucleic acid molecule containing several coding regions each coding for one of the amino acid chains of the antibody, preferably separated by regulatory elements such as IRES elements in order to generate separate amino acid chains, or the nucleic acid may be composed of several nucleic acid molecules wherein each nucleic acid molecule comprises one or more coding regions each coding for one of the amino acid chains of the antibody. Alternatively, the nucleic acid may be a single nucleic acid molecule containing one coding region which encodes for the heavy chain and the 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 recognition site. In addition to the coding regions encoding the antibody, the nucleic acid may also comprise further nucleic acid sequences or other modifications which, for example, may code for other proteins, may influence the transcription and / or translation of the coding region(s), may influence the stability or other physical or chemical properties of the nucleic acid, or may have no function at all.

[0160] In certain embodiments, the nucleic acid is a viral vector which can be used for the infection of human cells. These viral vectors may be suitable, for example, for therapy of humans, e.g. by directing infection and or replication of the virus to disease cells such as tumor cells, or be modifying T cells or NK cells in embodiments where the anti-CD24 antibody is in the form of a chimeric antigen receptor to obtain CAR T cells or CAR NK cells.

[0161] In a further aspect, the present invention provides an expression cassette or vector comprising a nucleic acid according to the invention and a promoter operatively connected with said nucleic acid. In addition, the expression cassette or vector may comprise further elements, in particular elements which are capable of influencing and / or regulating the transcription and / or translation of the nucleic acid, the amplification and / or reproduction 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 a host cell. Suitable expression cassettes and vectors comprising respective expression cassettes for expressing antibodies are well known in the prior art and thus, need no further description here.

[0162] 5. Host cells

[0163] Furthermore, the present invention provides a host cell comprising the nucleic acid according to the invention or the expression cassette or vector according to the invention. The host cell may be any host cell. It may be an isolated cell or a cell comprised in a tissue. Preferably, the host cell is a cultured cell, in particular a primary cell or a cell of an established cell line, preferably a tumor-derived cell. Suitable host cells are in particular 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, bird cells such as EB66 duck cell lines, 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.

[0164] In a preferred embodiment of the invention the host cell is a CHO cell or a cell derived from human myeloid leukaemia cells. Preferably, it is selected from the following cells or cell lines: K562, KG1 , MUTZ-3, CHO or a cell or cell line derived therefrom. The host cell is preferably selected from the group consisting of CHO, NM-H9D8, NM-H9D8-E6, NM- H9D8-E6Q12, and a cell or cell line derived from anyone of said host cells. These cell lines and their properties are described in detail in the PCT-application WO 2008 / 028686 A2. In certain embodiments, the host cell is optimized for expression of glycoproteins, in particular antibodies, having a specific glycosylation pattern. Preferably, the codon usage in the coding region of the nucleic acid according to the invention and / or the promoter and the further elements of the expression cassette or vector are compatible with and, more preferably, optimized for the type of host cell used. Preferably, the anti- CD24 antibody is produced by a host cell or cell line as described above.

[0165] The present invention further provides a host cell carrying an anti-CD24 CAR. Such a host cell is also called CAR cell herein. The CAR cell in particular comprises the nucleic acid according to the invention or the expression cassette or vector according to the invention, which encoding the anti-CD24 CAR. In certain embodiments, the CAR cell is engineered to express the anti-CD24 CAR, for example by introducing a vector comprising an expression cassette for the anti-CD24 CAR. In specific embodiments, the CAR cell is a white blood cell, especially a lymphocyte such as a T cell, a NK cell and a NKT cell, or a monocyte such as a macrophage. The CAR cell may be a primary white blood cell or a cell of an immune cell line. In certain embodiments, the CAR cell is selected from the group consisting of primary T cells, primary NK cells, primary NKT cells and primary macrophages, and especially is a primary T cell.

[0166] 6. Pharmaceutical compositions and therapeutic use

[0167] In another aspect, the present invention provides a composition comprising the anti- CD24 antibody, the nucleic acid, the expression cassette or vector, the host cell, or the conjugate according to the invention. The composition may also contain more than one of these components. Furthermore, the composition may comprise one or more further components selected from the group consisting of solvents, diluents, and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, the components of the composition preferably are all pharmaceutically acceptable. The composition may be a solid or fluid composition, in particular a - preferably aqueous - solution, emulsion or suspension or a lyophilized powder.

[0168] The anti-CD24 antibody or the conjugate thereof or the CAR cell in particular is useful in medicine, in particular in therapy, diagnosis, prognosis and / or monitoring of a disease, in particular a disease as described herein, for example cancer and infections, preferably cancer. Therefore, in a further aspect, the invention provides the anti-CD24 antibody, the nucleic acid, the expression cassette or vector, the host cell, the conjugate, or the composition for use in medicine. Preferably, the use in medicine is a use in the treatment, prognosis, diagnosis and / or monitoring of a disease, especially a disease associated with CD24. Exemplary diseases include diseases associated with abnormal cell growth such as cancer, bacterial and viral infections and vascular disease.

[0169] 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, cervix cancer, colorectal cancer, kidney cancer, stomach cancer, lung cancer, esophageal cancer, and pancreas cancer. The cancer may for example be breast cancer, endometrial cancer or ovarian cancer. The cancer is CD24 positive and in particular comprises cancer cells which carry CD24 on their cell surface.

[0170] In specific embodiments, the anti-CD24 antibody is used in combination with another anti-cancer therapeutic agent. Said further therapeutic agent may be any known anticancer drug and in particular may be an antibody against a cancer antigen including bispecific antibodies, especially immune cell recruiting bispecific antibodies. T reatment with the anti-CD24 antibody may further be combined with immunostimulatory agents, cytokines, chemokines, radiation therapy, therapeutic cells such as native 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, peptide or RNA vaccines, B-Raf inhibitors such as vemurafenib, dexametasone, protease inhibitors such as bortezomib, and lenalidomide.

[0171] For use in the treatment of cancer wherein the cells express CD24, the antibody may be coupled to a further agent as described above, wherein the further agent preferably is a cytotoxic agent such as a radionuclide or a cytotoxin. Cytotoxic agents also include precursor compounds which only develop cytotoxic activity upon activation, e.g., by irradiation with light or enzymatic reaction inside the body. Furthermore, the antibody may be engineered so as to enhance its ability to activate the patient's immune response, in particular the ability to activate ADCC (antibody-dependent cell-mediated cytotoxicity) and / or CDC (complement dependent cytotoxicity). For example, this may be achieved by optimizing the amino acid sequence and / or the glycosylation pattern of the antibody, in particular of its constant regions.

[0172] For use as detection agent in diagnosis, prognosis and / or monitoring of a disease, the antibody preferably is coupled to a labeling agent which is capable of producing a detectable signal. In particular, said labeling agent may be a radionuclide, a fluorophore or an enzyme.

[0173] FIGURES

[0174] Figure 1 shows a visualization of the process for antibody generation.

[0175] Figure 2 shows the biosynthesis pathways of the Tn and TF carbohydrate antigens as well as their structures. In the structures, a square represents GalNAc, a circle represents Gal, and a diamond represents a sialic acid.

[0176] Figure 3 shows binding of anti-CD24 clones to the differently glycosylated CD24 variants in an antigen ELISA. Binding to equimolar amounts of antigens (35 nM) was assessed in an antigen ELISA using 250 ng / ml (A + B + C) or 5 pg / ml (D) of anti-CD24 mAbs. Anti- CD24 (SWA-11), anti-glycan antibodies (data not shown) were used as controls. sTF-, TF- and T n-carrying proteins were recombinantly expressed and purified from NM-H9D8, NM-F9 and NM-F9-GalKO cells, respectively, and enzymatically treated for de-sialylation (siaA) or de-O-glycosylation to remove terminal sialic acids or non-sialylated TF and Tn glycans. A high O.D. signal indicates strong binding of the antibody to the antigen. Figure 4 shows the titration of anti-CD24 clones to their respective primary on-target protein CD24-TF. Clones 1 E9 (A), 1 E1 (B), 2C9 (C-H) and their humanized variants were titrated on a fixed amount of CD24-TF protein (35 nM). Binding was analyzed by ELISA, ECso values from dose-response curves were calculated by non-linear regression using GraphPad Prism 5 software.

[0177] Figure 5 shows binding of anti-CD24 clones 1 E1 (A), 1 E9 (A), 2C9 (A + B + C) and several of their humanized variants to differentially glycosylated CD24 expressed on 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 no O-glycans) and F9 (NM-F9 cells expressing low levels of CD24 and high levels of TF). Cells were stained with 10 pg / ml of anti-CD24 clones, protein-specific, glycosylation-independent control anti-CD24 mAb SWA-11 and anti-glycan control antibodies (data not shown). Shown is signal-to-noise ratio of stained cells: MFI (median fluorescence intensity) of stained cells divided by MFI of isotype-stained cells.

[0178] Figure 6 shows binding of anti-CD24 clone 1 E1 and its humanized variants to tumor cell lines expressing CD24 with (+ NA) and without neuraminidase treatment (wt). Shown are the binding data for tumor cell lines MCF7 (A) and SKOV-3 (B + C) Cancer cells were untreated or treated with neuraminidase (30 min with 5 mll / ml), washed and stained with 10 pg / ml anti-CD24 clones and fluorophore-coupled anti-human-IgG secondary reagent. Expression of CD24, TF and Tn was confirmed using control anti- CD24 (SWA-11), anti-TF and anti-Tn mAbs (data not shown). Shown is signal-to-noise ratio of stained cells: MFI of stained cells divided by MFI of isotype-stained cells.

[0179] Figure 7 shows binding of anti-CD24 clone 1 E9 and its humanized variants to tumor cell lines expressing CD24 with (+ NA) and without neuraminidase treatment (wt). Shown are the binding data for tumor cell lines MCF7 (A), SKOV-3 (B) and MDA-MB-468 (C). Cancer cells were untreated or treated with neuraminidase (30 min with 5 mll / ml), washed and stained with 10 pg / ml anti-CD24 clones and fluorophore-coupled anti- human-IgG secondary reagent. Expression of CD24, TF and Tn was confirmed using control anti-CD24 (SWA-11), anti-TF and anti-Tn mAbs (data not shown). Shown is signal-to-noise ratio of stained cells: MFI of stained cells divided by MFI of isotype- stained cells.

[0180] Figure 8 shows binding of anti-CD24 clone 2C9 and its humanized variants to tumor cell lines expressing CD24 with (+ NA) and without neuraminidase treatment (wt). Shown are the binding data for tumor cell lines MCF7 (A + D), SKOV-3 (B) and MDA-MB468 (C + D). Cancer cells were untreated or treated with neuraminidase (30 min with 5 mll / ml), washed and stained with 10 pg / ml anti-CD24 clones and fluorophore-coupled anti- human-IgG secondary reagent. Expression of CD24, TF and Tn was confirmed using control anti-CD24 (SWA-11), anti-TF and anti-Tn mAbs (data not shown). Shown is signal-to-noise ratio of stained cells: MFI of stained cells divided by MFI of isotype- stained cells

[0181] Figure 9 shows the results of the quantitative flow cytometry of the two clones 1 E9 and 2C9 on different cell lines, including exemplary humanized variants of each clone, and the glycan independent anti-CD24 antibody SWA-11. For the quantitative analysis of antibody binding capacity (ABC), Quantum Simply Cellular anti human IgG beads (Bangs Laboratories) were stained with the same secondary reagent, measured at the same instrument with the same settings on the same day as cells labelled with a saturated amount (100 pg / ml) of anti-CD24 clones. ABC values of the cells are calculated according to the standard curve of the quantum beads, isotype control is subtracted. The limit of detection is determined by the blank bead population.

[0182] Figure 10 shows titration of anti-CD24 clone 2C9 and exemplary humanized 2C9 variants on TF- and CD24-expressing MCF7 cells. Cells were stained with different concentrations of anti-CD24 clones and detected with fluorophore-coupled anti-human- IgG secondary reagent. Shown is the MFI of live cells.

[0183] Figure 11 shows binding of (A) anti-TF mAb to healthy blood cells with and without neuraminidase treatment and (B) binding of the glycan-independent anti-CD24 clone SWA-11 and exemplary anti-CD24 clones to blood cells of two healthy donors. Granulocytes and B cells from healthy donors were stained with 10 pg / ml of anti-TF mAb and fluorophore-coupled anti-IgG secondary reagent or CD24-positive Granulocytes and B cells were stained with 1 pg / ml DIG-conjugated anti-CD24 clones and fluorophore- coupled anti-DIG secondary reagent Expression of CD24 and the O-glycans TF and Tn was confirmed using control anti-CD24 (SWA-11) and anti-glycan antibodies (data not shown). Binding is shown as the median fluorescence intensity of live cells.

[0184] Figure 12 shows the capacity of anti-CD24 clones 1 E9 and 2C9 and the humanized variants 1 E9-2A04 and 2C9-3A12 to be internalized by TF- and CD24-positive MCF7 cells (A + B), CD24-F9 (TF) cells (C + D), CD24+ granulocytes (E + F) and B cells (G + H), both purified from healthy donors. The glycan-independent anti-CD24 antibody SWA- 11 was used as control Internalization was analyzed with an Incucyte S3 device. Test mAbs were labeled with an anti-human Fab fragment conjugated pH-sensitive fluorophore and incubated for 24 h at 37°C with cells. After internalization and entering acidic lysosomes, red fluorescence is induced and was quantified by Incucyte S3 (Sartorius). Percentage of lysosomal routing is expressed as the percentage of high red intensity of total cells.

[0185] Figure 13 exemplarily shows the ADCC (antibody-dependent cellular cytotoxicity) activity of clone 2C9 as additional mode of action as naked mAb. MCF-7 tumor cells were loaded with Europium and used as target cells. The human NK cell line KHYG- CD16aV was used as effector cells at an effector to target (E:T) ratio of 10:1 . Co-cultures were set up with titrated amounts of either 2C9 antibody, SWA-11 or hlgG1 as control. Specific lysis was analyzed after 4 h by quantification of released Europium via fluorescence readout.

[0186] Figure 14 shows the capacity of anti-CD24 clones to inhibit proliferation of CD24- expressing NM-F9 cells. Cells were incubated with different concentrations of anti-CD24 clones couples to MMAE via Protein G. Proliferation was measured after 4 days. Proliferation is shown in % relative to a medium control without antibody based on luminescent signals.

[0187] EXAMPLES

[0188] Example 1 : Generation of antigens

[0189] Different O-glycosylation variants of human CD24 were generated for an optimized selection of antibodies specifically binding to human CD24 with tumor-associated glycosylation pattern. The CD24 sequence was obtained from Uniprot accession number P25063.

[0190] The most relevant CD24 isoform 1 was chosen as target structure ("CD24"; SEQ ID NO: 58) for antibody generation with a high potential to yield glycosylation-dependent anti- CD24 antibodies in antibody generation approaches described in Example 2.

[0191] Cells of the cancer-derived cell lines NM-F9 and NM-H9D8 were transfected with DNA encoding CD24. Soluble protein constructs were recombinantly expressed as fusion proteins, e.g., with a tandem version of the Strep-tag® (Twin-Strep-tag®, IBA, Germany) and maltose binding lectin (“MBL”) to enable purification from cell supernatant by affinity chromatography and detection of protein via the fusion part independent of CD24 (StrMBL-CD24).

[0192] The NM-F9 cell line was used for expression of human CD24 proteins with a tumor- associated O-glycosylation pattern, predominantly producing CD24 carrying TF and lower amounts of Tn. Glycopeptide analysis showed that >99% of the CD24 peptide were glycosylated, with 87% of the glycan structures being TF structures and the remaining 12% being Tn structures. Soluble CD24 proteins with only Tn-glycosylation or without O- glycosylation were generated from proteins purified from NM-F9 transfectants by enzymatic digestion with the appropriate glycosidases or for Tn-glycosylation by expression in NM-F9-GalKO cells having a knockout of the glycosylation pathway responsible for attaching galactose to Tn to convert it to TF. Soluble CD24 proteins with mainly sTF structures and small amounts of TF structures were produced in NM-H9D8 cells.

[0193] For selection of antibody clones and binding analysis, a further construct with CD24 being fused to the N-terminus of a murine antibody Fc part was produced (CD24-mFc).

[0194] As off-target controls for ELISA binding studies, the extracellular domains of irrelevant glycoproteins were expressed in NM-F9 or NM-F9-de-O-glyc, either with Strep-tag® and MBL or fused to the N-terminus of a murine antibody Fc part. As control anti-CD24 antibody, the commercially available mouse anti-human CD24 lgG1 antibody SWA-11 was used.

[0195] Example 2: Generation of anti-CD24 antibodies

[0196] Monoclonal antibodies specifically recognizing hCD24 with tumor-associated glycosylation pattern were generated via immunization of animals.

[0197] HybriFree technology was used to isolate CD24-specific antibodies from splenocytes of chickens and rabbits immunized with purified CD24 from transfected NM-F9 cells and boosted with CD24. Spleen cells were isolated after final immunization from animals with confirmed antigen-specific antibody response in blood serum or chicken egg yolk preparations which was assessed by flow cytometry and / or ELISA assays. Splenic B cells with antigen-specificity for O-glycosylated CD24 were enriched by depletion of cells showing unwanted protein binding. This included binding to CD24 without glycosylation and / or binding to irrelevant glycoproteins similarly produced as O-glycosylated CD24 and by this containing identical protein fusion partners as well as similar glycan structures bound to non-homologous protein sequences. For this negative selection step, spleen cells were first incubated with off-target proteins immobilized on microtiter plates and / or present in the panning solution. Subsequently unbound cells were transferred to plates coated with O-glycosylated CD24 protein purified from NM-F9 cells to capture splenic B cells with target-specificity. cDNA of antibody variable domains was amplified from the captured cells, cloned into a plasmid with separate expression cassettes for the IgG heavy and light chain, respectively, for construction of a combinatorial mouse lgG1 encoding library in a mammalian expression vector. Plasmid DNA from resulting antibody library pools was transfected into CHO cells for transient production of chimeric antibodies. Antibody minipool cell culture supernatants were tested for target-specific binding in ELISA with various on- and off-target proteins. Single clones were generated from antibody pools that showed specific binding to O-glycosylated CD24. VH and VL cDNAs of antigenspecific single clones were sequenced, and antibodies with unique sequences were expressed in CHO cells. Antigen-specific antibody production in single clone supernatants was confirmed by ELISA as well as flow cytometry before antibody purification.

[0198] The following glycosylation-dependent anti-CD24 antibodies were obtained by immunization of chickens or rabbits:

[0199] Table 1

[0200] In addition, cysteine-modified versions of antibody 10B5 were generated, wherein serine 49 of the heavy chain is substituted for cysteine (SEQ ID NO: 37) and / or cysteine 80 of the light chain is substituted for serine (SEQ ID NO: 57). All selected antibodies were upscaled and expressed as chimeric IgG 1 with human backbone followed by purification via protein A affinity chromatography from mammalian cell culture supernatant. Purity and integrity of mAbs was confirmed with SDS-PAGE and analytical SEC.

[0201] Example 3: Humanization of the heavy and light chain variable regions of anti-CD24 antibodies

[0202] The humanization of chicken derived anti-CD24 antibodies was performed by CDR grafting. Different VH and VL variants per lead clone were combined in a Fab phage library, selected and screened for the desired glycosylation-dependent CD24 binding pattern by ELISA. Top variants were expressed as hlgG 1 in CHO and screened for fine- specificity and tumor cell binding. The following variants were further evaluated:

[0203] Table 2

[0204] Example 4: Antigen ELISA with on- and off-target controls

[0205] Antibodies were analyzed in antigen ELISA assays for specific binding to CD24 carrying tumor-associated glycans.

[0206] Briefly, differently glycosylated and non-glycosylated protein antigens were coated to 96- well plates overnight, unspecific binding was blocked and test antibody samples were added.

[0207] Glycosylation-dependent binding of the generated anti-CD24 antibodies, parental clones as well as humanized variants, to mature human CD24 was assessed in an antigen ELISA (see Table 3 for chimeric clones and Figure 3 for chimeric clones and all humanized variants of those clones, always in comparison). All antibodies showed significant binding to O-glycosylated CD24 proteins, confirming that the epitope recognized by these antibodies is present in the CD24 amino acid sequence. Absence of O-glycosylation on the tested CD24 proteins abrogates binding of the anti-CD24 antibodies. This clearly shows that an O-glycosylated CD24 epitope is required for antibody binding and thus confirms glycosylation-dependency of CD24-binding.

[0208] Results also showed, anti-CD24 antibody clones elicit diverse fine specificities regarding the type of CD24 O-glycosylation. This is evident from varying degrees of recognition of CD24 carrying mainly Tn-, TF, or sTF-glycosylation. In particular, antibody clones 1 E1 , 1 E9 and 2C9, showed strong recognition of CD24 carrying TF structures, while recognition of CD24 carrying Tn and sTF was weak and de-O-glycosylated CD24 was not bound at all. Clones 10B5 and 10F9 also showed strong recognition of CD24 carrying both TF and Tn structures, with little recognition of CD24 carrying sTF and little to no recognition of de-O-glycosylated CD24 (Table 3).

[0209] Table 3

[0210] TF: Thomsen-Friedenreich antigen; sTF: sialylated Thomsen-Friedenreich antigen; Tn: Thomsen nouvelle antigen; de-O: de-O-glycosylated; sTF+NA: sialylated Thomsen-Friedenreich antigen treated with neuraminidase resulting in TF

[0211] Furthermore, no off-target binding to unrelated proteins (off-target proteins) or the glycan structures alone was observed (Table 4).

[0212] Table 4

[0213] Example 5: Titration of anti-CD24 clones on CD24 proteins

[0214] As shown in Figure 4, anti-CD24 clones 1 E9 (A), 1 E1 (B) and 2C9 (C-H) and their humanized variants were titrated on a fixed amount of CD24-TF protein, variants always in comparison with the parental, chimeric clone. For all clones, parental and humanized sequences perform highly similar with EC50 values in a very narrow range, giving proof to a successful humanization of the parental, chicken derived sequences. Example 6: Binding to O-glycosylated and de-O-glycosylated CD24 expressed on cell lines

[0215] Binding of anti-CD24 clones to NM-F9, NM-F9 GalKO and HEK cell lines with different glycosylation status of CD24 was tested.

[0216] CD24-transfected NM-F9 cells expressing CD24 carrying mainly TF and little Tn (CD24- F9) and CD24-transfected HEK-O-glycKO cells expressing CD24 without O- glycosylation (CD24-HEK-non-glyc.) were stained with anti-CD24 clones and detected via fluorophore-coupled anti-human-IgG secondary reagent. Non-transfected NM-F9 (TF) cells which expressed only low amounts of CD24, as well as antibodies against TF and anti-CD24 antibody SWA-11 were used as controls.

[0217] The results showed that the tested parental and humanized anti-CD24 clones bound to CD24 on the different cell lines in an O-glycosylation-dependent manner (Figure 5). In particular, all clones showed strong binding to CD24 carrying TF structures, while none of the clones bound to non-glycosylated CD24 expressed by CD24-HEK cells.

[0218] In addition, the clones 1 E1 , 1 E9, 2C9, 10B5 and 10F9 were also tested for binding to Tn-glycosylated F9 GalKO cells (Table 5). Neither F9 GalKO cells, nor CD24-F9 GalKO cells (expressing high amounts of Tn-glycosylated CD24) were bound.

[0219] Table 5

[0220] Example 7: Binding of anti-CD24 clones to tumor cell lines

[0221] To examine binding of anti-CD24 clones to tumor cell lines, different cell lines with varying endogenous expression levels for CD24, TF, sTF, sTn and Tn were chosen: MCF7 cells expressing CD24 and showing a glycosylation pattern with high TF but low sTF, Tn or sTn, MDA-MB-468 and SKOV-3 cells with a higher sTF but lower 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 mainly TF- glycosylation

[0222] Tumor cell lines were stained with anti-CD24 clones and binding was detected using fluorophore-coupled anti-human-IgG secondary reagent. Expression of CD24, TF, and Tn was confirmed using control anti-CD24 antibodies (SWA-11) and anti-TF and anti-Tn antibodies (data not shown).

[0223] As shown in Figures 6, 7 and 8, all anti-CD24 clones showed strong binding to MCF7 cells independent of neuraminidase treatment due to their low sTF expression. In contrast, binding to SKOV-3 and MDA-MB-468 cells was increased after treatment of the tumor cells with neuraminidase due to release of additional TF-glycosylated CD24 epitope. This demonstrates that the anti-CD24 clones are specific for CD24 glycosylated with TF. CD24-negative tumor cell lines were not recognized (data not shown). Quantum analyses (Figure 9) of exemplary clones correlate with these binding data and confirm high numbers of binding sites per cell in a comparable range as the glycosylationindependent SWA-11 control antibody.

[0224] Example 8: Titration of anti-CD24 clones on tumor cells

[0225] To compare binding strengths of parental and humanized anti-CD24 clones, tumor cell line MCF7 expressing TF-glycosylated CD24 was stained with different concentrations of anti-CD24 clones. All anti-CD24 clones showed dose-dependent binding to CD24 on the cells, with clone 2C9 and its variants being the clone with the strongest binding to cells. Figure 10 shows the titration of 2C9 and the humanized 2C9 variants 2C9-3A12, 2C9-3A12-VL1VH, 2C9-3A12-VL5VH, 2C9-3A12-VL6VH and 2C9-3A12-VL7VH on MCF7 cells. Bound antibodies were detected with fluorophore-coupled anti-human-IgG secondary reagent by flow cytometry. EC50 values of all humanized variants (grey symbols) were similar to that of the parental clone 2C9 (black dots) and glycosylationindependent control antibody SWA-11 (black triangle).

[0226] Example 9: Anti-CD24 clones do not bind to CD24 expressed on human blood cells

[0227] To demonstrate that anti-CD24 clones do not bind to CD24 glycoforms mainly found on normal human blood cells, CD24 expressing human B cells and granulocytes from healthy donors were treated with excess hlgG (Octagam) to block Fc receptor mediated binding and stained with DIG-conjugated anti-CD24 clones followed by detection via fluorophore-coupled anti-DIG secondary reagent. Expression of CD24 was confirmed using the glycan-independent control anti-CD24 mAb SWA-11 (Figure 11 A). DAPI was used to discriminate live from dead cells. In addition, the glycosylation pattern of normal human blood cells from healthy donors was analyzed (Figure 11 B). It was demonstrated that human granulocytes and B cells carry a high amount of sTF structures as shown by binding of the anti-TF antibody to these blood cells only after treatment with neuraminidase. Therefore, fine-specificity of the anti-CD24 antibody clones for TF-carrying CD24, but not sTF-carrying CD24, enables specific targeting of tumor cells without causing off-target effects on normal blood cells.

[0228] As a result, none of the anti-CD24 clones showed binding to CD24 expressed on the human CD24+ B cells and only strongly reduced binding on highly CD24-positive granulocytes, while all both blood cell populations were strongly bound by the commercial glycosylation-independent anti-CD24 antibody SWA-11.

[0229] Example 10: Internalization of anti-CD24 antibodies

[0230] Internalization of the anti-CD24 clones 1 E9, 1 E9-2A04, 2C9 and 2C9-3A12 was analyzed by incubation of the antibodies with different cell lines and determination of the percentage of cells showing antibody uptake by fluorescence measure (Figure 12). As on-target cells, CD24-F9 cells (NM-F9 cells expressing TF-glycosylated CD24) and MCF7 cells were used to determine internalization. Additionally, purified granulocytes and B cells were analyzed to investigate potential internalization in CD24-positive blood cell populations of healthy donors. Internalization was analyzed using an Incucyte S3 device. Test mAbs were labeled with an anti-human Fab fragment conjugated pH- sensitive fluorophore. After internalization and entering acidic lysosomes, red fluorescence was induced and could be quantified. In brief, cells were harvested and seeded to 5x103cells per well of a 96-flat bottom plate in medium. The respective antibody was incubated with a 12-fold molar excess of anti-human Fabfluor-pH antibody labeling dye for 15 min at RT and then added to the cells. Phase contrast and red fluorescence were monitored over time (up to 24 h) in an Incucyte S3 at 37°C. Degree of internalization is given as percentage of high red intensity of total cells.

[0231] The results show strong internalization of anti-CD24 clones 1 E9 and 2C9 as well as the humanized clones 1 E9-2A04 and 2C9-3A12 in the tested CD24-F9 cells. All clones were also strongly internalized into TF- and CD24-expressing MCF7 cells, but - in contrast to control antibody SWA-11 which binds to CD24 in a glycosylation-independent manner - no internalization in CD24-positive granulocytes and B cells of healthy donors could be detected.

[0232] Example 11 : Anti-CD24 clones mediate ADCC

[0233] In a europium release assay, clone 2C9 was investigated for its potential to initiate antibody dependent cellular cytotoxicity. Dependent on the 2C9 concentration, NK cell line KHYG-CD16aV mediated the specific lysis of on-target tumor cell line MCF-7 (Figure 13). The results show, 2C9 mediated lysis is concentration dependent and in a very similar range as the specific lysis mediated by the glycan-independent anti-CD24 antibody SWA-11.

[0234] Example 12: Inhibition of proliferation using protein G-drug conjugated anti-CD24 antibodies

[0235] In order to investigate the potential of anti-CD24 clones to deliver cytotoxic drugs into target cells, protein G-drug conjugate assays were performed. In flat bottom 96-well plates, 5x103CD24-F9 cells (NM-F9 cells expressing predominantly TF-glycosylated CD24) per well were seeded in the presence of indicated test antibody dilutions as well as constant concentrations of Protein G preloaded with the toxin MMAE (Protein G- MMAE, Levena Biopharma). After incubation for 4 days, cell viability was assessed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions and analyzed in a microplate reader TECAN Infinite F200 (Tecan). Proliferation in percent was calculated relative to a medium control without antibody based on luminescent signals.

[0236] The results demonstrated that all anti-CD24 clones conjugated to protein G-MMAE inhibit proliferation of CD24-F9 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 lgG1 antibody (hlgG1 Iso) served as controls.

[0237] Example 13: Tissue binding of anti-CD24 antibodies

[0238] Formalin-fixed paraffin embedded tissue sections were stained with either with anti- CD24 clones and detected using secondary antibodies or stained with DIG-coupled anti- CD24 clones. The commercial anti-CD24 antibody SWA-11 was included to detect protein levels of CD24 in the respective tissues.

[0239] Staining of cancer tissue sections showed strong binding of anti-CD24 clones to e.g. breast cancer. Importantly, the reactivity to healthy tissue was absent or significantly reduced as compared to the glycosylation-independent control anti-CD24 antibody.

[0240] Strong binding of anti-CD24 clones could be detected on several tumor tissues, including breast cancer, esophagus cancer, head-and-neck cancer, kidney cancer, oral cancer, ovarian cancer, bladder cancer, colorectal, skin, endometrium, thyroid, prostate and cervix cancer (no data shown). SEQUENCE LISTING

[0241] IDENTIFICATION OF THE DEPOSITED BIOLOGICAL MATERIAL

[0242] The cell line DSM ACC 2606 was deposited on the date indicated in the following table at the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig (DE) by Nemod Biotherapeutics GmbH & Co. KG, Robert- Rbssle-Str. 10, 13125 Berlin (DE). Glycotope is entitled to refer to this biological material since it was in the meantime assigned from Nemod Biotherapeutics GmbH & Co. KG to Glycotope GmbH.

[0243] The cell lines DSM ACC 2806, DSM ACC 2807 and DSM ACC 2856 were deposited at the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, InhoffenstraBe 7B, 38124 Braunschweig (DE) by Glycotope GmbH, Robert-Rbssle-Str.

[0244] 10, 13125 Berlin (DE) on the dates indicated in the following table.

Claims

CLAIMS1. An antibody which is capable of binding to human CD24 glycosylated at one or more serine and / or threonine residues with Gaipi-3GalNAca1-.

2. The antibody according to claim 1 , which is capable of specifically binding to human CD24 glycosylated at one or more serine and / or threonine residues with Gaipi- 3GalNAca1-.

3. An antibody which is capable of binding to human CD24 and which comprises(i) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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) a heavy chain variable region comprising the 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 a light chain variable region comprising the 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) a heavy chain variable region and a light chain variable region according to any one of items (i) to (v), above, comprising 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences.

4. The antibody according to claim 3, comprising a heavy chain variable region comprising the 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 a light chain variable region comprising the 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 comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 38; or(ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 44; or(iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 47; or(iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55; or(v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 36, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 56; or(vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 56; or(vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 57; or(viii)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 36, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 57; or(ix) the heavy chain variable region and the light chain variable region comprise amino acid sequences which are at least 60% identical to the heavy chain variable region sequence and light chain variable region sequence, respectively, of any one of items (i) to (viii), above, over their entire length, wherein the antibody preferably is a humanized version of the antibody according to any one of items (i) to (viii), above.

6. The antibody according to claim 3, being a humanized antibody, wherein(i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39; or(ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40; or(iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 41 ; or(iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42; or(v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 43; or(vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 45; or(vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 46; or(viii)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48; or(ix) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48; or(x) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49; or(xi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 50; or(xii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51 ; or(xiii)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 52; or(xiv)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 53; or(xv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable region comprises 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 of the binding properties defined in claims 1 and 2.

8. The antibody according to any one of claims 1 to 7, being an lgG1-type, lgG2-type, lgG3-type or lgG4-type antibody, in particular an lgG1-type antibody.

9. A conjugate comprising the antibody according to any one of claims 1 to 8 conjugated to a further agent, wherein the further agent is optionally a cytotoxic agent, tumor-specific antibody or immune checkpoint blocking or activating antibody, or wherein the conjugate is optionally a chimeric antigen receptor.

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

11. An expression cassette or vector comprising the nucleic acid according to claim 10 and a promoter operatively connected with said nucleic acid.

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

13. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 8, the conjugate according to claim 9, the nucleic acid according to claim 10, the expression cassette or vector according to claim 11 , or the 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 antibody, the conjugate, the host cell or the composition for use in medicine according to claim 14 for use in the treatment of cancer, bacterial or viral infections, or vascular disease.

16. The antibody, the conjugate, the host cell or the composition for use in the treatment of cancer according to claim 15, 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, cervix cancer, colorectal cancer, kidney cancer, stomach cancer, lung cancer, esophageal cancer, pancreas cancer, in particular, wherein the cancer is breast cancer, endometrial cancer or ovarian cancer.

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