Antibodies against CD44

Abstract

The present invention pertains to anti-CD44 antibodies which bind CD44 at the C- terminal part of the extracellular domain in an O-glycosylation-dependent manner. Thereby, the antibodies are specific for tumor-associated CD44. The invention further provides pharmaceutical compositions containing said anti-CD44 antibodies and their use in cancer treatment.

Description

[0001] Pentixapharm AG

[0002] BismarckstraBe 13, 97080 Wurzburg

[0003] ..Antibodies against CD44“

[0004] FIELD OF THE INVENTION

[0005] The present invention pertains to the field of antibodies. In particular, anti-CD44 antibodies showing strong antigen binding in a tumor-specific manner are provided. In specific embodiments, the present invention is directed to anti-CD44 antibodies for therapeutic and diagnostic use.

[0006] BACKGROUND OF THE INVENTION

[0007] 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.

[0008] 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.

[0009] A possible cancer antigen is CD44. This protein is a polymorphic cell-surface glycoprotein synthesized in a variety of cells. CD44 interacts with actin-based cytoskeletons and co-localizes with ERM proteins at actin filament-plasma membrane interaction sites. CD44 is suspected to be involved in cell migration, adhesion and differentiation in normal cells, but also in metastasis in cancer cells. It is a receptor for extracellular matrix components, such as soluble or cell-bound hyaluronic acid, collagen, fibronectin and serglycin. The structure of the protein comprises a single membranespanning domain and a heavily glycosylated extracellular domain. The CD44 cytoplasmic domain is putatively associated with an ankyrin-like protein. In humans, CD44 is encoded by at least 10 and up to 19 exons, wherein exons 1-5 and 15-19 are conserved in all isoforms. CD44 comprising only the 10 conserved exons is also referred to as standard CD44 or CD44 short (CD44s) while CD44 comprising all 19 exons is referred to herein as “CD44 large”. The distinct CD44 variants (CD44v) comprise additional peptide motifs that can interact with and sequester growth factors and cytokines at the cell surface, thereby functioning as coreceptors to facilitate cell signaling.

[0010] While the distinct functional roles of the specific variants are not fully understood, overexpression of different CD44v has been detected in several types of cancer such as pancreatic cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma and gastrointestinal cancer. Elevated levels of CD44 variant 6 (CD44v6) and CD44 variant 2 (CD44v2) for example, were reported to play a promoting role in metastasis of pancreatic cancer. Similarly, overexpression of both CD44v6 and CD44 variant 8 (CD44v8) in breast cancer cells were reported to increase metastasis and in head and neck squamosa cell carcinoma, elevated levels of CD44 variant 3 (CD44v3) expression were associated with a significant increase in tumor cell migration. Also in gastrointestinal cancer, overexpression of CD44v6 is correlated with a poor prognosis, promoting migration and metastasis. In prostate cancer, CD44s was reported to be underexpressed while overexpression was detected for all other isoforms. Interestingly CD44v2 was found to be associated with a better recurrence-free survival rate when expressed independently.

[0011] Hence, CD44 and its alternate variants represent interesting targets for anti-tumor immunotherapy.

[0012] However, under normal physiologic conditions CD44 is expressed throughout most epithelial and lymphoid tissues. This may cause unwanted side effects in a cancer therapy with an antibody which cannot discriminate between cancer-associated CD44 and CD44 on normal tissue. To avoid off-target binding in cancer therapy, many antibodies were targeted to a specific variant of CD44 overexpressed in the specific type of cancer that was to be treated. While this approach solves the issue of off-target binding to some degree, it also results in the production of highly specific antibodies suitable for use in a specific therapy only, with correspondingly high costs for development and production.

[0013] Therefore, there is a need in the art to provide tumor-specific anti-CD44 antibodies which can be universally used in the treatment of many different cancer types irrespective of the CD44 variant overexpressed therein.

[0014] SUMMARY OF THE INVENTION

[0015] The present inventors have developed universal anti-CD44 antibodies with good tumor binding.

[0016] These antibodies bind to tumor-associated human CD44 independent of isoform, recognizing conserved structures present on CD44 of cancer cells.

[0017] The antibodies according to the present invention are targeted at a binding region which is present in every splicing variant of human CD44, enabling the antibodies to bind to a larger population of target receptors and thus, improving binding affinity to tumor target cells. Surprisingly, it was found that the epitope region in exons 15 and 16 of CD44 was especially well suited for targeting cancer cells. This epitope region is directly adjacent to the transmembrane domain of CD44 and thus, wedged between the plasma membrane of the cell and the N-terminal domains of CD44. The present inventors nevertheless demonstrated that this binding region is well accessible to antibodies and offers an advantageous target for tumor-specific anti-CD44 antibodies.

[0018] Additionally, the antibodies are able to discriminate between CD44 expressed by cancer cells and CD44 expressed by healthy tissue due to a glycosylation-dependent binding to CD44. O-glycosylation of cancer cells comprises high amounts of short chain structures, especially mono-, di- and trisaccharides such as the Thomsen-Friedenreich antigen (TF; Gaipi-3GalNAca1-), sialylated Thomsen-Friedenreich antigen (sTF), the Thomsen nouvelle antigen (Tn; GalNAcal-) and sialylated Thomsen nouvelle antigen (sTn). In contrast, in normal cells O-glycosylation encompasses much longer oligosaccharide chains. Since the developed antibodies specifically bind to CD44 carrying the short, cancer cell-derived O-glycosylation, they discriminate between tumor-associated CD44 (i.e. , CD44 present on cancer cells) and CD44 on cells of normal tissue. Thereby, the antibodies according to the present invention preferentially bind to CD44 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.

[0019] In view of the above, in a first aspect, the present invention is directed to an antibody which is capable of binding to human CD44 at a combined peptide and carbohydrate epitope located within a binding region at amino acid positions 204 to 248 of CD44s, wherein the binding region has the amino acid sequence of SEQ ID NO: 1 and is glycosylated at one or more serine and / or threonine residues with at least one oligosaccharide structure selected from the group consisting of GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, and sialylated Gaipi-3GalNAca1-.

[0020] In a second aspect, the present invention is directed to an antibody which is capable of binding to human CD44 and which comprises

[0021] (i) 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: 3 and CDR-H3 having the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the complementarity-determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 5, CDR-L2 having the amino acid sequence of SEQ ID NO: 6 and CDR-L3 having the amino acid sequence of SEQ ID NO: 7; or

[0022] (ii) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 13, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15; or

[0023] (iii) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 18, CDR-H2 having the amino acid sequence of SEQ ID NO: 19 and CDR-H3 having the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 21 , CDR-L2 having the amino acid sequence of SEQ ID NO: 22 and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or (iv) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 26, CDR-H2 having the amino acid sequence of SEQ ID NO: 27 and CDR-H3 having the amino acid sequence of SEQ ID NO: 28, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 29, CDR-L2 having the amino acid sequence of SEQ ID NO: 30 and CDR-L3 having the amino acid sequence of SEQ ID NO: 31 ; or

[0024] (v) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 34, CDR-H2 having the amino acid sequence of SEQ ID NO: 35 and CDR-H3 having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 37, CDR-L2 having the amino acid sequence of SEQ ID NO: 38 and CDR-L3 having the amino acid sequence of SEQ ID NO: 39; or

[0025] (vi) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 42, CDR-H2 having the amino acid sequence of SEQ ID NO: 43 and CDR-H3 having the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 45, CDR-L2 having the amino acid sequence of SEQ ID NO: 46 and CDR-L3 having the amino acid sequence of SEQ ID NO: 47; or

[0026] (vii) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 50, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15; or

[0027] (viii)a heavy chain variable region and a light chain variable region according to any one of items (i) to (vii), above, comprising 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences.

[0028] In certain embodiments, the antibody according to the first aspect is an antibody according to the second aspect.

[0029] 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. 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.

[0030] 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.

[0031] 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, arthritis, bacterial or viral infections, interstitial lung disease, vascular disease, or scoliosis.

[0032] 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.

[0033] DEFINITIONS

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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)2 fragment, 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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-CD44 antibody, in particular refers to a population of this kind of antibody.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] The term “capable of binding to” in particular refers to “capable of specifically binding to”. "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- CD44 antibody" refers to an antibody specifically binding CD44 and preferably is capable of binding to CD44 with an affinity having a Kaof at least 105M’1, preferably at least 106M'1, more preferably at least 107M’1.

[0047] 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.

[0048] The term "CD44" according to the present invention in particular refers to the human CD44 protein, especially the mature human CD44 protein, including all its variants. CD44 as used herein especially refers to the human CD44 protein according to the UniProt entry P16070, with UniProt entry P16070-1 showing the entire amino acid sequence of human CD44 with all exons (CD44 large) and the signal sequence present (SEQ ID NO: 66, with amino acid positions 1-20 being the signal sequence, and amino acid positions 223-604 being the different variable exons). CD44 in particular comprises the conserved exons 1-5 and 15-19 of human CD44, comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence which is at least 90%, especially at least 95% identical to SEQ ID NO: 67 over the entire length. In certain embodiments, “CD44” refers to human CD44s, the standard CD44 which consists of the amino acid sequence of SEQ ID NO: 67. CD44 in particular is post-translationally modified and may carry O- glycosylation and / or N-glycosylation. For example, CD44 may carry O-glycosylation structures at one or more positions corresponding to Thr207, Ser211 , Ser214, Thr216, Thr217, Ser220, Ser222, Ser226, Ser229, Thr236, Thr237, Ser238 and Thr243 of SEQ ID NO: 67. CD44 in particular comprises the binding region with the amino acid sequence of SEQ ID NO: 1 , corresponding to amino acid positions 204 to 248 of SEQ ID NO: 67 and / or amino acid positions 605 to 649 of SEQ ID NO: 66.

[0049] The term "GalNAccd-", 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.

[0050] 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-.

[0051] 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.

[0052] 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 (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. 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-.

[0053] 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 02, respectively, of the left monosaccharide and carbon atom 03 or 06, respectively, of the right monosaccharide, wherein the linkage can be in a- or p-position (shown in the following scheme for glucose):

[0054] The term "GalNAcal-" indicates that the GalNAc residue at the reducing end of the oligosaccharide is linked via its carbon atom 01 in a-configuration to the support structure.

[0055] 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.

[0056] The terms "glycan", "glycan structure", "carbohydrate", "carbohydrate chain" and "carbohydrate structure" are generally used synonymously herein. 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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, 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention is based on the development of universal anti-CD44 antibodies which bind to human CD44 on cancer cells at a binding region having the amino acid sequence of SEQ ID NO: 1. The binding region spans the C-terminal extracellular domain in close proximity to the transmembrane part of CD44 and is present in all CD44 variants.

[0069] Further, the present invention is based on the development of anti-CD44 antibodies which specifically bind tumor-associated CD44. These antibodies were generated using CD44 or CD44 fragments carrying O-glycan structures produced by cancer cells and selecting those antibodies which bind to CD44 in an O-glycosylation-dependent manner. O-glycosylation produced by normal cells significantly differs from O-glycosylation produced by cancer cells. For example, normal cells produce large O-glycan structures while CD44 on tumor cells predominantly carries mono-, di- and trisaccharides such as GalNAcal- (Tn), sialylated GalNAcal- (sTn), Gaipi-3GalNAca1- (TF) and sialylated Gaipi-3GalNAca1- (sTF). The antibodies according to the invention recognize and bind to these small O-glycan structures in the context of tumor-associated CD44. Therefore, the antibodies according to the present invention preferentially bind to CD44 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. Additionally, the antibodies according to the present invention are targeted at a binding region which is present in every splice variant of human CD44, enabling the antibodies to bind to a large population of target antigens and thus, improving recognition of and binding to target tumor cells. Surprisingly, it was found that the epitope region in exons 15 and 16 of CD44 was especially well suited for targeting cancer cells. This epitope region is directly adjacent to the transmembrane domain of CD44 and thus, wedged between the plasma membrane of the cell and the N-terminal domains of CD44. Although this binding region is putatively covered by the rest of the extracellular domain of CD44 and the plasma membrane, the present inventors nevertheless demonstrated that this binding region is well accessible to antibodies and offers an advantageous target for glycosylation-dependent antibodies to discriminate between cancer cells and cells of healthy tissue.

[0070] The term “binding region” as used herein refers to a region in the C-terminal extracellular domain of the CD44 protein comprises exons 15 to 16 of CD44 and consisting of the amino acid sequence of SEQ ID NO: 1.

[0071] 1. Anti-CD44 antibodies

[0072] In view of these findings, the present invention provides in a first aspect an antibody which is capable of binding to human CD44 at a combined peptide and carbohydrate epitope located within a binding region at amino acid positions 204 to 248 of CD44s, wherein the binding region has the amino acid sequence of SEQ ID NO: 1 and is glycosylated at one or more serine and / or threonine residues with at least one oligosaccharide structure selected from the group consisting of GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, and sialylated Gaipi-3GalNAca1-. In certain aspects, the antibodies according to the first aspect are capable of specifically binding to the binding region of human CD44.

[0073] In a specific embodiment, the antibody is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with any one of GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, sialylated Gaipi-3GalNAca1-, and mixtures thereof. Glycosylation within the binding region as referred to herein generally refers to glycan structures attached to serin and / or threonine residues of the binding region. These serin and / or threonine residues in particular are selected from Thr4, Ser8, Ser11 , Thr13, Thr14, Ser17, Ser19, Ser23, Ser26, Thr33, Thr34, Ser35 and Thr40 of SEQ ID NO: 1 or from Thr207, Ser211 , Ser214, Thr216, Thr217, Ser220, Ser222, Ser226, Ser229, Thr236, Thr237, Ser238 and Thr243 of SEQ ID NO: 67.

[0074] In certain embodiments, an antibody capable of binding to glycosylated human CD44 binds to glycosylated human CD44 with a higher binding affinity compared to non- glycosylated human CD44. In certain embodiments, an antibody capable of binding to glycosylated human CD44 binds to glycosylated human CD44 with a higher binding affinity compared to an unrelated protein carrying the same glycan structures and / or compared to a carrier molecule unrelated to CD44 carrying the same glycan structures. For example, the carrier molecule may be polyacrylamide (PAA) or a random peptide.

[0075] In certain embodiments, an antibody capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with any one of GalNAcal- , sialylated GalNAcal-, Gaipi-3GalNAca1-, sialylated Gaipi-3GalNAca1-, and mixtures thereof, binds to glycosylated human CD44 at said binding region with a higher binding affinity compared to human CD44 wherein the binding region is glycosylated with O- glycans of normal human cells from healthy tissue. 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 in particular refers to a difference in the dissociation constant of at least 3-fold, especially at least 5-fold, in particular at least 10-fold.

[0076] In certain embodiments, the antibody is capable of binding to human CD44 at the binding region comprising more than one oligosaccharide structure, for example 2, 3 or 4 oligosaccharide structures, which are attached to serine and / or threonine residues of the binding region. One or more of these further oligosaccharide structures may also be selected from the group consisting of GalNAcal-, sialylated GalNAcal-, Gaipi- 3GalNAca1-, and sialylated Gaipi-3GalNAca1-. In specific embodiments, the antibody is capable of binding to human CD44 at the binding region comprising at least two oligosaccharide structures, each selected from the group consisting of GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, and sialylated Gaipi-3GalNAca1-, and each being attached to a serine or threonine residue of the binding region. Especially, the antibody is capable of binding to human CD44 at the binding region comprising at least three oligosaccharide structures, each selected from the group consisting of GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, and sialylated Gaipi-3GalNAca1-, and each being attached to a serine or threonine residue of the binding region.

[0077] The antibody is capable of binding to human CD44 at a combined peptide and carbohydrate epitope located in the binding region. The epitope comprises one or more amino acids of SEQ ID NO: 1 , in particular 3 or more or especially 5 or more amino acids of SEQ ID NO: 1. 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: 1.

[0078] Preferably, all of the amino acids of the epitope are present within SEQ ID NO: 1.

[0079] The term "capable of binding to human CD44 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 CD44, or directly interacts only with amino acids of CD44. In certain aspects, the binding affinity of the antibody to CD44 is higher if one or more oligosaccharide structures selected from the group consisting of GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, and sialylated Gaipi-3GalNAca1- are attached to the amino acids of the binding region compared to the non-glycosylated binding region. In embodiments where the antibody directly interacts only with amino acids of CD44, but not with carbohydrate chains, the higher affinity for the O-glycosylated CD44 is based on conformational changes of the polypeptide chain induced by the oligosaccharides attached to the binding region.

[0080] In certain embodiments, the antibody specifically binds to tumor-associated CD44. In certain embodiments, the antibody is capable of binding tumor-associated CD44 with a higher binding affinity than CD44 expressed by cells of normal tissue.

[0081] In specific embodiments, the antibody is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with any one of GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, sialylated Gaipi-3GalNAca1-, and mixtures thereof. Thus, the antibody binds to CD44 if the binding region is glycosylated with GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, sialylated Gaipi-3GalNAca1-, or any mixture of two, three or all of these glycan structures. In these embodiments, the binding region may also carry other glycan structures as long as at least one of the above glycan structures is also present. In particular, these antibodies bind to human CD44 at the binding region glycosylated with any one or more of GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, and sialylated Gaipi- 3GalNAca1-.

[0082] In specific embodiments, the antibody is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with any one of GalNAcal-, Gaipi-3GalNAca1-, and mixtures thereof. Thus, the antibody binds to CD44 if the binding region is glycosylated with GalNAcal-, Gaipi-3GalNAca1-, or any mixture of these two glycan structures. In these embodiments, the binding region may also carry other glycan structures as long as at least one of the above glycan structures is also present. In particular, these antibodies bind to human CD44 at the binding region glycosylated with any one or both of GalNAcal- and Gaipi-3GalNAca1-. In specific embodiments, the antibody is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with any one of Gaipi- 3GalNAca1-, sialylated Gaipi-3GalNAca1-, and mixtures thereof. Thus, the antibody binds to CD44 if the binding region is glycosylated with Gaipi-3GalNAca1-, sialylated Gaipi-3GalNAca1-, or any mixture of these two glycan structures. In these embodiments, the binding region may also carry other glycan structures as long as at least one of the above glycan structures is also present. In particular, these antibodies bind to human CD44 at the binding region glycosylated with any one or both of Gaipi- 3GalNAca1- and sialylated Gaipi-3GalNAca1-.

[0083] In specific embodiments, the antibody is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with Gaipi-3GalNAca1- . Thus, the antibody binds to CD44 if the binding region is glycosylated with Gaipi- 3GalNAca1-. In these embodiments, the binding region may also carry other glycan structures as long as Gaipi-3GalNAca1- is also present. In particular, these antibodies bind to human CD44 at the binding region glycosylated with Gaipi-3GalNAca1-.

[0084] In specific embodiments, the antibody is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with GalNAcal-. Thus, the antibody binds to CD44 if the binding region is glycosylated with GalNAcal-. In these embodiments, the binding region may also carry other glycan structures as long as GalNAcal- is also present. In particular, these antibodies bind to human CD44 at the binding region glycosylated with GalNAcal-.

[0085] In specific embodiments, the antibody is capable of binding to human CD44 at the binding region, wherein the binding region is glycosylated with any one of Gaipi- 3GalNAca1-, sialylated GalNAcal-, GalNAcal-, sialylated Gaipi-3GalNAca1-, and mixtures thereof or is not glycosylated. Thus, the antibody binds to CD44 if the binding region is glycosylated with GalNAcal-, sialylated GalNAcal-, Gaipi-3GalNAca1-, sialylated Gaipi-3GalNAca1-, or any mixture of two or all of these glycan structures, or if the binding region is not glycosylated. In particular, the antibody binds to human CD44 at said binding region with a higher binding affinity compared to human CD44 wherein the binding region is glycosylated with O-glycans of normal human cells from healthy tissue.

[0086] In a second aspect, the present invention provides an anti-CD44 antibody which comprises

[0087] (i) 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: 3 and CDR-H3 having the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the complementarity-determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 5, CDR-L2 having the amino acid sequence of SEQ ID NO: 6 and CDR-L3 having the amino acid sequence of SEQ ID NO: 7; or

[0088] (ii) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 13, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15; or

[0089] (iii) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 18, CDR-H2 having the amino acid sequence of SEQ ID NO: 19 and CDR-H3 having the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 21 , CDR-L2 having the amino acid sequence of SEQ ID NO: 22 and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or

[0090] (iv) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 26, CDR-H2 having the amino acid sequence of SEQ ID NO: 27 and CDR-H3 having the amino acid sequence of SEQ ID NO: 28, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 29, CDR-L2 having the amino acid sequence of SEQ ID NO: 30 and CDR-L3 having the amino acid sequence of SEQ ID NO: 31 ; or

[0091] (v) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 34, CDR-H2 having the amino acid sequence of SEQ ID NO: 35 and CDR-H3 having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 37, CDR-L2 having the amino acid sequence of SEQ ID NO: 38 and CDR-L3 having the amino acid sequence of SEQ ID NO: 39; or (vi) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 42, CDR-H2 having the amino acid sequence of SEQ ID NO: 43 and CDR-H3 having the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 45, CDR-L2 having the amino acid sequence of SEQ ID NO: 46 and CDR-L3 having the amino acid sequence of SEQ ID NO: 47; or

[0092] (vii) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 50, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15.

[0093] In specific embodiments, the anti-CD44 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-CD44 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.

[0094] 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.

[0095] 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

[0096] (i) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 8 over its entire length and 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: 3 and CDR-H3 having the amino acid sequence of SEQ ID NO: 4, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 9 over its entire length and comprising the complementarity-determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 5, CDR-L2 having the amino acid sequence of SEQ ID NO: 6 and CDR-L3 having the amino acid sequence of SEQ ID NO: 7,

[0097] (ii) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 16 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR- H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 17 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 13 or 50, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15;

[0098] (iii) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 24 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 18, CDR- H2 having the amino acid sequence of SEQ ID NO: 19 and CDR-H3 having the amino acid sequence of SEQ ID NO: 20, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 25 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 21 , CDR-L2 having the amino acid sequence of SEQ ID NO: 22 and CDR-L3 having the amino acid sequence of SEQ ID NO: 23;

[0099] (iv) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 32 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 26, CDR- H2 having the amino acid sequence of SEQ ID NO: 27 and CDR-H3 having the amino acid sequence of SEQ ID NO: 28, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 33 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 29, CDR-L2 having the amino acid sequence of SEQ ID NO: 30 and CDR-L3 having the amino acid sequence of SEQ ID NO: 31 ;

[0100] (v) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 40 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 34, CDR- H2 having the amino acid sequence of SEQ ID NO: 35 and CDR-H3 having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 41 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 37, CDR-L2 having the amino acid sequence of SEQ ID NO: 38 and CDR-L3 having the amino acid sequence of SEQ ID NO: 39; and

[0101] (vi) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 48 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 42, CDR- H2 having the amino acid sequence of SEQ ID NO: 43 and CDR-H3 having the amino acid sequence of SEQ ID NO: 44, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 49 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 45, CDR-L2 having the amino acid sequence of SEQ ID NO: 46 and CDR-L3 having the amino acid sequence of SEQ ID NO: 47.

[0102] 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.

[0103] In certain embodiments, the anti-CD44 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-CD44 antibody retains the antigen specificity of the antibody without said amino acid substitutions.

[0104] 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

[0105] (i) a heavy chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 over its entire length and 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: 3 and CDR-H3 having the amino acid sequence of SEQ ID NO: 4, and a light chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 9 over its entire length and comprising the complementarity-determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 5, CDR-L2 having the amino acid sequence of SEQ ID NO: 6 and CDR-L3 having the amino acid sequence of SEQ ID NO: 7,

[0106] (ii) a heavy chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 16 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR- H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 13 or 50, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15;

[0107] (iii) a heavy chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 24 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 18, CDR- H2 having the amino acid sequence of SEQ ID NO: 19 and CDR-H3 having the amino acid sequence of SEQ ID NO: 20, and a light chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 25 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 21 , CDR-L2 having the amino acid sequence of SEQ ID NO: 22 and CDR-L3 having the amino acid sequence of SEQ ID NO: 23;

[0108] (iv) a heavy chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 32 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 26, CDR- H2 having the amino acid sequence of SEQ ID NO: 27 and CDR-H3 having the amino acid sequence of SEQ ID NO: 28, and a light chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 33 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 29, CDR-L2 having the amino acid sequence of SEQ ID NO: 30 and CDR-L3 having the amino acid sequence of SEQ ID NO: 31 ; (v) a heavy chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 40 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 34, CDR- H2 having the amino acid sequence of SEQ ID NO: 35 and CDR-H3 having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 41 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 37, CDR-L2 having the amino acid sequence of SEQ ID NO: 38 and CDR-L3 having the amino acid sequence of SEQ ID NO: 39; and

[0109] (vi) a heavy chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 48 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 42, CDR- H2 having the amino acid sequence of SEQ ID NO: 43 and CDR-H3 having the amino acid sequence of SEQ ID NO: 44, and a light chain variable region having an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 49 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 45, CDR-L2 having the amino acid sequence of SEQ ID NO: 46 and CDR-L3 having the amino acid sequence of SEQ ID NO: 47.

[0110] In the above embodiments the sequence identity may in particular be at least 95%. In certain embodiments, the anti-CD44 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-CD44 antibody retains the antigen specificity of the antibody without said amino acid substitutions.

[0111] In particular, the antibody according to the invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of

[0112] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;

[0113] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

[0114] 16, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17; (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

[0115] 24, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25;

[0116] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 33;

[0117] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 41 ;

[0118] (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49;

[0119] (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53;

[0120] (viii)a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 54;

[0121] (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

[0122] 51 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55;

[0123] (x) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

[0124] 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53;

[0125] (xi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 54;

[0126] (xii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55; (xiii)a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57;

[0127] (xiv)a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59;

[0128] (xv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 ;

[0129] (xvi)a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62;

[0130] (xvii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64; and

[0131] (xviii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65.

[0132] In certain embodiments, the antibody is a humanized version of any one of the antibodies according to items (i) to (vi), 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.

[0133] 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: 8, 9, 16, 17, 24, 25, 32, 33, 40, 41 , 48 or 49, 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.

[0134] In specific embodiments, the antibody is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with any one of Gaipi- 3GalNAca1-, sialylated Gaipi-3GalNAca1-, and mixtures thereof, and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of (i) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 26, CDR-H2 having the amino acid sequence of SEQ ID NO: 27 and CDR-H3 having the amino acid sequence of SEQ ID NO: 28, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 29, CDR-L2 having the amino acid sequence of SEQ ID NO: 30 and CDR-L3 having the amino acid sequence of SEQ ID NO: 31 ;

[0135] (ii) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 32 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 26, CDR- H2 having the amino acid sequence of SEQ ID NO: 27 and CDR-H3 having the amino acid sequence of SEQ ID NO: 28, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 33 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 29, CDR-L2 having the amino acid sequence of SEQ ID NO: 30 and CDR-L3 having the amino acid sequence of SEQ ID NO: 31 ; and

[0136] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 33.

[0137] In specific embodiments, the antibody is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is 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) heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 3 and CDR-H3 having the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 5, CDR-L2 having the amino acid sequence of SEQ ID NO: 6 and CDR-L3 having the amino acid sequence of SEQ ID NO: 7;

[0139] (ii) heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 13 or 50, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15;

[0140] (iii) heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 8 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 3 and CDR-H3 having the amino acid sequence of SEQ ID NO: 4, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 9 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 5, CDR-L2 having the amino acid sequence of SEQ ID NO: 6 and CDR-L3 having the amino acid sequence of SEQ ID NO: 7;

[0141] (iv) heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 16 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 17 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 13 or 50, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15;

[0142] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;

[0143] (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17;

[0144] (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53; (viii)a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 54;

[0145] (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

[0146] 51 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55;

[0147] (x) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

[0148] 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53;

[0149] (xi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 54; and

[0150] (xii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55.

[0151] In these embodiments, said antibody is in particular capable of specifically binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with Gaipi-3GalNAca1-.

[0152] In these embodiments, the heavy chain variable region and the light chain variable region may especially be selected from the group consisting of

[0153] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53;

[0154] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 54;

[0155] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55;

[0156] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

[0157] 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

[0158] 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 54; and

[0159] (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

[0160] 53, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55.

[0161] In specific embodiments, the antibody is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with GalNAcal-, and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of

[0162] (i) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 34, CDR-H2 having the amino acid sequence of SEQ ID NO: 35 and CDR-H3 having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 37, CDR-L2 having the amino acid sequence of SEQ ID NO: 38 and CDR-L3 having the amino acid sequence of SEQ ID NO: 39;

[0163] (ii) a heavy chain variable region comprising CDR-H 1 having the amino acid sequence of SEQ ID NO: 18, CDR-H2 having the amino acid sequence of SEQ ID NO: 19 and CDR-H3 having the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 21 , CDR-L2 having the amino acid sequence of SEQ ID NO: 22 and CDR-L3 having the amino acid sequence of SEQ ID NO: 23;

[0164] (iii) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 40 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 34, CDR- H2 having the amino acid sequence of SEQ ID NO: 35 and CDR-H3 having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 41 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 37, CDR-L2 having the amino acid sequence of SEQ ID NO: 38 and CDR-L3 having the amino acid sequence of SEQ ID NO: 39; (iv) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 24 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 18, CDR- H2 having the amino acid sequence of SEQ ID NO: 19 and CDR-H3 having the amino acid sequence of SEQ ID NO: 20, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 25 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 21 , CDR-L2 having the amino acid sequence of SEQ ID NO: 22 and CDR-L3 having the amino acid sequence of SEQ ID NO: 23;

[0165] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 41 ;

[0166] (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25;

[0167] (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57;

[0168] (viii)a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59;

[0169] (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64; and

[0170] (x) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65.

[0171] In these embodiments, said antibody is in particular capable of specifically binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with GalNAcal-.

[0172] In these embodiments, the heavy chain variable region and the light chain variable region may especially be selected from the group consisting of (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57;

[0173] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59;

[0174] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64; and

[0175] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65.

[0176] In specific embodiments, the antibody is capable of binding to human CD44 at the binding region, wherein the binding region is glycosylated with any one of GalNAcal-, Gaipi-3GalNAca1-, sialylated GalNAcal-, sialylated Gaipi-3GalNAca1-, and mixtures thereof or is not glycosylated, and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of

[0177] (i) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 42, CDR-H2 having the amino acid sequence of SEQ ID NO: 43 and CDR-H3 having the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 45, CDR-L2 having the amino acid sequence of SEQ ID NO: 46 and CDR-L3 having the amino acid sequence of SEQ ID NO: 47;

[0178] (ii) a heavy chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 48 over its entire length and comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 42, CDR- H2 having the amino acid sequence of SEQ ID NO: 43 and CDR-H3 having the amino acid sequence of SEQ ID NO: 44, and a light chain variable region having an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 49 over its entire length and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 45, CDR-L2 having the amino acid sequence of SEQ ID NO: 46 and CDR-L3 having the amino acid sequence of SEQ ID NO: 47; (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49;

[0179] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 ; and

[0180] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.

[0181] In these embodiments, said antibody is in particular capable of specifically binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with any one of GalNAcal-, Gaipi-3GalNAca1- and mixtures thereof or is not glycosylated.

[0182] In these embodiments, the heavy chain variable region and the light chain variable region may especially be selected from the group consisting of

[0183] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 ; and

[0184] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.

[0185] 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.

[0186] In certain embodiments, the anti-CD44 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-CD44 antibody retains the antigen specificity of the antibody without said amino acid substitutions.

[0187] 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-CD44 antibody is a chimeric, humanized or human antibody.

[0188] In further embodiments, the anti-CD44 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-CD44 antibody does not comprise an Fc region.

[0189] The present invention further provides an anti-CD44 antibody which competes for binding to CD44 with an antibody as described herein, especially with an antibody according to the second aspect of the invention. In certain embodiments, the competitive anti-CD44 antibody competes with an antibody comprising a heavy chain variable region and a light chain variable region selected from the group consisting of

[0190] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;

[0191] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17;

[0192] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25;

[0193] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 33;

[0194] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 41 ; and

[0195] (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49. Assays for determining competitive binding of two antibodies are well known in the art. For example, an ELISA can be used wherein CD44 is immobilized and the first antibodies is labeled and added with an excess of the second antibody to the immobilized CD44. If the label can be detected in the sample with the immobilized CD44 after washing, no competitive binding was observed. In preferred embodiments, control experiments wherein the second antibody is labeled and the first antibody is added in excess are performed. Alternatively, also one of the antibodies may be immobilized and CD44 may be labeled and added with an excess of the other antibody.

[0196] 2. Production of the anti-CD44 antibodies

[0197] The anti-CD44 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.

[0198] In certain embodiments, the anti-CD44 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-CD44 antibody as well as suitable production procedures are described in WO 2008 / 028686 A2. In a specific embodiment, the anti-CD44 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 GlycNAc, 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-CD44 antibody is produced recombinantly in CHO cells, especially in CHO dhfr cells. 3. Conjugates of the anti-CD44 antibodies

[0199] In specific embodiments, the anti-CD44 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.

[0200] 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-CD44 antibody, enzymes, interaction domains, detectable labels, toxins, cytolytic components, immunomodulators, immunoeffectors, cytokines, chemokines, MHC class I or class II antigens, and liposomes.

[0201] 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-CD44 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-CD44 antibody. In embodiments wherein the anti-CD44 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-CD44 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. 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.

[0202] The conjugate comprising the anti-CD44 antibody conjugated to a further agent in particular is a chimeric antigen receptor (CAR). Such conjugates are also referred herein as anti-CD44 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-CD44 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.

[0203] In these embodiments, the anti-CD44 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.

[0204] An exemplary CAR may comprise, from N terminus to C terminus, (i) the anti-CD44 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.

[0205] Alternatively, the anti-CD44 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-CD44 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.

[0206] 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.

[0207] 4. Nucleic acids encoding the anti-CD44 antibodies

[0208] In a further aspect, the present invention provides a nucleic acid encoding the anti-CD44 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.

[0209] If the anti-CD44 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. 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-CD44 antibody is in the form of a chimeric antigen receptor to obtain CAR T cells or CAR NK cells.

[0210] 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.

[0211] 5. Host cells

[0212] 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.

[0213] 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- CD44 antibody is produced by a host cell or cell line as described above.

[0214] The present invention further provides a host cell carrying an anti-CD44 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-CD44 CAR. In certain embodiments, the CAR cell is engineered to express the anti-CD44 CAR, for example by introducing a vector comprising an expression cassette for the anti-CD44 CAR.

[0215] 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.

[0216] 6. Pharmaceutical compositions and therapeutic use

[0217] In another aspect, the present invention provides a composition comprising the anti- CD44 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.

[0218] The anti-CD44 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-CD44 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 CD44. Exemplary diseases include diseases associated with abnormal cell growth such as cancer, arthritis, bacterial and viral infections, interstitial lung disease, vascular disease and scoliosis. In a preferred embodiment, the disease is cancer. Preferably the cancer is selected from the group consisting of esophagus cancer, head and neck cancer, oral cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, cervix cancer, brain cancer and gastrointestinal cancer. The cancer may for example be head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, oral squamous cell carcinoma, breast carcinoma, cervix carcinoma, glioblastoma, or non-small cell lung carcinoma. The cancer is CD44 positive and in particular comprises cancer cells which carry CD44 on their cell surface.

[0219] In specific embodiments, the anti-CD44 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.

[0220] Treatment with the anti-CD44 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, etc.), vaccines such as protein, peptide or RNA vaccines, B-Raf inhibitors such as vemurafenib, dexametasone, protease inhibitors such as bortezomib, and lenalidomide.

[0221] For use in the treatment of cancer wherein the cells express CD44, 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.

[0222] 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. FIGURES

[0223] Figure 1 shows a schematic overview of the exon structure of CD44 indicating the diverse splicing variants based on alternative splicing of Exons 5a to 14 as well as the conserved exons 1 to 5 forming the N-terminal domain and exons 15 to 19 forming the C-terminal domain.

[0224] 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.

[0225] Figure 3 shows binding of anti-CD44 clones and the humanized variants of those clones to the differently glycosylated CD44is / i6 protein variants (on-target) in an antigen ELISA. Binding to equimolar amounts of antigens (35 nM) was assessed in an antigen ELISA using 250 ng / ml of anti-CD44 mAbs. Anti-MBL was used as coating control. sTF-, TF- and Tn-carrying proteins were recombinantly expressed and purified from NM-H9D8, NM-F9 and NM-F9-GalKO (F9cosmc) cells, respectively, and enzymatically treated for de-sialylation (siaA) or de-O-glycosylation. A high O.D. signal indicates strong binding of the antibody to the antigen.

[0226] Figure 4 shows the titration of anti-CD44 clones to their respective primary on-target protein. Clones 2A9 (A) and 1 B5 (B) and their humanized variants were titrated on a fixed amount of CD44-TF protein (35 nM). Clones 2B5 (C) and 42F6 (D) and their humanized variants were titrated on a fixed amount of CD44-T n 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.

[0227] Figure 5 shows binding of anti-CD44 clones to differentially glycosylated CD44 expressed on transgenic cell lines. A and B show binding of clones 1 B5 and 2A9 and their humanized variants, C and D shows binding of clones 2B5 and 42F6 and their humanized variants. Cell lines recombinantly expressing CD44v9 with different glycosylation (NM-F9 cells expressing TF-glycosylated proteins, NM-F9 (cosmc) expressing Tn-glycosylated proteins and HEK cells expressing de-O-glycosylated proteins), were stained with 10 pg / ml of anti-CD44 clones, protein-specific, glycosylation independent control anti-CD44 mAb (RG7356) and anti-TF and anti-Tn mAbs (data not shown). Shown is the signal-to-noise ratio of stained cells: MFI (median fluorescence intensity) of stained cells divided by MFI of isotype-stained cells.

[0228] Figure 6 shows binding of clone 1 B5 and its humanized variants to tumor cell lines expressing CD44 with and without neuraminidase (NA) treatment. A and C shows binding to CaOV3 and MDA-MB-468 cells, B shows binding to HCC-15 cells. Cancer cells were treated with NA (30 min with 5 mll / ml) or kept untreated (wt), washed and stained with 10, 1 or 0.1 pg / ml anti-CD44 clones and fluorophore-coupled anti-human- IgG secondary reagent. Expression of CD44 was confirmed using control anti-CD44 antibody RG7356. Shown is signal-to-noise ratio of stained cells: MFI (median fluorescence intensity) of stained cells divided by MFI of isotype-stained cells.

[0229] Figure 7 shows binding of clone 2A9 and its humanized variants to tumor cell lines expressing CD44 with and without neuraminidase (NA) treatment. A and C shows binding to CaOV3 and MDA-MB-468 cells, B shows binding to HCC-15 cells. Cancer cells were treated with NA (30 min with 5 mll / ml) or kept untreated (wt), washed and stained with 10, 1 or 0.1 pg / ml anti-CD44 clones and fluorophore-coupled anti-human- IgG secondary reagent. Expression of CD44 was confirmed using control anti-CD44 antibody RG7356. Shown is signal-to-noise ratio of stained cells: MFI (median fluorescence intensity) of stained cells divided by MFI of isotype-stained cells.

[0230] Figure 8 shows binding of clone 42F6 and its humanized variants to tumor cell lines expressing CD44 with and without neuraminidase (NA) and galactosidase (GA) treatment. A and B shows binding to CaOV3 and MDA-MB-468 cells. Cancer cells were treated with GA (25 U / ml) and NA (2.5 mU / ml) over night or kept untreated (wt), washed and stained with 10, 1 or 0.1 pg / ml anti-CD44 clones and fluorophore-coupled anti- human-IgG secondary reagent. Expression of CD44 was confirmed using control anti- CD44 antibody RG7356. Shown is signal-to-noise ratio of stained cells: MFI (median fluorescence intensity) of stained cells divided by MFI of isotype-stained cells.

[0231] Figure 9 shows binding of clone 2B5 and its humanized variants to tumor cell lines expressing CD44 with and without neuraminidase (NA) and galactosidase (GA) treatment. A and B shows binding to CaOV3 and MDA-MB-468 cells. Cancer cells were treated with GA (25 U / ml) and NA (2.5 mU / ml) over night or kept untreated (wt), washed and stained with 10, 1 and 0.1 pg / ml anti-CD44 clones and fluorophore-coupled anti- human-IgG secondary reagent. Expression of CD44 was confirmed using control anti- CD44 antibody RG7356. Shown is signal-to-noise ratio of stained cells: MFI (median fluorescence intensity) of stained cells divided by MFI of isotype-stained cells.

[0232] Figure 10 shows the results of the quantitative flow cytometry of clones 1B5 (A), 2A9 (B), 2B5 (C), 42F6 (D) on different tumor cell lines, including an exemplary humanized variant of each clone, and the glycan independent anti-CD44 antibody RG7356 (E). 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-CD44 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.

[0233] Figure 11 shows binding of anti-CD44 clones to healthy human immune cells of two donors (A and B). Monocytes, B cells, T cells and CD3' CD19' lymphocytes from healthy donors were stained with 1 pg / ml DIG-conjugated anti-CD44 clones and fluorophore- coupled anti-DIG secondary reagent. Expression of CD44 was confirmed using the glycan independent anti-CD44 mAb RG7356. Human lgG1 served as isotype control. Shown is the mean fluorescence intensity of live cells for the different PBMC subpopulations.

[0234] Figure 12 shows the capacity of anti-CD44 clones 1 B5 (A and B) and 2B5 (C and D) to be internalized by CD44-expressing cells. Shown is the internalization into CD44 transgenic target cells CD44v9-F9 (TF) (A) and CD44v9-F9 GalKO (Tn) (C) as well as internalization into PBMC of a healthy human donor (B and D). Test mAbs were labeled with an anti-human Fab fragment conjugated pH-sensitive fluorophore and incubated at 37°C with cells. After internalization and entering acidic lysosomes, red fluorescence is induced and can be quantified by Incucyte S3 device (Sartorius). Given are the percentages of total cells in high red intensity over time (60 h).

[0235] EXAMPLES

[0236] Example 1 : Generation of antigens

[0237] Different O-glycosylation variants of human CD44 were generated for an optimized selection of antibodies specifically binding to human CD44 with a tumor-associated glycosylation pattern. The CD44 sequence was obtained from uniport accession number P16070.

[0238] The C-terminal part of the CD44 extracellular domain consisting of exons 15 and 16 was chosen as target structure ("CD44is / i6") for antibody generation with a high potential to yield glyco-dependent anti-CD44 antibodies in antibody generation approaches described in Example 2.

[0239] Cells of the cancer-derived cell line NM-F9 were transfected with DNA encoding CD44i5 / i6. Soluble protein constructs were recombinantly expressed as fusion proteins, e.g., with a tandem version of the Strep-tag® (Twin-Strep-tag®, I BA, 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 CD44. The NM-F9 cell line was used for expression of human CD44is / i6 proteins with tumor- associated O-glycosylation pattern, predominantly producing CD44is / i6 carrying TF and lower amounts of Tn. Glycopeptide analysis showed that 90% to 95% of the glycan structures on the CD44is / i6 peptide were TF structures, with the remaining 5% to 10% being Tn structures. Soluble CD44is / i6 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 CD44is / i6 proteins with sTF structures were produced in NM-H9D8 cells, and sTn-glycosylated CD44is / i6 was obtained from NM-H9D8-GalKO cells with a corresponding knockout as in the NM- F9-GalKO cells.

[0240] For selection of antibody clones and binding analysis, a further construct with CD44is / i6 being fused to the N-terminus of a murine antibody Fc part was produced and different cell lines expressing CD44v9, a human CD44 variant comprising exons 1-5, 13 and 15- 19, were constructed.

[0241] As off-target controls for ELISA binding studies, the extracellular domain of irrelevant glycoproteins were expressed in NM-F9 or NM-F9-GalKO, either with His-tag or fused to the N-terminus of a murine antibody Fc part.

[0242] Example 2: Generation of anti-CD44 antibodies

[0243] Monoclonal antibodies specifically recognizing the C-terminal part of the extracellular domain of hCD44 with tumor-associated glycosylation pattern were generated via immunization of animals.

[0244] HybriFree technology was used to isolate CD44-specific antibodies from splenocytes of chickens immunized with purified CD44is / i6 from transfected NM-F9 cells and boosted with CD44i5 / i6. Spleen cells were isolated after final immunization from animals with confirmed antigen-specific antibody response in chicken egg yolk preparations which was assessed by flow cytometry and / or ELISA assays. Splenic B cells with antigenspecificity for O-glycosylated CD44is / i6 were enriched. 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 human or 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 mini-pool 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 CD44is / i6. VH and VL cDNAs of antigen-specific 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.

[0245] The following glycosylation-dependent anti-CD44 antibodies were obtained by immunization of chicken:

[0246] 1 B5 1 H1 2B5 2A9 42F6 2F2

[0247] All selected antibodies were upscaled and expressed as chimeric IgG 1 with human and mouse backbones 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. Full comparability of both formats was confirmed in biochemical QC and binding studies on off- and on-target proteins and cells. Final binding studies on proteins and tumor cells were performed with the therapeutically relevant hlgG1 format, whereas mlgG1 format was used for binding studies on primary human immune cells and tissues to enable indirect detection method by anti-mouse sec Ab.

[0248] Example 3: Humanization of the murine heavy and light chain variable regions of anti-CD44 antibodies

[0249] The humanization of chicken derived anti-CD44 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 CD44 binding pattern by ELISA. Top variants were expressed as hlgG1 in CHO and screened for fine- specificity and tumor cell binding.

[0250] Example 4: Antigen ELISA with on- and off-target controls

[0251] Antibodies were analyzed in antigen ELISA assays for specific binding to CD44is / i6 carrying tumor-associated glycans.

[0252] Briefly, differently glycosylated and non-glycosylated protein antigens or glycans conjugated to carbohydrate-polyacrylamide (carbohydrate PAA-conjugates) were coated to 96-well plates overnight, unspecific binding was blocked and test antibody samples were added. To control off-target binding, an exemplary irrelevant protein, the Strep-Tag-MBL construct without CD44is / i6, and glycans attached to PAA were used.

[0253] Glyco-dependent binding of the generated anti-CD44 antibodies to the CD44 protein used for the generation of the antibodies (exons 15+16) was assessed in an antigen ELISA (Table 1 and 2; Figure 3). All antibodies showed significant binding to O-glycosylated CD44 proteins, confirming that the epitope recognized by these antibodies is present in the CD44is / i6 amino acid sequence. Absence of O-glycosylation on the tested CD44 proteins abrogates binding of the anti-CD44 antibodies in most cases, except for antibody 2A9. This clearly shows that an O-glycosylated CD44is / i6 epitope is required for antibody binding and thus confirms glyco-dependency of CD44i5 / i6-binding.

[0254] Furthermore, anti-CD44 antibody clones elicit diverse fine specificities regarding the type of CD44 O-glycosylation. This is evident from varying degrees of recognition of CD44is / i6 carrying mainly Tn-, TF, or sTF-glycosylation.

[0255] Table 1

[0256] 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

[0257] Table 2

[0258] The analyzed antibodies showed strong, highly specific binding to O-glycosylated CD44, but no significant binding to unrelated O-glycosylated proteins or pure carbohydrate antigens. Thus, binding of the anti-CD44 antibody clones also specifically relies on the CD44i5 / i6 protein backbone, independent of the attached carrier protein (mFc or StrMBL). Example 5: Titration of anti-CD44 clones on CD44 proteins

[0259] As shown in Figure 4, anti-CD44 clones 2A9 (A) and 1 B5 (B) and their humanized variants were titrated on a fixed amount of CD44-TF protein. Clones 2B5 (C) and 42F6 (D) and their humanized variants were titrated on a fixed amount of CD44-Tn protein. For all clones, parental and humanized sequence perform highly similar with EC50 values in a very narrow range, giving proof to a successful humanization of the parental, chicken derived sequences.

[0260] Example 6: Binding to O-glycosylated and de-O-glycosylated CD44 expressed on transgenic cell lines

[0261] Binding of anti-CD44 clones to NM-F9 and HEK cell lines with different glycosylation status of CD44 was tested.

[0262] CD44v9-transfected NM-F9 cells expressing CD44v9 carrying mainly TF (CD44-F9), CD44v9-transfected NM-F9-GalKO cells expressing CD44v9 carrying predominantly Tn (CD44-F9 cosmc), and CD44v9-transfected HEK-O-glycKO cells expressing CD44v9 without O-glycosylation (CD44-HEK) were stained with anti-CD44 clones and detected via fluorophore-coupled anti-human-IgG secondary reagent. Non-transfected NM-F9 (F9) cells and NM-F9-GalKO (CD44F9 cosmc) cells which do not express CD44, as well as antibodies against TF or Tn (data not shown) and commercial antibodies against CD44 were used as controls.

[0263] The results showed that the tested anti-CD44 clones bound to CD44 on the different cell lines in an O-glycosylation-dependent manner (Figure 5).

[0264] Example 7: Binding of anti-CD44 clones to tumor cell lines

[0265] To examine binding of anti-CD44 clones to tumor cell lines, different cell lines with varying endogenous expression levels for CD44, TF, sTF, and Tn were chosen: MDA- MB-468 (breast adenocarcinoma cell line), CaOV-3 (ovarian adenocarcinoma cell line), and HOC- 15 (lung carcinoma cell line) all express CD44 and show a glycosylation pattern with sTF and TF, but low or no Tn. Treatment of these cell lines with neuraminidase (+ NA) removed sialic acid from the cell surface and generated mainly TF-glycosylation.

[0266] Tumor cell lines were stained with anti-CD44 clones and binding was detected using fluorophore-coupled anti-human-IgG secondary reagent. Expression of CD44, TF, and Tn was confirmed using control anti-CD44 antibodies (RG7356) and anti-TF and anti-Tn antibodies (data not shown). Anti-CD44 clone 1 B5 showed a strong increase of binding after treatment of the tumor cells with neuraminidase, transforming sTF glycosylation into TF glycosylation (Figure 6). This demonstrates that 1 B5 is specific for CD44is / i6 glycosylated with TF. In contrast, both anti-CD44 clones 2A9 and 1 H1 showed a strong binding to the tumor cells before and after neuraminidase treatment, demonstrating specific binding of these antibodies to TF- and sTF-glycosylated CD44is / i6 (Figure 7). NA / GA treatment transforms sTF glycosylation into TF- and Tn- glycosylation. Clones 2B5 and 42F6 do not show tumor cell binding of untreated wildtype (wt) tumor cells, but show strong binding of the cells after treatment with neuraminidase and galactosidase (NA + GA), confirming their TF / Tn- glycan dependency (Figure 8, Figure 9, Figure 10 C and D).

[0267] Example 8: Anti-CD44 clones do not bind to CD44 expressed on human immune cells

[0268] To demonstrate that anti-CD44 clones do not bind to CD44 glycoforms mainly found on normal human immune cells, human monocytes, B cells, T cells and CD3'CD19_lymphocytes from two healthy donors (Figure 11) were treated with excess hlgG (Octagam) to block Fc receptor mediated binding and stained with DIG-conjugated anti- CD44 clones followed by detection via fluorophore-coupled anti-DIG secondary reagent. Expression of CD44 was confirmed using control anti-CD44 mAb RG7356. Additionally, mAbs against TF and Tn demonstrated that the immune cells did not carry the tumorspecific glycosylation structures TF and Tn (data not shown). DAPI was used to discriminate live from dead cells.

[0269] All human immune cells expressed high levels of CD44 as shown by the strong binding of the glycan independent anti-CD44 antibody RG7356. None of the anti-CD44 clones showed detectable binding to CD44 expressed on B cells, T cells and CD3 / CD19- negative lymphocytes. Clone 2A9 did show weak, but strongly reduced binding to granulocytes and monocytes, while clones 1 B5, 2B5 and 42F6 were also completely negative for these cell populations with the highest CD44 expression.

[0270] Example 9: Internalization of anti-CD44 antibodies

[0271] Internalization of the anti-CD44 clones 1 B5 and 2B5 was analyzed by incubation of the antibodies with different cell lines and determination of the percentage of cells showing antibody uptake. CD44-F9 cells (NM-F9 cells expressing TF-glycosylated CD44v9 or NM-F9-GalKO cells expressing CD44v9 carrying predominantly Tn) and PBMCs of a healthy donor were analyzed. Internalization was analyzed by 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 is induced and can be quantified. In brief, cells were harvested and seeded to 5x103cells per well of a 96-flat bottom plate in medium. The respective antibody is 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 are monitored over time (up to 60 h) in an Incucyte S3 at 37°C. Internalization was assessed as the percentage of total cells with a very high red fluorescence intensity. The results show strong internalization of the exemplary anti-CD44 clones 1 B5 and 2B5 in the tested on-target cell lines, but - in contrast to control antibody RG7356 which binds to CD44 in a glycosylation-independent manner - no unwanted internalization in healthy PBMCs (Figure 12).

[0272] Example 10: Tissue binding of anti-CD44 antibodies (data not shown) Formalin-fixed paraffin embedded tissue sections were stained with anti-CD44 clones and detected using secondary antibodies. A commercial anti-CD44 antibody was included to detect protein levels of CD44 in the respective tissues.

[0273] Staining of cancer tissue sections showed binding of anti-CD44 clones to head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, oral squamous cell carcinoma, breast cancer and lung cancer. Importantly, the reactivity to healthy tissue was absent or significantly reduced as compared to the glycosylation-independent control anti-CD44 antibody. In conclusion, anti-CD44 clones were found to react with several cancer tissue sections, but not their healthy counterparts.

[0274] SEQUENCE LISTING

[0275]

[0276] IDENTIFICATION OF THE DEPOSITED BIOLOGICAL MATERIAL

[0277] 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.

[0278] 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.

[0279] 10, 13125 Berlin (DE) on the dates indicated in the following table.

Claims

CLAIMS1. An antibody which is capable of binding to human CD44 at a combined peptide and carbohydrate epitope located within a binding region at amino acid positions 204 to 248 of CD44s, wherein the binding region has the amino acid sequence of SEQ ID NO: 1 and is glycosylated at one or more serine and / or threonine residues with at least one oligosaccharide structure selected from the group consisting of GalNAcal- , sialylated GalNAcal-, Gaipi-3GalNAca1-, and sialylated Gaipi-3GalNAca1-.

2. The antibody according to claim 1 , which is additionally capable of binding to the non-glycosylated binding region.

3. An antibody which is capable of binding to human CD44 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: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 3 and CDR-H3 having the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the complementarity-determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 5, CDR-L2 having the amino acid sequence of SEQ ID NO: 6 and CDR-L3 having the amino acid sequence of SEQ ID NO: 7; or(ii) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 13, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15; or(iii) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 18, CDR-H2 having the amino acid sequence of SEQ ID NO: 19 and CDR-H3 having the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 21 , CDR-L2 having the amino acid sequence of SEQ ID NO: 22 and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or(iv) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 26, CDR-H2 having the amino acid sequence of SEQ ID NO: 27 and CDR-H3 having the amino acid sequence of SEQ ID NO: 28, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 29, CDR-L2 having the amino acid sequence of SEQ ID NO: 30 and CDR-L3 having the amino acid sequence of SEQ ID NO: 31 ; or(v) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 34, CDR-H2 having the amino acid sequence of SEQ ID NO: 35 and CDR-H3 having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 37, CDR-L2 having the amino acid sequence of SEQ ID NO: 38 and CDR-L3 having the amino acid sequence of SEQ ID NO: 39; or(vi) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 42, CDR-H2 having the amino acid sequence of SEQ ID NO: 43 and CDR-H3 having the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 45, CDR-L2 having the amino acid sequence of SEQ ID NO: 46 and CDR-L3 having the amino acid sequence of SEQ ID NO: 47; or(vii) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 50, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15; or(viii)a heavy chain variable region and a light chain variable region according to any one of items (i) to (vii), above, comprising 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences.

4. The antibody according to claim 3, wherein(i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9; or(ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 17; or(iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25; or(iv) 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: 33; or(v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 40, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 41 ; or(vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 48, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49; or(vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 53; or(viii)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54; or(ix) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55; or(x) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 53; or(xi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54; or(xii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55; or(xiii)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 56, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 57; or(xiv)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 59; or(xv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 60, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61 ; or(xvi)the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 60, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or(xvii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 64; or(xviii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 65; or(xix)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 (vi), above, over their entire length, wherein the antibody preferably is a humanized version of the antibody according to any one of items (i) to (vi), above.

5. The antibody according to claim 3 or 4, wherein(i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 53; or(ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54; or(iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51 , and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55; or(iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 53; or(v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54; or(vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55; or(vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 56, 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: 58, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 59; or(ix) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 60, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61 ; or(x) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 60, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or(xi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 64; or(xii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 65.

6. The antibody according to claim 3 or 4, which is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with any one of Gaipi-3GalNAca1-, sialylated Gaipi-3GalNAca1- or mixtures thereof; wherein the antibody comprises(i) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 26, CDR-H2 having the amino acid sequence of SEQ ID NO: 27 and CDR-H3 having the amino acid sequence of SEQ ID NO: 28, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 29, CDR-L2 having the amino acid sequence of SEQ ID NO: 30 and CDR-L3 having the amino acid sequence of SEQ ID NO: 31 ; or(ii) a heavy chain variable region and a light chain variable region according to item (i), above, comprising 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences.

7. The antibody according to claim 3 or 4, which is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with Gaipi-3GalNAca1-, wherein the antibody comprises(i) heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 2, CDR-H2 having the amino acid sequence of SEQ ID NO: 3 and CDR-H3 having the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 5, CDR-L2 having the amino acid sequence of SEQ ID NO: 6 and CDR-L3 having the amino acid sequence of SEQ ID NO: 7; or(ii) heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 13, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15; or(iii) heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 11 and CDR-H3 having the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 50, CDR-L2 having the amino acid sequence of SEQ ID NO: 14 and CDR-L3 having the amino acid sequence of SEQ ID NO: 15; or(iv) a heavy chain variable region and a light chain variable region according to any one of items (i) to (iii), above, comprising 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences.

8. The antibody according to claim 3 or 4, which is capable of binding to glycosylated human CD44 at the binding region, wherein the binding region is glycosylated with GalNAcal-, wherein the antibody comprises(i) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 34, CDR-H2 having the amino acid sequence of SEQ ID NO: 35 and CDR-H3 having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 37, CDR-L2 having the amino acid sequence of SEQ ID NO: 38 and CDR-L3 having the amino acid sequence of SEQ ID NO: 39; or(ii) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 18, CDR-H2 having the amino acid sequence of SEQ ID NO: 19 and CDR-H3 having the amino acid sequence of SEQ ID NO: 20, anda light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 21 , CDR-L2 having the amino acid sequence of SEQ ID NO: 22 and CDR-L3 having the amino acid sequence of SEQ ID NO: 23; or(iii) a heavy chain variable region and a light chain variable region according to item (i) or (ii), above, comprising 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences.

9. The antibody according to claim 3 or 4, which is capable of binding to human CD44 at the binding region, wherein the binding region is glycosylated with any one of GalNAcal-, Gaipi-3GalNAca1-, sialylated GalNAcal-, sialylated Gaipi- 3GalNAca1-, or mixtures thereof or is not glycosylated, wherein the antibody comprises(i) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 42, CDR-H2 having the amino acid sequence of SEQ ID NO: 43 and CDR-H3 having the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 45, CDR-L2 having the amino acid sequence of SEQ ID NO: 46 and CDR-L3 having the amino acid sequence of SEQ ID NO: 47; or(ii) a heavy chain variable region and a light chain variable region according to item (i), above, comprising 1 , 2 or 3 amino acid substitutions in total in the six CDR sequences.

10. The antibody according to any one of claims 1 to 9, being an lgG1-type, lgG2-type, lgG3-type or lgG4-type antibody, in particular an lgG1-type antibody.

11. A conjugate comprising the antibody according to any one of claims 1 to 10 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.

12. A nucleic acid encoding the antibody according to any one of claims 1 to 10 or the conjugate according to claim 11 , wherein the further agent is a polypeptide or protein fused to the antibody.

13. An expression cassette or vector comprising the nucleic acid according to claim 12 and a promoter operatively connected with said nucleic acid.

14. A host cell comprising the nucleic acid according to claim 12 or the expression cassette or vector according to claim 13.

15. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 10, the conjugate according to claim 11 , the nucleic acid according to claim 12, the expression cassette or vector according to claim 13, or the host cell according to claim 14, wherein the pharmaceutical composition optionally further comprises one or more components selected from the group consisting of solvents, diluents and excipients.

16. The antibody according to any one of claims 1 to 10, the conjugate according to claim 11, the host cell according to claim 14, or the composition according to claim15 for use in medicine.

17. The antibody, the conjugate, the host cell or the composition for use in medicine according to claim 16 for use in the treatment of cancer, arthritis, bacterial or viral infections, interstitial lung disease, vascular disease, or scoliosis.

18. The antibody, the conjugate, the host cell or the composition for use in the treatment of cancer according to claim 17, wherein the cancer is selected from the group consisting of esophagus cancer, head and neck cancer, oral cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, cervix cancer, and gastrointestinal cancer.