CD44-specific antibodies

Abstract

Antibodies having binding specificity for CD44 v7 / 8 (also known as CD44 variant 7 / 8) and antibody-drug conjugates (ADCs) or chimeric antigen receptors (CARs) comprising the same. Also provided herein are uses of immune cells expressing anti-CD44 v7 / 8 antibodies, ADCs, or chimeric antigen receptors for therapeutic or diagnostic purposes.

Description

[Technical field]

[0001] 2. Background of the Invention CD44, also known as HCAM (homing cell adhesion molecule), Pgp-1 (phagocyte glycoprotein-1), Hermes antigen, lymphocyte homing receptor, ECM-III, or HUTCH-1, is a cell surface glycoprotein that functions primarily in cell adhesion and cell-cell interactions. Several splice variants of CD44 are known, including CD44 v7 / 8, a variant that contains exons 7 and 8. CD44 is a protein receptor for hyaluronan and has been further shown to bind or otherwise interact with osteopontin, collagens, matrix metalloproteinases, and other similar ligands. Summary of the Invention

[0002] SUMMARY OF THE PRESENT APPLICATION The present disclosure is based, at least in part, on the development of antibodies with high binding affinity and specificity for the CD44 v7 / 8 splice variant. Such antibodies are capable of binding to cells expressing CD44 v7 / 8 (CD44 + The results show inhibitory activity against IL-1 and IL-2 cells. Accordingly, one aspect of the disclosure features an isolated antibody that binds to CD44 v7 / 8 (anti-CD44 v7 / 8 antibody), where the antibody specifically binds to an epitope on human CD44 v7 / 8, where the epitope comprises the amino acid sequence of QAGRRMDMDSSHSIT (SEQ ID NO: 13). In some embodiments, the anti-CD44 v7 / 8 antibody binds to the same epitope on human CD44 v7 / 8 as the exemplary antibody HIS029, the structural features of which are provided herein. In some embodiments, the anti-CD44 v7 / 8 antibody does not bind to the peptide PISHPMGRGHQAGRR (SEQ ID NO: 12) or the peptide SHSITLQPTANPNTG (SEQ ID NO: 14).

[0003] In some embodiments, the anti-CD44 v7 / 8 antibody comprises a heavy chain variable region (VH ), where heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) are collectively at least 85% (e.g., at least 90%, at least 95%, at least 98% or more) identical to the HC CDR1, HC CDR2, and HC CDR3 of antibody HIS029. In some cases, the antibody may comprise a V domain that contains the same HC CDR1, HC CDR2, and HC CDR3 as antibody HIS029. H In other embodiments, the anti-CD44 v7 / 8 antibodies described herein may comprise a V sequence including HC CDR1, HC CDR2, and HC CDR3 that collectively contain 8 amino acid residue variations (up to 7, 6, 5, 4, 3, 2, or 1 mutation(s)) compared to the HC CDR1, HC CDR2, and HC CDR3 of antibody HIS029. H may also include

[0004] Alternatively or additionally, the anti-CD44 v7 / 8 antibodies described herein comprise a light chain variable region (V L ), where the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) are collectively at least 85% (e.g., at least 90%, at least 95%, at least 98% or more) identical to the LC CDR1, LC CDR2, and LC CDR3 of antibody HIS029. In some cases, the antibody may comprise the same LC CDR1, LC CDR2, and LC CDR3 as antibody HIS029. In other embodiments, the anti-CD44 v7 / 8 antibodies described herein may comprise LC CDR1, LC CDR2, and LC CDR3 that collectively contain up to 8 amino acid residue variations (7, 6, 5, 4, 3, 2, or 1 mutation(s)) compared to the LC CDR1, LC CDR2, and LC CDR3 of the reference antibody.

[0005] In some instances, the anti-CD44 v7 / 8 described herein comprises the same heavy and / or light chain CDRs as the antibody HIS029. In some instances, such anti-CD44 v7 / 8 antibodies comprise the same V and / or V CDRs as the antibody HIS029. H and / or V L may also include Any of the anti-CD44 v7 / 8 antibodies described herein may specifically bind to human CD44 v7 / 8. The antibody may be a human or humanized antibody. In some examples, it may be a chimeric antibody. In some embodiments, the anti-CD44 v7 / 8 antibody is selected from the group consisting of the V7 / 8 antibody shown in SEQ ID NO: 10. H and / or V as shown in SEQ ID NO:11 L It is a humanized antibody comprising: In some embodiments, the anti-CD44 v7 / 8 antibody may be a full-length antibody (e.g., an IgG molecule) or an antigen-binding fragment thereof. Alternatively, it may be a single chain antibody.

[0006] In another aspect, the disclosure features a nucleic acid or a set of nucleic acids (e.g., two nucleic acids) collectively encoding any of the anti-CD44 v7 / 8 antibodies described herein, and a vector or a set of vectors (e.g., two vectors) comprising a nucleic acid(s) encoding an anti-CD44 v7 / 8 antibody. In some cases, the vector or vector set can be an expression vector(s). Also provided herein is a host cell comprising the nucleic acid(s) or vector(s). Additionally, the disclosure provides a method for producing an anti-CD44 v7 / 8 as described herein, comprising culturing a host cell comprising a vector or vector set comprising a coding sequence for the antibody, wherein the coding sequence is operably linked to a suitable promoter, and collecting the antibody thus produced, for example, from the host cell or culture medium.

[0007] In addition, the disclosure provides antibody-drug conjugates (ADCs) comprising any of the anti-CD44 v7 / 8 antibodies described herein and at least one therapeutic agent covalently conjugated to the antibody. In some examples, the therapeutic agent can be a cytotoxic agent, such as monomethylauristatin E. In some embodiments, the antibody and the therapeutic agent may be conjugated via a linker. In some examples, the linker may be a cleavable linker, such as a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. In some cases, the linker may be a protease-sensitive linker that may include a peptide having 2-5 amino acids. The peptide may include naturally occurring amino acid residues, non-naturally occurring amino acid residues, or a combination thereof. In one example, the peptide may include valine-citrulline. In another example, the linker may be a non-cleavable linker. Such non-cleavable linkers may include an optionally substituted alkane or thioether.

[0008] In some embodiments, the linker may comprise a functional group that forms a covalent bond between the antibody and the linker. Exemplary functional groups include, but are not limited to, maleimide groups, iodoacetamide groups, vinylsulfone groups, acrylate groups, acrylamide groups, acrylonitrile groups, and methacrylate groups. In one example, the linker is represented by Formula I: [ka] During the ceremony, R1 is an optionally substituted C1-6 alkyl, an optionally substituted phenyl, an optionally substituted C2-6 alkylene, an optionally substituted C2-6 alkenylene, an optionally substituted C2-6 alkynylene, or an optionally substituted triazole; and X is O, S, or N. The compound may further comprise a molecular spacer of

[0009] Further, the present disclosure provides a chimeric antigen receptor (CAR), which may include (i) an extracellular domain comprising an antigen-binding fragment that binds to CD44 v7 / 8, (ii) a transmembrane domain, and (iii) one or more intracellular stimulatory domains. The antigen-binding fragment may specifically bind to an epitope of human CD44 v7 / 8, where the epitope comprises the amino acid sequence of QAGRRMDMDSSHSIT (SEQ ID NO: 13). In some embodiments, the antigen-binding fragment does not bind to the peptide PISHPMGRGHQAGRR (SEQ ID NO: 12) or the peptide SHSITLQPTANPNTG (SEQ ID NO: 14). In some examples, the antigen-binding fragment may include the same HC CDRs and / or LC CDRs as any of the reference antibodies. Such antigen-binding fragments may include the same V CDRs as the antibody HIS029. H and V L In some examples, the antigen-binding fragment may be a single chain antibody (scFv).

[0010] In any of the CARs described herein, the transmembrane domain may comprise a transmembrane domain derived from CD28 or CD8. Alternatively or in addition, the one or more intracellular stimulatory domains may comprise a signaling domain from CD3zeta, a costimulatory domain, or a combination thereof. In some embodiments, the costimulatory signaling domain may be from 4-1BB, CD7, CD27, CD28, CD40, OX40, ICOS, GITR, HVEM, TIM1, or LFA-1. The nucleic acid encoding any of the CARs described herein, the vector comprising the same, and the host cell expressing the CAR are also within the scope of the present disclosure. In some examples, the host cell expressing the CAR is an immune cell, such as a T cell.

[0011] In another aspect, the disclosure provides a pharmaceutical composition comprising one or more of: (i) an anti-CD44 v7 / 8 antibody described herein, a nucleic acid or set of nucleic acids encoding same, an antibody-drug conjugate comprising an anti-CD44 v7 / 8 antibody described herein, or a host cell expressing any of the nucleic acids or CAR constructs described herein; and (ii) a pharmaceutically acceptable carrier.

[0012] Moreover, the present disclosure features a method of inhibiting cells expressing CD44 v7 / 8, the method comprising administering an effective amount of any of the pharmaceutical compositions described herein to a subject in need thereof. In some embodiments, the subject may be a human patient having or suspected of having cancer, such as head and neck cancer, cervical cancer, and breast cancer. Also within the scope of the present disclosure are pharmaceutical compositions as described herein for use in the treatment of any of the target diseases described herein (e.g., cancers such as head and neck cancer, cervical cancer, and breast cancer) or for use in the manufacture of a medicament for the treatment of a target disease.

[0013] In addition, the disclosure features a method for detecting the presence of cells expressing CD44 v7 / 8, the method comprising: (i) detecting a cell expressing CD44 v7 / 8; + The method includes contacting a sample suspected of having cells with any of the anti-CD44 v7 / 8 antibodies described herein conjugated to a labeling agent; and (ii) detecting the presence of cells expressing CD44 v7 / 8 in the sample based on binding of the antibody to cells in the sample. In some cases, the sample is derived from a human patient at risk of having or suspected of having cancer, such as head and neck cancer, cervical cancer, and breast cancer. The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following drawings and detailed description of several embodiments, and from the appended claims. [Brief description of the drawings]

[0014] Detailed Description of the Drawings [Figure 1] Figures 1A-1B include diagrams showing the binding specificity of antibody HIS029 to the epitope of the CD44 v7 / 8 splice variant. Figure 1A: The table shows the CD44 peptide sequences (CD44 v7; CD44 v7 / 8; and CD44 v8; SEQ ID NOs: 12, 13, and 14, respectively). Figure 1B: The graph shows that CD44 v7 / 8 peptide can inhibit / block binding between antibody HIS029 and CD44+ Detroit 562 (D562) cells, as evidenced by the loss of fluorescence upon addition of CD44 v7 / 8 peptide to a mixture of antibody HIS029 and Detroit 562 cells expressing CD44 v7 / 8 (CD44+ cells). [Diagram 2] FIG. 2 is a diagram showing anti-CD44 v7 / 8 antibodies (HIS029 and humanized HIS029) capable of binding to A253 cells expressing CD44 v7 / 8 (CD44+ cells).

[0015] [Diagram 3] Figure 3 includes a diagram showing that the anti-CD44 v7 / 8 antibody-drug conjugate (HIS029 conjugated to MMAE using a Val-Cit linker (HIS029-MMAE)) can specifically target CD44+ cancer cell lines. HIS029-MMAE demonstrates potent inhibition against CD44+ Detroit 562 (D562) cells (right) with limited inhibition against A375 cells. [Figure 4] FIG. 4 includes a diagram showing the transduction efficiency of anti-CD44 v7 / 8 CAR lentivirus into CD3+ T cells.

[0016] [Figure 5A]Figures 5A-5B include graphs showing the efficacy of CD44 v7 / 8 CAR+ T cells in inducing the secretion of cytokines when co-cultured with CD44+ A523 cells and A549 cells (control cells). Figure 5A: The graph shows that anti-CD44 v7 / 8 CAR+ T cells are effective in inducing the secretion of interferon-gamma (IFN-g or IFNγ) when co-cultured with CD44+ A523 cells. [Figure 5B] Figure 5B: The graph shows that anti-CD44 v7 / 8 CAR+ T cells are effective in inducing the secretion of interleukin-2 (IL2) when co-cultured with CD44+ A523 cells.

[0017] [Figure 6] Figure 6 includes graphs showing the efficacy of anti-CD44 v7 / 8 CAR+T cells in inducing antigen-dependent cytotoxicity against CD44+ A523 cells and A549 cells (control cells). Anti-CD44 v7 / 8 CAR+T cells are effective in inducing antigen-dependent cytotoxicity against CD44+ A523 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention CD44 variants are overexpressed in several cancers, for example, CD44 v7 / 8 is positively expressed in 64% of patients with head and neck squamous cell carcinoma (HNSCC), 11.5% of patients with cutaneous squamous cell carcinoma, and 5% of patients with cervical cancer. Consequently, agents that target the CD44 v7 / 8 variant (e.g., anti-CD44 v7 / 8 antibodies) are predicted to be effective in both cancer treatment and diagnosis. Disclosed herein are antibodies having binding specificity for the CD44 v7 / 8 splice variant, and antibody-drug conjugates or chimeric antigen receptors comprising the same. The anti-CD44 v7 / 8 antibodies disclosed herein have high binding affinity and specificity for the CD44 v7 / 8 antigen, and / or high binding affinity to the CD44 v7 / 8 splice variant. +It showed excellent characteristics, including high inhibitory activity against cells.

[0019] Accordingly, provided herein are antibodies capable of binding to CD44 v7 / 8, nucleic acids encoding same, antibody-drug conjugates (ADCs) and chimeric antigen receptors (CARs) comprising anti-CD44 v7 / 8 antibodies, and their uses for both therapeutic and diagnostic purposes. Also provided herein are kits for the therapeutic and / or diagnostic use of the antibodies and / or ADCs and CARs comprising same, as well as methods for producing anti-CD44 v7 / 8 antibodies.

[0020] Antibody that binds CD44 v7 / 8 The present disclosure provides antibodies that bind to CD44, also known as HCAM (homing cell adhesion molecule), Pgp-1 (phagocyte glycoprotein-1), Hermes antigen, lymphocyte homing receptor, ECM-III, or HUTCH-1. CD44 is encoded by the CD44 gene (NCBI gene ID: 960). In particular, the antibodies described herein have high binding specificity to CD44 v7 / 8, a splice variant that contains both exon 7 and exon 8. CD44 is a cell surface protein receptor involved in cell adhesion, cell-cell interaction, lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis. CD44 exists in several variant forms resulting from alternative splicing. Of particular interest is the CD44 variant CD44 v7 / 8, which contains the region of CD44 transcribed from exons 7 and 8 and is typically expressed in several types of cancer, including head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma, cervical cancer, and breast cancer patients. Thus, this receptor can serve as a target and / or biomarker for targeted cancer treatment and diagnosis.

[0021] Consequently, the anti-CD44 v7 / 8 antibodies disclosed herein can be used in treating and / or diagnosing target cancers as described herein, either by themselves or by conjugation to other moieties, e.g., conjugated to therapeutic agents to form antibody-drug conjugates, or by being extracellular antigen-binding domains in chimeric antigen receptors. + Cell-targeted cellular therapies are also within the scope of this disclosure.

[0022] Antibodies (used interchangeably in the plural) are immunoglobulin molecules capable of specific binding to targets such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact (i.e., full-length) polyclonal or monoclonal antibodies, as well as antigen-binding fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single chains (scFv), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of immunoglobulin molecule that contains an antigen recognition site of the required specificity (including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies).

[0023] Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of a specific class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be classified into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0024] A typical antibody molecule consists of a heavy chain variable region (V H ) and the light chain variable region (V L ), which are mostly involved in antigen binding. H and V L The regions can be further divided into regions of hypervariability, also known as "complementarity determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). H and V LTypically, a framework region is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of framework regions and CDRs can be precisely identified using methodologies known in the art, such as the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0025] In some embodiments, the anti-CD44 v7 / 8 antibodies described herein bind to CD44 v7 / 8 and are capable of inhibiting the activity of CD44 v7 / 8 by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more). The apparent inhibition constant (Ki) provides a measure of inhibitor potency. app or K i,app ) is related to the concentration of inhibitor required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-CD44 v7 / 8 antibodies described herein can be determined by routine methods known in the art.

[0026] Antibody K i, app The Ki value can be determined by measuring the inhibitory effect of various concentrations of antibody on the extent of the reaction (e.g., enzyme activity); fitting the change in the pseudo-first-order rate constant (v) as a function of inhibitor concentration to a modified Morrison equation (Equation 1) yields an estimate of the apparent Ki value. For competitive inhibitors, the Ki app is K i, app It can be obtained from the y-intercept extracted from linear regression analysis of a plot of concentration versus substrate.

number

[0027] In some embodiments, the anti-CD44 v7 / 8 antibodies described herein have a Ki of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less for a CD44 epitope, a CD44 v7 / 8 antigen or an antigenic epitope thereof. app In some embodiments, the anti-CD44 v7 / 8 antibody may have a lower Ki value for a first target compared to a second target. app It may have Ki app The difference in (e.g., for specificity or other comparison) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 It could be double.

[0028] The antibodies described herein may be of murine, rat, human, or any other origin (including chimeric or humanized antibodies). Such antibodies are not naturally occurring, i.e., would not be produced in an animal without human action (e.g., immunizing such an animal with the desired antigen or a fragment thereof).

[0029] Any of the antibodies described herein can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous antibody population, and a "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of the antibody or the manner in which the antibody is made.

[0030] In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibody refers to a non-human form of an antibody (e.g., mouse) that is a specific chimeric immunoglobulin, immunoglobulin chain, or antigen-binding fragment thereof that contains minimal sequence derived from a non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's complementarity determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, that has the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences, but are included to further improve and optimize antibody performance.

[0031] Generally, a humanized antibody comprises substantially all, at least one, and typically two variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being of a human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have a modified Fc region as described in WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, and / or six) that have been altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. A humanized antibody may also include affinity maturation.

[0032] In another example, the antibody described herein may be a chimeric antibody, which may include heavy and light chain constant regions from a human antibody. Chimeric antibody refers to an antibody that has a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable region of an antibody from one mammal (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant region is homologous to the sequence of an antibody from another mammal, such as a human. In some embodiments, amino acid modifications may be made in the variable region and / or the constant region.

[0033] In some embodiments, the anti-CD44 v7 / 8 antibodies described herein specifically bind to the corresponding target antigen or epitope thereof. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with a higher affinity to a particular target antigen than it reacts with alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to a CD44 v7 / 8 antigen or an epitope of that antigen is an antibody that binds to this target antigen with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other antigens or other epitopes in the same antigen.

[0034] With this definition, for example, it is also understood that an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. In some instances, an antibody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes in the same antigen. In some embodiments, the anti-CD44 v7 / 8 antibodies described herein specifically bind to human CD44. In some embodiments, the anti-CD44 v7 / 8 antibodies described herein specifically bind to human CD44 v7 / 8. In some instances, the binding activity to a non-human CD44 v7 / 8 antigen is not detectable in conventional assays or is so low that it would not have significant biological significance known to those skilled in the art. In other examples, the anti-CD44 v7 / 8 antibodies described herein may cross-react with CD44 v7 / 8 from different species, for example, between human CD44 v7 / 8 and non-human CD44 v7 / 8 (e.g., CD44 v7 / 8 from an experimental animal such as a non-human primate, mouse or rat).

[0035] As used herein, the terms "CD44", "CD44 v7 / 8", or "HCAM" refer to CD44 of any suitable species, e.g., human, non-human mammalian such as non-human primate, or rodent (e.g., mouse or rat). CD44 v7 / 8 is a splice variant of a cell surface glycoprotein that functions primarily in cell adhesion, cell-cell interactions, and as a protein receptor for ligands such as hyaluronic acid. The amino acid sequence of an exemplary human CD44 v7 / 8 splice variant is provided below, with the v7 / 8 epitope targeted by the exemplary antibodies described herein marked in bold (see also NCBI Accession No. NP_000601.3): [ka]

[0036] CD44 v7 / 8 molecules from other species are known in the art and their amino acid sequences can be retrieved from publicly available databases, such as GenBank or NCBI. In some embodiments, an anti-CD44 v7 / 8 antibody as described herein has suitable binding affinity for a target antigen (e.g., human CD44 v7 / 8) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K A Refers to. A is the dissociation constant (K D The anti-CD44 v7 / 8 antibodies described herein have a binding affinity of at least 10 for the target antigen or antigenic epitope. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or less binding affinity (K D The increased binding affinity may be expressed as a decreased K D A higher binding affinity of an antibody for a first antigen compared to a second antigen corresponds to a K A(or a number K D ) for binding to the first antigen A (or a smaller number K D )

[0037] In such cases, the antibody has specificity for a first antigen (e.g., a first protein or a mimetic thereof in a first conformation) compared to a second antigen (e.g., the same first protein or a mimetic thereof in a second conformation; or a second protein). In some embodiments, the anti-CD44 v7 / 8 antibodies described herein have a higher binding affinity to human CD44 v7 / 8 (greater K A or smaller K D The difference in binding affinity (e.g., for specificity or other comparisons) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 In some embodiments, any of the anti-CD44 v7 / 8 antibodies may be further affinity matured to increase the binding affinity of the antibody to a target antigen or antigenic epitope thereof.

[0038] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (using, for example, a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). Using these techniques, the concentration of bound binding protein can be measured as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation: [Bound] = [Free] / (Kd+[Free])

[0039] However, the exact K A It is not necessary to perform a determination of K since sometimes it is sufficient to obtain a quantitative measure of affinity determined using methods such as, for example, ELISA or FACS analysis, which may be used to A and can thus be used for comparison, such as determining whether a higher affinity is, e.g., 2-fold higher, e.g., by activity in a functional assay, e.g., in vitro or in vivo assay, to obtain a quantitative measure of affinity or to obtain an inference of affinity.

[0040] In some embodiments, the anti-CD44 v7 / 8 antibodies described herein bind to the same epitope of the CD44 v7 / 8 antigen (e.g., human CD44 v7 / 8) as one of the reference antibodies provided herein, or compete against a reference antibody that binds to the CD44 v7 / 8 antigen. The reference antibodies provided herein include HIS029, the structural features of which are provided herein. An antibody that binds to the same epitope as a reference antibody described herein may bind to the exact same epitope as the reference antibody or to a substantially overlapping epitope (e.g., containing less than 3 non-overlapping amino acid residues, less than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue).

[0041] Whether two antibodies compete with each other to prevent binding to cognate antigens can be determined by competitive assays well known in the art. Such antibodies can be identified as known to those skilled in the art (e.g., those with substantially similar structural features (e.g., complementarity determining regions) and / or those identified by assays known in the art). For example, a competitive assay can be performed using one of the reference antibodies to determine whether the candidate antibody binds to the same epitope as the reference antibody or competes for its binding to the CD44 v7 / 8 antigen. In some embodiments, the anti-CD44 v7 / 8 antibodies described herein specifically bind to an epitope of human CD44 v7 / 8, wherein the epitope comprises the amino acid sequence of QAGRRMDMDSSHSIT (SEQ ID NO: 13). In some cases, the anti-CD44 v7 / 8 antibodies described herein do not bind to the peptide PISHPMGRGHQAGRR (SEQ ID NO: 12) or the peptide SHSITLQPTANPNTG (SEQ ID NO: 14). An antibody that does not bind to an antigen means that no significant binding can be detected using conventional assays (e.g., ELISA or surface plasmon resonance) (e.g., only background binding or no binding at all).

[0042] The anti-CD44 v7 / 8 antibodies described herein comprise a heavy chain variable region (V H (c) heavy chain complementarity determining region 3 (HC CDR3) as in HIS029 or humanized HIS029; or (d) a combination of any one of (a)-(c). In some cases, the antibody may comprise HC CDR1 of (a), HC CDR2 of (b), and HC CDR3 of (c).

[0043] Table 1 provides the amino acid sequences of the heavy chain CDRs (according to the IMGT definition) of exemplary anti-CD44 v7 / 8 antibodies. Antibodies having the same heavy chain CDR1, CDR2, and CDR3 regions as these exemplary anti-CD44 v7 / 8 antibodies are within the scope of this disclosure. Table 1: Heavy chain CDR sequences of reference anti-CD44 v7 / 8 antibodies [Table 1]

[0044] Alternatively or additionally, the anti-CD44 v7 / 8 antibodies described herein comprise a light chain variable region (V L ) containing a light chain variable domain (V L ), which may include: (a) light chain complementarity determining region 1 (LC CDR1) as in HIS029 or humanized HIS029; (b) light chain complementarity determining region 2 (LC CDR2) as in HIS029 or humanized HIS029; (c) light chain complementarity determining region 3 (LC CDR3) as in HIS029 or humanized HIS029; or (d) any combination of (a)-(c).

[0045] Table 2 provides the amino acid sequences of the light chain CDRs of exemplary anti-CD44 v7 / 8 antibodies. Antibodies having the same light chain CDR1, CDR2, and CDR3 regions as these exemplary anti-CD44 v7 / 8 antibodies are within the scope of this disclosure. Table 2: Light chain CDR sequences of reference anti-CD44 v7 / 8 antibodies [Table 2]

[0046] The heavy and light chain CDRs of the reference antibodies provided herein are determined based on the IMGT approach, which is well known in the art. In some cases, the anti-CD44 v7 / 8 antibodies disclosed herein may contain the same heavy and light chain CDRs as any of the reference antibodies disclosed herein. H and / or V LTwo antibodies that have CDRs mean that their CDRs are identical when determined by the same approaches (e.g., those described herein and / or known in the art).

[0047] In some instances, the anti-CD44 v7 / 8 antibodies disclosed herein have the same V H and / or V L Sequences, which are provided below (CDRs in bold): [ka]

[0048] Also within the scope of the present disclosure are functional variants of any of the reference anti-CD44 v7 / 8 antibodies (e.g., those listed in Tables 1 and 2 above) as disclosed herein. Functional variants contain up to eight (e.g., seven, six, five, four, three, two, or one) amino acid residue variations in one or more of the heavy and light chain CDR regions of the reference antibody and have substantially similar affinities (e.g., K of the same order). D The functional variants can bind to the same epitope of the CD44 v7 / 8 antigen (having a 1:1 ratio (a) or 1:2 ratio (b)). In some cases, each of the heavy and / or light chain CDRs of the functional variant contains no more than two amino acid residue variations compared to the counterpart CDR of the reference antibody. In some examples, each of the heavy and / or light chain CDRs in the functional variant contains no more than one amino acid residue variation compared to the counterpart CDR in the reference antibody.

[0049] In one example, the amino acid residue variation is a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, and such methods can be found in references that compile such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made within the following groups of amino acids: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0050] In some embodiments, the anti-CD44 v7 / 8 antibody comprises heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) individually or collectively identical to the heavy chain CDRs of a reference antibody (e.g., HIS029), and / or light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) individually or collectively identical to the light chain CDRs of the reference antibody. In some embodiments, the anti-CD44 v7 / 8 antibody comprises a heavy chain variable region (V) that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain variable region of either reference antibody. H ) and / or a light chain variable region (V) that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain variable region of the reference antibody. L ).

[0051] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. "Individually" means that one particular heavy or light chain CDR of the antibody shares the recited sequence identity with the corresponding heavy or light chain CDR of the exemplary antibody, e.g., HIS029. "Collectively" means that the three heavy or light chain CDRs of the antibody share the recited sequence identity with the corresponding heavy or light chain CDRs of the exemplary antibody, e.g., HIS029.

[0052] The present disclosure also provides a germline variant of any of the reference anti-CD44 v7 / 8 antibodies disclosed herein. A germline variant contains one or more mutations in the framework region compared to its parent antibody to the corresponding germline sequence. To generate a germline variant, the heavy or light chain variable region sequence of a parent antibody or a portion thereof (e.g., framework sequence) can be used as a query sequence against an antibody germline sequence database (e.g., bioinfo.org.uk / abs / , vbase2.org, or imgt.org) to identify the corresponding germline sequence used by the parent antibody and the amino acid residue variation in one or more of the framework regions between the germline sequence and the parent antibody. One or more amino acid substitutions can then be introduced into the parent antibody based on the germline sequence to produce a germline variant.

[0053] In some cases, a functional variant of a reference antibody disclosed herein is a humanized version of the reference antibody. Examples include: [ka]

[0054] In some embodiments, the heavy chain of any of the anti-CD44 v7 / 8 antibodies described herein may further comprise a heavy chain constant region (HC) or a portion thereof (e.g., HC1, HC2, HC3, or a combination thereof). The heavy chain constant region may be of any suitable origin, e.g., human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is from human IgG (gamma heavy chain). If necessary, the anti-CD44 v7 / 8 antibody as described herein may comprise a modified constant region. For example, it may comprise a modified constant region that is immunologically inert, e.g., does not trigger complement-mediated lysis or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity may be assessed using the methods described in U.S. Pat. No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT / GB99 / 01441; and / or UK Patent Application No. 9809951.8.

[0055] Any of the anti-CD44 v7 / 8 antibodies described herein may further comprise a light chain comprising a light chain variable region and optionally a light chain constant region, which may be any LC known in the art. In some examples, the LC is a kappa light chain. In other examples, the LC is a lambda light chain. Antibody heavy and light chain constant regions are well known in the art (e.g., those provided in the IMGT database (imgt.org) or vbase2.org / vbstat.php., both of which are incorporated herein by reference).

[0056] Preparation of anti-CD44 v7 / 8 antibody Antibodies capable of binding to CD44 v7 / 8 as described herein can be made by any method known in the art, see, e.g., Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0057] In some embodiments, antibodies specific for a target CD44 v7 / 8 antigen (e.g., human CD44) can be made by conventional hybridoma technology. A full-length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal to generate antibodies that bind to the antigen. The route and schedule of immunization of the host animal generally follows established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for the production of mouse, humanized, and human antibodies are known in the art and described herein. It is contemplated that any mammalian subject, including humans, or antibody-producing cells therefrom, can be engineered to serve as the basis for the production of mammalian hybridoma cell lines, including humans. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or intradermally with an amount of immunogen, including those described herein.

[0058] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497, or as modified by Buck, DW, et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653, and from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used in hybridization. In general, the technique involves fusing myeloma cells and lymphoid cells using membrane fusion such as polyethylene glycol, or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to remove unhybridized parent cells. Any of the media described herein, supplemented or not with serum, can be used to culture hybridomas secreting monoclonal antibodies. As another alternative to cell fusion techniques, EMB immortalized B cells can be used to produce anti-CD44 v7 / 8 monoclonal antibodies described herein. Hybridomas are expanded and subcloned, and if desired, supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

[0059] Hybridomas that can be used as a source of antibodies include all derivatives, progeny cells of the parent hybridoma that produce monoclonal antibodies capable of interfering with CD44 v7 / 8 activity. Hybridomas producing such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids, if desired, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesirable activity, if present, can be removed, for example, by running the preparation over an adsorbent made from the immunogen attached to a solid phase, and eluting or releasing the desired antibody from the immunogen. Immunization of a host animal with a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species being immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional or derivatizing agent (e.g., maleimidobenzoyl, sulfosuccinimide esters (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R1N=C=NR (where R and R1 are different alkyl groups) can generate a population of antibodies (e.g., monoclonal antibodies).

[0060] If desired, the antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in the vector in a host cell, and the host cell can then be expanded and frozen for future use. In the alternative, the polynucleotide sequence can be used for genetic engineering to "humanize" the antibody or improve affinity (affinity maturation) or other characteristics of the antibody. For example, the constant region can be engineered to be more similar to human constant regions to avoid immune responses when the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically engineer the antibody sequence to obtain greater affinity for the target antigen and greater efficacy in inhibiting the activity of CD44. It will be apparent to one of skill in the art that one or more polynucleotide alterations can be made to an antibody and still maintain its binding specificity for the target antigen.

[0061] In other embodiments, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desired (e.g., fully human antibodies) or more robust immune response can also be used for the generation of humanized or human antibodies. Examples of such technology are Xenomouse® from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, NJ). In another alternative, antibodies can be made recombinantly by phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0062] In some embodiments, antibodies capable of binding to the CD44 v7 / 8 antigen can be isolated from an antibody library, such as a phage display antibody library or a yeast display antibody library. In one example, the anti-CD44 v7 / 8 antibodies described herein can be isolated from a monoclonal antibody library, for example, according to the methods disclosed in US2015 / 0153356, the relevant disclosure of which is incorporated herein by reference for purposes or subject matter referenced herein. Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared through routine methods, for example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2.

[0063] Genetically modified antibodies, such as humanized antibodies, chimeric antibodies, single chain antibodies, and bispecific antibodies, can be produced, for example, through conventional recombinant technology. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be easily isolated and sequenced using conventional procedures (for example, using an oligonucleotide probe that can specifically bind to genes encoding the heavy and light chains of a monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is placed into one or more expression vectors, which can then be transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in recombinant host cells. For example, see PCT Publication No. WO87 / 04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, genetically altered antibodies with the binding specificity of a target antigen, such as "chimeric" or "hybrid" antibodies, can be prepared.

[0064] Techniques developed for the production of "chimeric antibodies" are well known in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.

[0065] Methods for constructing humanized antibodies are well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the V of a parent non-human antibody is H and V L The variable regions of the human V antibody are subjected to three-dimensional molecular modeling analysis according to methods known in the art. Framework amino acid residues predicted to be important for the formation of correct CDR structures are then identified using the same molecular modeling analysis. In parallel, human V antibody having amino acid sequences homologous to those of the parent non-human antibody are subjected to three-dimensional molecular modeling analysis. H and V L The strand is the parent V H and V L The sequence is used to identify the human V H and V L An acceptor gene is then selected.

[0066] The CDR regions in the selected human acceptor gene may be replaced with CDR regions from a parent non-human antibody or a functional variant thereof. If necessary, residues in the framework regions of the parent chain predicted to be important in interacting with the CDR regions (see above) may be used to substitute for the corresponding residues in the human acceptor gene.

[0067] Single chain antibodies can be prepared via recombinant technology by linking the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, the techniques described for producing single chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce phage or yeast scFv libraries, and CD44 v7 / 8 specific scFv clones can be identified from the libraries according to routine procedures. Positive clones can be subjected to further screening to identify those that inhibit CD44 v7 / 8 activity.

[0068] The antibodies known in the art and obtained according to the methods described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope that the antigen binds to, or "epitope mapping". There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including, for example, analyzing the crystal structure of the antibody-antigen complex as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999, competitive assays, gene fragment expression assays, and synthetic peptide-based assays. In an additional example, epitope mapping can be used to determine the sequence that the antibody binds to. The epitope can be a linear epitope (i.e., can be contained in a single stretch of amino acids) or a conformational epitope formed by the three-dimensional interaction of amino acids that are not necessarily contained in a single stretch (primary structure linear sequence). Peptides of varying lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used in binding assays with the antibody. In another example, the epitope to which an antibody binds can be determined in a systematic screen using overlapping peptides derived from the target antigen sequence and determining binding by the antibody.

[0069] According to gene fragment expression assay, the open reading frame encoding the target antigen is fragmented either randomly or by specific gene constructs, and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined. The gene fragments can be produced, for example, by PCR, and then in vitro transcribed and translated into proteins in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled antigen fragments is determined by immunoprecipitation and gel electrophoresis. An epitope can also be identified by using a large library of random peptide sequences (phage libraries) displayed on the surface of phage particles. Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In an additional example, mutagenesis of the antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutants of a target antigen in which various fragments of the CD44 v7 / 8 polypeptide are replaced (swapped) with sequences from a closely related but antigenically distinct protein. By assessing antibody binding to mutant CD44, the importance of specific antigen fragments to antibody binding can be assessed.

[0070] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibody. Competition assays are well known to those of skill in the art. In some instances, anti-CD44 v7 / 8 antibodies are prepared by recombinant techniques, as exemplified below.

[0071] The nucleic acids encoding the heavy and light chains of the anti-CD44 v7 / 8 antibody as described herein can be cloned into one expression vector, with each nucleotide sequence operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter, such that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0072] In some examples, the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cell in which it is expressed, and the isolated heavy and light chains are mixed and incubated under suitable conditions to allow the formation of the antibody.

[0073] In general, the nucleic acid sequence encoding one or all chains of the antibody can be cloned into a suitable expression vector, operably linked to a suitable promoter, using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other, and then linked together using a ligase. Alternatively, synthetic nucleic acid linkers can be linked to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell for use in producing the antibody.

[0074] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) immediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0075] Regulatable promoters can also be used, including those that use the lac repressor from E. coli as a transcriptional modulator to regulate transcription from mammalian cell promoters carrying the lac operator [Brown, M. et al., Cell, 49:603-612 (1987)], those that use the tetracycline repressor (tetR) [Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using estradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0076] Regulatable promoters including repressors with operons can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator and regulate transcription from mammalian promoters carrying the lac operator [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)], and the tetracycline repressor (tetR) is combined with a transcriptional activator (VP16) to create the tetR mammalian transcriptional activator fusion protein tTa (tetR-VP16), combined with the tetO carrying minimal promoter from the human cytomegalovirus (hCMV) major immediate early promoter to create the tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. When the tetracycline operator is appropriately positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivative, can function as a strong transmodulator and control gene expression in mammalian cells (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins to obtain its regulatable effect, which in some cases may be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992);Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0077] In addition, the vectors may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transductants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; the SV40 polyoma origin of replication and ColE1 for correct episomal replication; an internal ribosome binding site (IRES), a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0078] Examples of polyadenylation signals useful in practicing the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0079] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into suitable host cells for producing the antibodies. The host cells can be cultured under suitable conditions for expression of the antibodies or their polypeptide chains. Such antibodies or their polypeptide chains can be recovered from the cultured cells (from the cells or culture supernatant) via conventional methods, e.g., affinity purification. If necessary, the antibody polypeptide chains can be incubated under suitable conditions for a suitable period of time to allow production of the antibodies.

[0080] In some embodiments, the method for producing the antibodies described herein involves a recombinant expression vector encoding both the heavy and light chains of the anti-CD44 v7 / 8 antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, e.g., calcium phosphate-mediated transfection. Positive transformant host cells are selected and cultured under suitable conditions that allow the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow the formation of the antibody.

[0081] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-CD44 v7 / 8 antibody and the other encoding the light chain of an anti-CD44 v7 / 8 antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, e.g., calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants are selected and cultured under suitable conditions that allow the expression of the polypeptide chains of the antibody. If the two expression vectors are introduced into the same host cell, the antibody produced herein can be recovered from the host cell or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cell or from the culture medium and then incubated under suitable conditions that allow the formation of the antibody. If the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or from the corresponding culture medium. The two polypeptide chains can then be incubated under suitable conditions for the formation of the antibody.

[0082] Standard molecular biology techniques are used to prepare the recombinant expression vector, transform the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a protein A or protein G-binding matrix.

[0083] Any nucleic acid encoding the heavy chain, light chain, or both, of the anti-CD44 v7 / 8 antibodies described herein, vectors (e.g., expression vectors) containing the same; and host cells containing the vectors are within the scope of this disclosure.

[0084] Antibody-drug conjugates The present disclosure also provides an antibody-drug conjugate comprising any of the anti-CD44 v7 / 8 antibodies described herein, which is covalently linked to a therapeutic agent. The term "antibody-drug conjugate" or "ADC" as used herein refers to a conjugate in which an anti-CD44 v7 / 8 antibody described herein and a therapeutic agent are covalently linked. In general, the antibody-drug conjugate may include an anti-CD44 v7 / 8 antibody, a therapeutic agent, and optionally a linker between the antibody and the therapeutic agent. An ADC is a conjugate that binds a therapeutic agent to CD44. + The therapeutic effect can be increased by delivering to cells, which includes antibodies, particularly CD44 + Targeted by cancer cells. The antibody-drug conjugate can be prepared by various methods known in the art for preparing antibody-drug conjugates.

[0085] The therapeutic agent in the ADC described herein can be a toxin, a chemotherapeutic agent, an antibiotic, an ADP-ribosyltransferase, a radioisotope, or a nuclease.In some cases, the therapeutic agent is a cytotoxic agent.Examples include, but are not limited to, anthracyclines, auristatins (e.g., auristatin E or monomethylauristatin E (MMAE)), camptothecins, combretastatins, dolastatins, duocarmycins, enediynes, geldanamycins, indolino-benzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulysins, hemiasterlins, splicostatins, pladienolides, and calicheamicins.

[0086] In some embodiments, the anti-CD44 v7 / 8 antibody and the therapeutic agent are connected via a linker. Such a linker may be a cleavable linker, for example, cleavable under certain pH conditions (pH-sensitive linker), cleavable by a protease (protease-sensitive linker), or cleavable in the presence of glutathione (glutathione-sensitive linker). In some examples, the linker includes a protease cleavage site, which may contain 2-5 amino acid residues recognizable and / or cleavable by a suitable protease. Such peptides may include naturally occurring amino acid residues, non-naturally occurring amino acid residues, or combinations thereof. In one example, the peptide linker may be a dipeptide linker. Examples include a valine-citrulline (val-cit) linker, a phenylalanine-lysine (phe-lys) linker, or a maleimidocaproic acid-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker. Alternatively, the linker can be a non-cleavable linker, such as a linker comprising an optionally substituted alkane or a thioether.

[0087] In some instances, the linker may comprise a functional group capable of forming a covalent bond with the antibody. Exemplary functional groups include, but are not limited to, maleimide, iodoacetamide, vinylsulfone, acrylate, acrylamide, acrylonitrile, or methacrylate. In some instances, the linker may contain one or more reactive amines, including, but not limited to, acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC) or aminoPEG6-propionyl. For example, see WO2012 / 059882. Other exemplary linkers include sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation occurs through a maleimide group that reacts with sulfhydryls (thiols, --SH), while the sulfo-NHS ester is reactive toward primary amines (such as those found at lysines and protein or peptide N-termini).

[0088] In some instances, the linker is a molecular spacer, such as that represented by Formula I: [ka] wherein R1 is an optionally substituted C 1-6 Alkyl (e.g., C 1-3 alkyl), optionally substituted phenyl, optionally substituted C 2-6 Alkylene, optionally substituted C 2-6 Alkenylene, optionally substituted C 2-6 alkynylene, or an optionally substituted triazole; and / or X can be O, S, or N.

[0089] Methods for conjugating cytotoxic agents or other therapeutic agents to antibodies are known in the art and have been described in various publications. For example, chemical modifications can be made to antibodies either through lysine side chain amines or through activated cysteine ​​sulfhydryl groups by reducing interchain disulfide bonds, whereby conjugation reactions occur. See, for example, Tanaka et al., FEBS Letters 579:2092-2096, (2005), and Gentle et al., Bioconjug. Chem. 15:658-663, (2004). Reactive cysteine ​​residues engineered at specific sites of antibodies for specific drug conjugation with defined stoichiometry have been described. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, (2008). Conjugation using acyl donor glutamine-containing tags and / or endogenous glutamines made reactive by engineering of the polypeptide in the presence of transglutaminase and amines, e.g., cytotoxic agents modified with reactive amines, has also been described in WO2012 / 059882, Strop et al., Chem. Biol. 20(2):161-167 (2013), and Farias et al., Bioconjug. Chem. 25(2):245-250 (2014). The relevant disclosures of such publications are incorporated by reference for the purposes and subject matter referred to herein.

[0090] Chimeric antigen receptors (CARs) and immune cells expressing them The present disclosure also features chimeric antigen receptors that target CD44 v7 / 8 and immune cells expressing same. Chimeric antigen receptors (CARs) as disclosed herein target the binding specificity of immune cells expressing them (e.g., T cells) to CD44 v7 / 8, such as cancer cells from head and neck cancer, cervical cancer, and breast cancer. +CAR is an artificial cell surface receptor that redirects CD44 v7 / 8 to cells, thereby eliminating targeted disease cells, for example, via the effector activity of immune cells. CAR constructs often include an extracellular antigen binding domain fused to at least one intracellular signaling domain. Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010. The extracellular antigen binding domain, which may be a single-chain antibody fragment (scFv), is specific for the CD44 v7 / 8 antigen, and the intracellular signaling domain may mediate cell signaling leading to immune cell activation. Thus, immune cells expressing a CAR construct specific for CD44 v7 / 8 may bind to disease cells expressing CD44 (for example, tumor cells), leading to immune cell activation and elimination of disease cells.

[0091] Any of the anti-CD44 v7 / 8 antibodies described herein can be used to produce a CAR construct, also described herein. For example, the V H and V L The domain can be fused to an intracellular signaling domain(s) and the CAR construct can be produced using conventional recombinant techniques. In some examples, the V of anti-CD44 v7 / 8 H and V L The domains are connected via peptide linkers to form the scFv fragment.

[0092] The CAR constructs disclosed herein may comprise one or more intracellular signaling domains. In some examples, the CAR comprises an intracellular signaling domain that includes an immunoreceptor tyrosine-based activation motif (ITAM). Such an intracellular signaling domain may be from CD3zeta. In addition, the CAR constructs may further comprise one or more costimulatory signaling domains, which may be from costimulatory receptors, for example, from 4-1BB (CD137), CD7, CD27, CD28, CD40, OX40, ICOS, GITR, HVEM, TIM1, or LFA-1. The CAR constructs disclosed herein may further comprise a transmembrane hinge domain, which can be derived from a suitable cell surface receptor, e.g., CD28 or CD8.

[0093] Also provided are isolated nucleic acid molecules and vectors encoding any of the anti-CD44 v7 / 8 CARs as disclosed herein, and host cells, such as host immune cells (e.g., T cells and natural killer cells), that contain the nucleic acid molecules or vectors. Immune cells expressing anti-CD44 v7 / 8 CARs, including CD44-specific antigen-binding fragments, can be used to treat cancers that express CD44. Thus, also provided herein are methods for treating cancers that express CD44 by selecting a subject with a cancer that expresses CD44, and administering to the subject a therapeutically effective amount of immune cells expressing a CD44-targeted CAR. + A method of treating a subject with cancer.

[0094] Pharmaceutical Compositions As described herein, the anti-CD44 v7 / 8 antibody, the nucleic acid or nucleic acid set encoding it, the vector containing it, or the host cell containing the vector, as well as the ADC containing the anti-CD44 v7 / 8 antibody and / or the immune cell expressing the CD44-targeted CAR can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably be able to stabilize the active ingredient) and not harmful to the subject being treated. Pharmaceutically acceptable excipients (carriers), including buffers, are well known in the art. For example, see Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0095] The pharmaceutical compositions used in the methods of the invention may contain pharma- ceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides. ; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0096] In some examples, the pharmaceutical compositions described herein include liposomes containing the antibody (or nucleic acid or ADC encoding same), which can be prepared by methods known in the art, such as those described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are described in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes with the desired diameter.

[0097] The antibody, the nucleic acid(s) encoding it, or the ADC can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, and can be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are known in the art. For example, see Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0098] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release form.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, such as films, or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0099] Pharmaceutical compositions used for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through a sterile filtration membrane. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or a vial having a stopper that can be pierced by a hypodermic injection needle. The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0100] To prepare solid compositions such as tablets, the primary active ingredient can be mixed with pharmaceutical carriers, e.g., corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, calcium hydrogen phosphate, or gum, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When these formulation compositions are referred to as homogenous, it is meant that the active ingredient is evenly dispersed throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form that provides the advantage of sustained action. For example, the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0101] Suitable surfactants include inter alia non-ionic agents such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions containing a surfactant will conveniently contain 0.05-5% surfactant, and may be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, may be added, if required.

[0102] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient can be dissolved in the emulsion composition before mixing, or alternatively, it can be dissolved in the emulsion formed when mixed with oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and phospholipids (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be understood that other ingredients, such as glycerol or glucose, may be added to adjust the osmotic pressure of the emulsion. Suitable emulsions will typically contain up to 20% oil, e.g., 5-20%.

[0103] The emulsion composition may be one that may be prepared by mixing the antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water). Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable, aqueous or organic solvents, or mixtures and powders thereof. The liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients, as set forth above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0104] Compositions in preferred sterile pharma- ceutically acceptable solvents can be nebulized by the use of gases. Nebulized solutions can be inhaled directly from the nebulizing device, or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0105] Treatment and diagnostic methods As described herein, the anti-CD44 v7 / 8 antibody, a nucleic acid or set of nucleic acids encoding the same, a vector comprising the same, an ADC comprising an anti-CD44 v7 / 8 antibody, and an immune cell (e.g., a T cell or an NK cell) expressing a CD44-targeted CAR are capable of expressing CD44. + CD44 on cancer cells + are useful in inhibiting and / or eliminating disease cells, thereby inhibiting CD44 + This would be beneficial in treating diseases or disorders associated with diseased cells.

[0106] To practice the methods described herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical route. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, antibodies as described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and milled powders.

[0107] The subject to be treated by the methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. Human subjects in need of treatment may be those that express CD44 + The target disease / disorder may be a human patient having, at risk of having, or suspected of having, a target disease / disorder associated with disease cells. In some embodiments, the target disease / disorder may be a human patient having, at risk of having, or suspected of having, a target disease / disorder associated with disease cells. +The diseased cells are cancer cells, such as head and neck cancer, cervical cancer, and breast cancer cells.Subjects with target diseases or disorders can be identified by routine medical tests, such as laboratory tests, organ function tests, CT scans, or ultrasound.Subjects suspected of having any of these target diseases / disorders can show one or more symptoms of the disease / disorder.Subjects at risk of disease / disorder can be subjects with one or more risk factors for the disease / disorder.

[0108] As used herein, "effective amount" refers to the amount of each active agent, either alone or in combination with one or more other active agents, required to confer a therapeutic effect on a subject. In some embodiments, the therapeutic effect is an increase in CD44 v7 / 8 activity or CD44 + The reduction of cell activity. It will be clear to those skilled in the art how to determine whether the amount of antibody or therapeutic agent (for example, ADC or CAR-T cell) containing it achieves therapeutic effect. As recognized by those skilled in the art, the effective amount varies depending on the specific condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, size, sex and weight), duration of treatment, nature of concurrent treatment (if any), specific route of administration, and similar factors within the knowledge and experience of health practitioners. These factors are well known to those skilled in the art and can be addressed with a minimum of routine experimentation. In general, it is preferred to use the maximum dose of each component or combination thereof, i.e., the highest safe dose according to sound medical judgment.

[0109] Empirical considerations such as half-life will generally contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted according to the course of treatment and is generally, but not necessarily, based on the treatment and / or suppression and / or recovery and / or delay of the target disease / disorder. Alternatively, sustained continuous release formulations of antibodies may be appropriate. A variety of formulations and devices for achieving sustained release are known in the art.

[0110] In one example, the dosage of the antibody as described herein can be empirically determined in an individual given one or more antibody administrations. The individual is given increasing dosages of the antagonist. To assess the effectiveness of the antagonist, disease / disorder indications can be followed.

[0111] Generally, for administration of any of the anti-CD44 v7 / 8 antibodies or ADCs comprising the same as described herein, the initial candidate dosage may be about 2 mg / kg. For purposes of this disclosure, typical daily dosages may range from approximately any of 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, up to 30 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or more, depending on the condition, treatment is sustained until a desired suppression of symptoms occurs or sufficient therapeutic levels are achieved to alleviate the target disease or disorder, or its symptoms. An exemplary dosing regimen includes administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of the antibody of about 1 mg / kg, or followed by a biweekly maintenance dose of about 1 mg / kg. However, other dosage regimens may be useful depending on the pattern of pharmacokinetic decay the practitioner wishes to achieve. For example, dosing 1-4 times per week is contemplated. In some embodiments, dosing in the range of about 3 μg / mg to about 2 mg / kg (such as about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg) can be used. In some embodiments, the dosing frequency is once every week, once every 2 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, or once every 10 weeks; or once every month, once every 2 months, or once every 3 months, or longer. The progress of this treatment is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) can be changed over time.

[0112] For purposes of this disclosure, appropriate dosages of antibodies as described herein will depend on the specific antibody, antibody, and / or non-antibody peptide (or compositions thereof) used, the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antagonist, and the discretion of the attending physician. Typically, the clinician will administer the antibody until the dosage achieves the desired result. In some embodiments, the desired result is a reduction in thrombosis. Methods for determining whether a dosage results in the desired result will be apparent to one of skill in the art. Administration of one or more antibodies can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to the skilled practitioner. Administration of the antibody can be essentially continuous over a preselected period of time, or can be a series of spaced doses, for example, either before, during, or after the onset of the target disease or disorder.

[0113] As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition towards a disease / disorder, for the purpose of curing, healing, alleviating, mitigating, altering, treating, ameliorating, improving, or affecting the disorder, the symptom of the disease, or the predisposition towards a disease or disorder.

[0114] Alleviating the target disease / disorder encompasses delaying the onset or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of the target disease or disorder means to postpone, prevent, delay, retard, stabilize, and / or prolong the progression of the disease. This delay can be of various lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease, or delaying the onset of a disease, is a method that reduces the likelihood of the onset of one or more symptoms of the disease in a given time frame and / or reduces the severity of symptoms in a given time frame, compared to when the method is not used. Such comparisons are typically based on clinical studies using a sufficient number of other subjects to obtain statistically significant results.

[0115] "Onset" or "progression" of a disease refers to the first signs of disease and / or subsequent progression. Onset of a disease can be detected and evaluated using standard clinical techniques well known in the art. However, onset also refers to progression that may not be detectable. For the purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a target disease or disorder includes initial onset and / or recurrence.

[0116] In some embodiments, the antibodies described herein are administered to a subject in need of treatment in an amount sufficient to inhibit the activity of one or both of the target antigens by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) in vivo. In other embodiments, the antibodies are administered in an amount effective to reduce the activity level of the target antigen by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0117] Conventional methods known to those skilled in the art of medicine can be used to administer the pharmaceutical composition to a subject, depending on the type of disease or site of disease to be treated. The composition can also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. In addition, it can be administered to a subject via administration of an injectable depot route, such as using a 1-month, 3-month or 6-month depot injectable or biodegradable material and method. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

[0118] Injectable compositions may contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by drip infusion, whereby a pharmaceutical formulation containing the antibody and a pharmaceutically acceptable excipient is injected. Pharmaceutically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0119] In one embodiment, antibody is administered via site-specific or targeted local delivery technique.Examples of site-specific or targeted local delivery technique include various implantable depot sources or local delivery catheters such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application of antibody.For example, see PCT Publication No. WO00 / 53211 and U.S. Patent No. 5,981,568.

[0120] Targeted delivery of therapeutic compositions containing antisense polynucleotides, expression vectors or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol. (1993) 11:202;Chiou et al., Gene Therapeutics: Methods and Applications Of Direct Gene Transfer (JA Wolff, ed.) (1994);Wu et al., J. Biol. Chem. (1988) 263:621;Wu et al., J. Biol. Chem. (1994) 269:542;Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655;Wu et al., J. Biol. Chem. (1991) 266:338.

[0121] Therapeutic compositions containing polynucleotides (e.g., those encoding the antibodies described herein) are administered in the range of about 100 to about 200 mg of DNA for local administration in gene therapy protocols. In some embodiments, concentrations ranging from about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg or more of DNA may also be used during gene therapy protocols.

[0122] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of coding sequences can be either constitutive or regulated.

[0123] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO90 / 07936; WO94 / 03622; WO93 / 25698; WO93 / 25234; WO93 / 11230; WO93 / 10218; WO91 / 02805; U.S. Patent Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242), alphavirus-based vectors (see, e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1247). VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94 / 12649, WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984 and WO95 / 00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be used.

[0124] Non-viral delivery vehicles and methods can also be used, including, but not limited to, polycation-condensed DNA, either linked or not linked to killed adenovirus alone (see, for example, Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, for example, Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicle cells (see, for example, U.S. Pat. No. 5,814,482; PCT Publication Nos. WO95 / 07994; WO96 / 17072; WO95 / 30763; and WO97 / 42338) and nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA transfer methods are described in PCT Publication No. WO90 / 11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO95 / 13796; WO94 / 23697; WO91 / 14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581. The specific dosage regimens, ie, dosage, timing and repetition, used in the methods described herein will depend on the specific subject and the subject's medical history.

[0125] When immune cells expressing CD44-targeted CARs are used to treat disease, patients receive approximately 10 5 ~10 10 Patients may be treated by injecting a therapeutically effective dose of immune cells, such as T lymphocytes or NK cells, in the range of 100 mg / kg or more of cells (cells / Kg). Often, the infusions may be repeated as many times as the patient can tolerate until the desired response is achieved. Appropriate infusion doses and schedules will vary from patient to patient, but can be determined by the physician treating the particular patient. Typically, infusions are administered in doses of 10 mg / kg or more of cells (cells / Kg). 8or up to more than this number of cells / Kg, approximately 10 6 An initial dose of cells / Kg will be injected. IL-2 will be co-administered to expand the injected cells. The amount of IL-2 is approximately 1-5 x 10 per square meter of body surface. 6 It can be an international unit.

[0126] In some embodiments, more than one antibody or a combination of antibodies and another suitable therapeutic agent may be administered to a subject in need of treatment. Antibodies, ADCs and / or CAR-T cells comprising same may be used in combination with other agents that function to enhance and / or complement the effectiveness of the agents.

[0127] Efficacy of a treatment for a target disease / disorder can be assessed by methods well known in the art. Any of the anti-CD44 v7 / 8 antibodies described herein may be used to detect CD44 in a sample. + The diagnostic assays may be used to detect the presence or levels of cells. Such diagnostic assays may be performed in vitro or in vivo.

[0128] For diagnostic applications, anti-CD44 v7 / 8 antibodies as described herein may be conjugated to a detectable label (e.g., an imaging agent, such as a contrast agent) in vivo or in vitro for diagnostic purposes. As used herein, "conjugated" or "attached" preferably means that two entities are associated with sufficient affinity that the therapeutic / diagnostic benefit of the association between the two entities is realized. The association between the two entities may be either direct or via a linker, such as a polymeric linker. Conjugated or attached may encompass covalent or non-covalent bonds as well as other forms of association, such as, for example, entrapment of one entity on or within the other entity, or entrapment of either or both entities on or within a third entity, such as a micelle.

[0129] In one example, anti-CD44 v7 / 8 antibodies as described herein can be attached to a detectable label, which is a compound that can emit a detectable signal, either directly or indirectly, so that the aptamer can be detected, measured, and / or quantified in vitro or in vivo. Examples of such "detectable labels" are intended to include, but are not limited to, fluorescent labels, chemiluminescent labels, colorimetric labels, enzyme markers, radioisotopes, and affinity tags such as biotin. Such labels can be conjugated to the aptamer directly or indirectly by conventional methods.

[0130] In some embodiments, the detectable label is a label that detects CD44 in vivo. + A suitable agent for imaging cells, which may be a radioactive molecule, a radiopharmaceutical, or an iron oxide particle. Suitable radioactive molecules for in vivo imaging include, but are not limited to: 122 I, 123 I, 124 I, 125 I, 131 I, 18 F, 75 Br, 76 Br, 76 Br, 77 Br, 211 At, 225 Ac, 177 Lu, 153 Sm, 186 Re, 188 Re, 67 Cu, 213 Bi, 212 Bi, 212 Pb, and 67 Exemplary radiopharmaceuticals suitable for in vivo imaging include: 111 Inoxyquinoline, 131 I sodium iodide, 99m Tc mebrophenin, and 99m Tc red blood cells, 123 I sodium iodide, 99m Tc Examethadime, 99m Tc macroaggregated albumin, 99m Tc medronate, 99m Tc mertiazide,99m Tc oxidronate, 99m Tc pentatate, 99m Tc pertechnetate, 99m Tc sestamibi, 99m Tc sulfur colloid, 99m Reporting agents include Tc tetrofosmin, thallium-201, and xenon 133. The reporting agent can also be a dye, such as a fluorophore, which detects CD44 in tissue samples. + It is useful in detecting cell mediated diseases.

[0131] To perform an in vitro diagnostic assay, anti-CD44 v7 / 8 antibodies were used to detect CD44 + The antibody may be contacted with a sample suspected of containing cells. The antibody and the sample may be incubated under suitable conditions for a suitable period of time to allow the antibody to bind to the CD44 v7 / 8 antigen. Such interaction may then be detected by routine methods, for example, via ELISA or FACS. To perform a diagnostic assay in vivo, a suitable amount of anti-CD44 v7 / 8 antibody conjugated to a label may be administered to a subject in need of investigation. The presence of the labeled antibody may be detected based on the signal emitted from the label by routine methods.

[0132] Kits for Use in Treatment and Diagnostics The present disclosure also provides a method for the preparation of a method for the preparation of a compound comprising administering to a subject a subject, comprising administering to said subject a compound comprising administering to said subject a ... + Kits are provided for use in inhibiting and / or eliminating disease and thus alleviating diseases / disorders associated with such diseased cells. Such kits may include one or more containers containing anti-CD44 v7 / 8 antibodies, ADCs comprising same, or immune cells expressing CD44-targeted CAR polypeptides, such as any of those described herein.

[0133] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administration of anti-CD44 v7 / 8 antibodies, ADCs, or immune cells to treat, delay onset, or ameliorate the target disease, as described herein. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibodies, ADCs, or immune cells to an individual at risk for the target disease.

[0134] The instructions for use of anti-CD44 v7 / 8 antibody, ADC comprising it, or immune cells expressing CD44-targeted CAR generally include information on dosage, dosing schedule, and administration route for intended treatment. Containers can be unit dose, bulk package (e.g., multi-dose package) or sub-unit dose. The instructions provided in the kit of the present invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0135] The label or package insert may indicate that the composition is an antibody that inhibits CD44 + It is indicated that the method is used to treat, delay the onset of, and / or ameliorate a cell-related disease or disorder. Instructions may be provided for practicing any of the methods described herein.

[0136] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, soft packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated is a package for use in combination with a specific device, such as an inhaler, a nasal administration device (e.g., an atomizer) or an injection device, such as a minipump. The kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an anti-CD44 v7 / 8 antibody, an ADC comprising the same, or an immune cell expressing a CD44-targeted CAR, as described herein.

[0137] The kit may optionally provide additional components such as buffers and interpretive information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described herein.

[0138] Also provided herein is a method for detecting CD44 in a sample. +A kit for use in detecting cells. Such a kit may include any of the anti-CD44 v7 / 8 antibodies described herein. In some cases, the anti-CD44 v7 / 8 antibody may be conjugated to a detectable label such as those described herein. As used herein, "conjugated" or "attached" means that the two entities are associated, preferably with sufficient affinity, until the therapeutic / diagnostic benefit of the association between the two entities is realized. The association between the two entities may be either direct or via a linker, such as a polymeric linker. Conjugated or attached may include covalent or non-covalent binding, as well as other forms of association, such as entrapment, for example, entrapment of one entity on or within the other entity, or entrapment of either or both entities on or within a third entity, such as a micelle. Alternatively or in addition, the kit may include a secondary antibody capable of binding to the anti-CD44 v7 / 8 antibody. + , and in particular includes instructions for using the anti-CD44 v7 / 8 antibody to detect CD44 v7 / 8.

[0139] Common techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1989) Academic Press; 1987);Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons;Methods in Enzymology (Academic Press, Inc.);Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);Current Protocols in Molecular Biology (FM Ausubel, et al. eds. 1987);PCR: The Polymerase Chain Reaction, (Mullis, et al., eds.(1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985)); Transcription and translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells and Enzymes (lRL Press, (1986)); and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al.(eds.) etc. are fully described in the literature.

[0140] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific aspects are therefore to be construed as merely illustrative, and not limiting of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0141] example Example 1: Generation of anti-CD44 v7 / 8 antibodies Reagents and general methods Hybridoma cell culture medium (PFHM-II Protein-Free Hybridoma Medium; #12040077) was purchased from Thermo Fisher. RNA was isolated using standard protocols and TRIzol reagent from Thermo Fisher (#15596018). The resulting cDNA molecules were generated using a cDNA synthesis kit from Takara (PrimeScript II 1st strand cDNA synthesis kit; #6210A). Antibody V-region amplification was performed using premixed Taq from Takara (#RR901A). Standard PCR primer sets (Ig-Primer Sets #TB326) were obtained from Novagen. Genes were cloned into pET28a (Novagen; #69864) using standard techniques, including the use of EcoRI, HindIII, SalI, and T4 ligase (all from NEB). Some, but not all, oligonucleotide molecules were purified utilizing a QIAEX II gel extraction kit (QIAgen #20021). A253, A375, A549, and Detroit 562 (D562) cell cultures were maintained in vitro as independent monolayer cultures in an atmosphere of 5% CO2 at 37°C. These tumor cells were regularly passaged at any time.

[0142] Screening of antibody libraries to identify anti-CD44 v7 / 8 antibodies A large library of monoclonal antibodies (>100,000 in total) was generated using a mixture of proteomic and peptide antigens as previously described in US 2015 / 0153356. The library was partitioned into a series of high-density antibody arrays before screening against cancer tumor samples and an FDA normal tissue panel. A number of isolated antibodies from the library, including HIS029, were cloned into the D562 cell line (CD44 + D44 v7 / 8 was identified as the target antigen via antibody immunoprecipitation followed by mass spectrometry.

[0143] Production of antibody clones Individual hybridoma clones were cultured in T25 flasks containing 10 mL hybridoma cell culture medium (PFHM-II protein-free hybridoma medium). Cells were cultured at 37° C. until 80% confluent. Culture medium was then removed and cells were washed twice with 1×PBS. TRIzol reagent (1 mL vol.) was added directly to the flask and cells were lysed by pipette mixing. Cell lysates were then harvested from the T25 flasks and total RNA was isolated using standard methods. RNA concentrations were subsequently measured using a Nanodrop 2000 (Thermo Fisher). Standard cDNA was then generated from the isolated RNA following the Takara PrimeScript II 1st strand cDNA synthesis kit protocol. Amplification of the hybridoma V region of the resulting cDNA was performed following Novagen user protocol TB326. Primer pairs were used for amplification as shown in Table 3 below:

[0144] [Table 3]

[0145] The PCR products were checked using a 1% agarose gel. Positive PCR products were recovered using a QIAgen gel extraction kit and subsequently cloned into pET28a vector using restriction enzymes corresponding to the primer sequences (from NEB). The pET28a vectors with the PCR product inserts were transformed into DH5α bacterial cells and grown on ampicillin positive agar plates. Each bacterial clone was sent for Sanger sequencing using MuIgGVH3'-2, MuIgkVL3'-1 or MuIglVL3'-1 primers. The resulting sequences were compared for consistency and were identical to the target VH3'-2, MuIgkVL3'-1 or MuIglVL3'-1 primers, respectively. H and V L The sequence was confirmed. H and V L The sequences were then analyzed based on the IGMT database (imgt.org / ) and the V regions, frame and V H and V L The CDR elements were obtained.

[0146] Example 2: In vitro evaluation of anti-CD44 v7 / 8 antibodies Binding activity to CD44 variants Peptides derived from CD44 v7 (PISHPMGRGHQAGRR; SEQ ID NO: 12), CD44 v7 / 8 (QAGRRMDMDSSHSIT; SEQ ID NO: 13), and CD44 v8 (SHSITLQPTANPNTG; SEQ ID NO: 14) were used to assess the binding specificity of the antibody clone HIS029.

[0147] Samples of D526 cells were harvested using trypsin-EDTA partial digestion followed by centrifugation at 1000 rpm for 5 min. Cells were resuspended in cold PBS and anti-CD44 v7 / 8 antibody, HIS029, was added. The cell solution was mixed, incubated at 4°C in the dark, and washed with PBS prior to the addition of a secondary antibody conjugate (for detection purposes). After incubation, cells were washed with PBS before mixing with one of the three CD44 peptides mentioned above. After incubation of D562 / HIS029 samples with CD44 peptides, the samples were fixed with fixative and then subjected to flow cytometry (FACS) analysis. IgG antibody incubated with D562 cells was used as a negative control. As shown in Figure 1B, anti-CD44 v7 / 8 antibody (HIS029) inhibited CD44 in the absence of any peptide or in the presence of either CD44 v7 or CD44 v8 peptides. + The anti-CD44 v7 / 8 peptide showed saturable binding to the D562 cell samples. Conversely, the CD44 v7 / 8 peptide completely disrupted the binding interaction between HIS029 and D562 cells. These data suggest that the anti-CD44 v7 / 8 antibodies described herein specifically target CD44 v7 / 8.

[0148] Using the same FACS-based assay as above, anti-CD44 v7 / 8 antibodies (HIS029 and humanized HIS029) were shown to mediate CD44 + It was further demonstrated that HIS029 binds to CD44 with a binding affinity of 35 nM. + Humanized HIS029 binds to CD44 with a binding affinity of 36 nM. + A253 cells (Fig. 2).

[0149] Example 3: Preparation and in vitro evaluation of anti-CD44 v7 / 8 antibody-drug conjugates An anti-CD44 v7 / 8 antibody-drug conjugate (ADC) (HIS029-vc-MMAE) was generated that contains the antibody HIS029 and monomethyl auristatin E. A linker molecule (maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate) was first coupled to MMAE using an amide coupling reaction. After purification of this first compound, it was then coupled to the antibody HIS029 using a thiol-based reaction.

[0150] HIS029-vc-MMAE was cloned into CD44 + Cell samples (A253, D562, NCI-H520, and BXPC3) were added to determine the ability of anti-CD44 v7 / 8 ADC to be encapsulated in CD44+ cells and induce cytotoxicity. A375 cells, which have low expression levels of CD44, served as a negative control cell line. All cell lines were treated with either HIS029-vc-MMAE or a control ADC (IgG-vc-MMAE) at various concentrations. After treatment, cell viability was assessed using standard methods. CD44 + In the D562 cell line, HIS029-vc-MMAE had an IC of 630 pM, as shown in Figure 3. 50 In the control cell line (A375), HIS029-vc-MMAE caused a minimal decrease in cell viability at high ADC concentrations, but not at IC 50 The value was not determined. The IgG control antibody did not lead to a significant loss of cell viability. HIS029-vc-MMAE inhibited other CD44 + Cell lines also demonstrated a decrease in cell viability, with IC of 797 pM (A253), 588 pM (NCI-H520), and 189 pM (BXPC3) (data not shown). 50 was the value.

[0151] Example 4: Anti-CD44 v7 / 8 CAR + Preparation of T cells and in vitro evaluation Humanized HIS029 antibody (V H and V L) was inserted into a chimeric antigen receptor vector in frame with the CD8 hinge region, the CD8 transmembrane region, the intracellular domain of the costimulatory molecule 4-1BB, and the intracellular domain of CD3ζ. This vector further contains the EF1α promoter. Anti-CD44 v7 / 8 CAR + The nucleic acid encoding the anti-CD44 v7 / 8 CAR was transfected using lentiviral packaging into 239T cells. Human CD3+ T cells were then activated and transduced with the lentivirus to generate anti-CD44 v7 / 8 CAR+ T cells. The anti-CD44 v7 / 8 CAR was transfected using lentiviral packaging into 239T cells. + Following T cell generation, the transduction efficiency for each CAR construct was determined using FACS (Figure 4). The T cell population transduced with anti-CD44 v7 / 8 CAR was >55.8% CAR + Contained cells.

[0152] CD44 + Anti-CD44 v7 / 8 CAR induces antigen-dependent cytotoxicity and cytokine (IFNγ and IL2) secretion in A253 cell line + The ability of T cells to express CD44 was tested. A549 cells, which have a low expression level of CD44, were used as a negative control cell line. Each cell line was co-transfected with anti-CD44 v7 / 8 CAR at various ratios of T cells (effector cells) to target cells. + T cells or controls (CD3 + T cells) were administered.

[0153] After incubation of T cells with target cells, specific lysis / cytotoxicity of target cells was assessed using standard methods (Figures 5A, 5B, 6). + Anti-CD44 v7 / 8 CAR in A253 cells + Addition of T cells induced antigen-dependent cytotoxicity at a T cell:target cell ratio of 0.25:1 or greater. Control experiments (incubation with CD3 T cells without CAR; and incubation with CAR + Incubation of A549 with T cells did not lead to significant induction of specific cytotoxicity. +Addition of T cells induced IFNγ and IL2 levels at or above a T cell:target cell ratio of 0.25:1. Control experiments (incubation with CD3 T cells without CAR; and CAR + Incubation of A549 with T cells did not lead to significant secretion of IFNγ.

[0154] Other Aspects All features disclosed herein may be used in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a generic series of equivalent or similar features. From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from the spirit and scope thereof. Therefore, other embodiments are also within the scope of the claims.

[0155] Equivalent While several embodiments of the invention have been described and illustrated herein, those of ordinary skill in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the invention described herein. More generally, those of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the invention are used. Those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Thus, it should be understood that the above embodiments are provided by way of example only, and that within the scope of the appended claims and their equivalents, embodiments of the invention may be practiced other than as specifically described and claimed. The inventive aspects of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is encompassed within the scope of the present disclosure, except where such features, systems, articles, materials, kits, and / or methods are not mutually consistent.

[0156] All definitions defined and used herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, and in some cases may include the entirety of the document. The indefinite articles "a" and "an," as used herein and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0157] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so conjoined (i.e., elements that are conjunctively present in some cases and disjunctively present in other cases). Multiple elements listed with "and / or" should be interpreted in the same manner (i.e., "one or more" elements so conjoined). Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", may refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in another embodiment to both A and B (optionally including other elements); and so forth.

[0158] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be understood to be inclusive (i.e., the inclusion of at least one of a number or list of elements (but may include more than one), and optionally additional items not listed). Only terms clearly indicating the contrary, such as "only one of" or "exactly one of," or when used in the claims, "consisting of" refers to the inclusion of exactly one element of a number or list of elements. In general, the term "or," as used herein, should only be understood to refer to exclusive alternatives (i.e., one or the other, but not both) when preceded by a term of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, should have its ordinary meaning as used in the field of patent law.

[0159] As used herein in the specification and claims, the phrase "at least one" refers to a list of one or more elements and means at least one element selected from any one or more elements in the list of elements, but it should be understood that it does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or not to the specifically identified elements, may optionally be present. Thus, as non-limiting examples, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") refers, in one embodiment, to at least one A (optionally including more than one), and no B (and optionally including elements other than B); in another embodiment, to at least one B (optionally including more than one), and no A (and optionally including elements other than A); in another embodiment, to at least one A (optionally including more than one), and at least one B (optionally including more than one) (and optionally including other elements); and so forth.

[0160] Unless expressly indicated to the contrary, it is to be understood that in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

Claims

1. 1. An isolated antibody that binds to human CD44, comprising: A heavy chain variable region (V CDR1) comprising a heavy chain complementarity determining region 1 (HC CDR1) represented by the amino acid sequence of SEQ ID NO:2, a HC CDR2 represented by the amino acid sequence of SEQ ID NO:3, and a HC CDR3 represented by the amino acid sequence of SEQ ID NO:

4. Ｈ ), and a light chain variable region (V CDR1) comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO:5, a LC CDR2 represented by the amino acid sequence of SEQ ID NO:6, and a LC CDR3 represented by the amino acid sequence of SEQ ID NO:

7. Ｌ ) The isolated antibody comprising:

2. 2. The isolated antibody of claim 1, which does not bind to peptide PISHPMGRGHQAGRR (SEQ ID NO: 12) or peptide SHSITLQPTANPNTG (SEQ ID NO: 14).

3. V of isolated antibody Ｈ is at least 85% identical to the amino acid sequence of SEQ ID NO: 8, and / or the V Ｌ The isolated antibody of claim 1 or 2, which is at least 85% identical to the amino acid sequence of SEQ ID NO:

9.

4. V of isolated antibody Ｈ comprises the amino acid sequence of SEQ ID NO: 8, and / or the V Ｌ The isolated antibody of any one of claims 1 to 3, comprising the amino acid sequence of SEQ ID NO:

9.

5. The antibody, (a) is a full-length antibody or an antigen-binding fragment thereof; (b) is a single chain antibody (scFv); and / or (c) a humanized antibody or a chimeric antibody; The antibody according to claim 1 or 2.

6. The antibody of any one of claims 1 to 5, wherein the antibody is a full-length antibody that is an IgG molecule.

7. The antibody comprises the V Ｈ and / or the V shown in the amino acid sequence of SEQ ID NO:11 Ｌ The antibody of claim 1 or 2, which is a humanized antibody comprising:

8. A nucleic acid or set of nucleic acids encoding an antibody according to any one of claims 1 to 7.

9. A vector or a set of vectors comprising the nucleic acid or set of nucleic acids of claim 8.

10. A host cell comprising the vector or set of vectors according to claim 9.

11. i. an antibody according to any one of claims 1 to 7; and ii. at least one therapeutic agent for treating cancer; wherein the antibody is covalently conjugated to at least one therapeutic agent. Antibody-drug conjugates (ADCs).

12. The antibody-drug conjugate of claim 11, wherein the antibody and the therapeutic agent are conjugated via a linker, the linker being a cleavable or non-cleavable linker.

13. The linker: (a) comprises a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker; or (b) a non-cleavable linker that includes an alkane or a thioether; and / or (c) comprises a functional group that forms a covalent bond with the antibody and the linker; and / or (d) further comprising Formula I: 【Chemistry 1】 During the ceremony, R １ is C1-6 alkyl, phenyl, C2-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, or triazole; and X is O, S, or N; The molecular spacer is represented by The antibody-drug conjugate of claim 12.

14. The antibody-drug conjugate of claim 13, wherein the protease-sensitive linker comprises a peptide sequence of 2 to 5 amino acids, wherein the 2 to 5 amino acids comprise naturally occurring amino acid residues, non-naturally occurring amino acid residues, or a combination thereof.

15. The antibody-drug conjugate of claim 13, wherein the functional group comprises a maleimide group, an iodoacetamide group, a vinylsulfone group, an acrylate group, an acrylamide group, an acrylonitrile group, or a methacrylate group.

16. The antibody-drug conjugate of any one of claims 11 to 15, wherein at least one therapeutic agent is a cytotoxic agent.

17. The antibody-drug conjugate of claim 16, wherein the cytotoxic agent is monomethylauristatin E.

18. (i) an extracellular domain comprising an antigen-binding fragment that specifically binds to CD44 v7 / 8, wherein the antigen-binding fragment comprises A heavy chain variable region (V CDR1) comprising a heavy chain complementarity determining region 1 (HC CDR1) represented by the amino acid sequence of SEQ ID NO:2, a HC CDR2 represented by the amino acid sequence of SEQ ID NO:3, and a HC CDR3 represented by the amino acid sequence of SEQ ID NO:

4. Ｈ ), and a light chain variable region (V CDR1) comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO:5, a LC CDR2 represented by the amino acid sequence of SEQ ID NO:6, and a LC CDR3 represented by the amino acid sequence of SEQ ID NO:

7. Ｌ ) The extracellular domain comprising: (ii) a transmembrane domain, and (iii) one or more intracellular signaling domains. A chimeric antigen receptor comprising:

19. (a) the antigen-binding fragment is a single chain antibody fragment (scFv), the scFv comprising a VH and a VL according to claim 3 or 4; and / or (b) the antigen-binding fragment specifically binds to a peptide comprising the amino acid sequence QAGRRMDMDSSHSIT (SEQ ID NO: 13); and / or (c) the transmembrane domain of (ii) comprises a transmembrane domain derived from the CD28 or CD8 receptor; and / or (d) one or more domains (iii) comprise a signaling domain, a costimulatory domain, or a combination thereof from CD3ζ; The chimeric antigen receptor of claim 18.

20. 20. The chimeric antigen receptor of claim 19, wherein the antigen-binding fragment does not bind to the peptide PISHPMGRGHQAGRR (SEQ ID NO: 12) or the peptide SHSITLQPTANPNTG (SEQ ID NO: 14).

21. 20. The chimeric antigen receptor of claim 19, wherein (iii) comprises a costimulatory domain that is from a 4-1BB, CD7, CD27, CD28, CD40, OX40, ICOS, GITR, HVEM, TIM1, or LFA-1 receptor.

22. A nucleic acid comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 18 to 21.

23. A vector comprising the nucleic acid of claim 22.

24. A host cell expressing the chimeric antigen receptor of any one of claims 18 to 21.

25. A pharmaceutical composition comprising: (i) an antibody according to any one of claims 1 to 7, a nucleic acid according to claim 8 or 22, a set of nucleic acids according to claim 8, a host cell according to claim 10 or 24, or an antibody-drug conjugate according to any one of claims 11 to 17; and (ii) a pharma- ceutically acceptable carrier.

26. 26. The pharmaceutical composition of claim 25 for use in a method of inhibiting cells expressing CD44 v7 / 8 comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof, wherein the subject has or is suspected of having cancer.

27. 27. The pharmaceutical composition of claim 26, wherein the cancer is selected from the group consisting of head and neck cancer, cervical cancer, and breast cancer.

28. 1. A method for detecting the presence of a cell expressing surface CD44 v7 / 8, comprising the steps of: i. contacting a sample suspected of having cells expressing surface CD44 v7 / 8 with an antibody according to any one of claims 1 to 7, wherein the antibody is conjugated to a labeling agent; and ii. detecting the presence of cells in the sample that express surface CD44 v7 / 8 based on binding of the antibody to cells in the sample. The method comprising:

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