Anti-CD44V6 antibody and its use in the treatment of CD44V6-overexpressing cancers

Abstract

The present disclosure relates to a binding protein that binds to human CD44v6. The binding protein can be linked to an agent, such as a contrast agent or a therapeutic agent, which can be a radioactive isotope, to form a conjugate. The binding protein or conjugate, or a pharmaceutical composition thereof, can be used in medical treatments, such as cancer treatment, or in diagnosis and medical imaging. The binding protein can also be used to engineer cells to express a chimeric antigen receptor having the binding protein of the present disclosure as an antigen-binding domain.

Description

Technical Field

[0001] The present disclosure relates to a binding protein that binds to an epitope of human CD44v6. In some embodiments, the binding protein comprises a CD44v6-binding antibody or a fragment thereof, or a conjugate binding protein or monoclonal antibody that carries a therapeutic agent such as a contrast agent or an anti-tumor agent. In some embodiments, the binding protein, antibody, conjugate binding protein / antibody, or a pharmaceutical composition thereof is used for a medical treatment such as cancer treatment. In some embodiments, the binding protein, antibody, or conjugate binding protein / antibody is used for diagnosis or medical imaging. In other embodiments, the binding protein is used to engineer cells to express a chimeric antigen receptor having the binding protein of the present disclosure as an antigen-binding domain.

Background Art

[0002] Cancer is one of the most common fatal diseases and accounts for a significant number of deaths every year despite recent advances in diagnosis and treatment. Monoclonal antibodies (mAbs) that modulate the immune response, or conjugate mAbs that carry anti-tumor agents, provide very effective and promising treatments for many cancers.

[0003] CD44 is a cell surface glycoprotein involved in cell-cell interaction, cell proliferation, differentiation, adhesion, and migration. The standard CD44 isoform contains exons 1-5 and 16-20, while CD44 splice variants that contain variable exons are designated as CD44v. A selective splice variant of CD44 that contains exon 6 is called CD44v6. Since exon v6 is involved in cancer and has been suggested to be associated with the spread of metastasis, CD44v6 has been identified as a potential target for cancer treatment.

[0004] mAbs (BIWA-1 or VFF-18) against CD44v6 have been previously developed for the treatment of squamous cell carcinoma (WO97 / 21104), and humanized mAbs (BIWA-4 or bevacizumab) have been used as conjugate mAbs in the treatment of inoperable recurrent or metastatic head and neck cancers against CD44v6 (Postema EJ, et al. Journal of Nuclear Medicine 2003, 44(10):1690-9).

[0005] Despite previous developments, there is a need for new and improved binding proteins that target CD44v6 and can be used in medical procedures such as diagnosis and cancer treatment.

Summary of the Invention

[0006] The object of the present disclosure is to provide novel and enhanced binding molecules that can be used in medical procedures, diagnosis, and medical imaging. This object is achieved by a binding protein that specifically binds to CD44v6 and contains an antibody binding domain, the binding domain including a heavy chain variable domain (VH) and a light chain variable domain (VL), each containing three complementarity determining regions (CDRs), the amino acid sequences of the CDRs being selected from the group consisting of VHCDR1 defined by SEQ ID NO: 1, VHCDR2 defined by SEQ ID NO: 2, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 4, VLCDR2 defined by X1AS where X1 is T, A, or S, VLCDR3 defined by SEQ ID NO: 6, and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto, and the binding protein recognizes an epitope of CD44v6 defined by SEQ ID NO: (7). In some embodiments, the amino acid sequences of the CDRs of the binding protein are VHCDR1 defined by SEQ ID NO: 11, VHCDR2 defined by SEQ ID NO: 19, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 26, VLCDR2 defined by SEQ ID NO: TAS, and VLCDR3 defined by SEQ ID NO: 6.

[0007] In some embodiments, VHCDR1, VHCDR2 and VLCDR2 of the binding protein are present adjacent to specific framework amino acids, and the CDR and framework amino acid (faa) sequences are VHCDR1 and faa defined by SEQ ID NO: 8, VHCDR2 and faa defined by SEQ ID NO: 9, VLCDR2 and faa defined by SEQ ID NO: 10, and a CDR sequence having at least 95% identity, such as 96%, 97%, 98%, 99% or more, thereto.

[0008] In some aspects, the binding protein is a monoclonal antibody or an antigen-binding fragment selected from the group consisting of Fv fragments, Fab-like fragments and domain antibodies. In some embodiments, the Fv fragment is a scFv fragment. In some embodiments, the Fab-like fragment is a Fab or F(ab’)2 fragment. In some embodiments, the binding molecule is a monoclonal antibody of the IgG1 isotype such as an IgG1 LALA antibody or an IgG1 IAHA antibody. Typically, the binding protein is of human or human origin.

[0009] In some aspects, a conjugate binding protein is provided. The conjugate binding protein comprises (i) at least one binding protein and (ii) at least one agent.

[0010] The agent may be a therapeutic agent such as a cytotoxic agent. The cytotoxic agent is selected from radioisotopes, cell growth inhibitors, toxins, and chemotherapeutic agents. In some embodiments, the therapeutic agent is 177 a Lu radioisotope.

[0011] The agent may be a detectable agent such as a radioisotope, an enzyme, a fluorescent molecule, a pigment, digoxigenin, or biotin. In some embodiments, the detectable agent is 111 a radioisotope.

[0012] In some embodiments, the agent is linked to the binding protein via a linker in the form of a chelating agent. The chelating agent may be selected from the group consisting of derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), derivatives of deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), derivatives of (tBu)4(1-(1-carboxy-3-carboxy tert-butoxypropyl)-4,7,10-(carboxy tert-butoxymethyl)-1,4,7,10-tetraazacyclododecane) (DOTAGA), derivatives of 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), derivatives of 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0013] In some embodiments, engineered cells are provided. The cells may be engineered to express a chimeric antigen receptor (CAR), which includes an antigen-binding domain, a transmembrane domain connected to the antigen-binding domain by a hinge region, and an intracellular domain optionally connected to one or more co-stimulatory domains, and the antigen-binding domain includes the scFv fragment of the binding protein of the present disclosure.

[0014] According to some embodiments, a pharmaceutical composition is provided. The pharmaceutical composition includes the binding protein, composite binding protein, or engineered cells described above, and a pharmaceutically acceptable carrier or excipient.

[0015] The above-described binding protein, composite binding protein, engineered cell, or pharmaceutical composition may be for use in therapy. In some embodiments, the therapy is for the treatment of cancer, including advanced thyroid cancer, head and neck cancer, pancreatic cancer, squamous cell carcinoma, Hodgkin lymphoma, colorectal cancer, liver cancer, cervical cancer, gastric cancer, ovarian cancer, lung cancer, bladder cancer, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, breast cancer, hepatocellular carcinoma, and esophageal cancer, as well as metastatic cancer of the brain. In some embodiments, the cancer is advanced thyroid cancer.

[0016] According to some aspects, the present disclosure provides an in vitro (including in cellulo and ex vivo) method for detecting the expression of CD44 variant CD44v6, the method comprising: (i) contacting the above-described binding protein or composite binding protein with a biological sample such as a tissue sample or a liquid obtained from a subject, such that when the binding protein or composite binding protein is present in the biological sample, it binds to the epitope of CD44v6 defined by SEQ ID NO: 7; (ii) washing the biological sample to remove unbound binding protein or composite binding protein; and (iii) detecting any binding protein or composite binding protein bound to the epitope in the biological sample.

[0017] According to some aspects, the present disclosure provides an in vivo method for detecting the expression of CD44 variant CD44v6, the method comprising: (i) administering a composite binding protein to a subject, wherein the composite binding protein binds to the epitope of CD44v6 defined by SEQ ID NO: 7; and (ii) detecting that the composite binding protein has bound to cells expressing the epitope.

[0018] Other objects and advantages will become apparent to those skilled in the art upon consideration of the following detailed description, taken in conjunction with the accompanying exemplary drawings and the appended claims.

[0019] The above, as well as additional objects, features, and advantages of the concepts of the present invention, will be better understood through the following illustrative and non-limiting detailed description of different embodiments of the concepts of the present invention with reference to the accompanying drawings.

Brief Description of the Drawings

[0020]

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DETAILED DESCRIPTION OF THE INVENTION

[0021] The figures are not necessarily to scale and generally show only the parts necessary to illustrate the concept of the present invention, and other parts may be omitted or merely suggested.

[0022] The present disclosure relates to novel binding proteins such as monoclonal antibodies or antigen-binding fragments thereof that selectively bind to CD44v6, and composite binding proteins carrying therapeutic agents such as antibody-drug conjugates (ADCs). The binding protein, antibody, or binding antibody can be used for medical treatments such as cancer treatment or for imaging applications in in vitro and in vivo diagnosis. The binding protein can also be used to engineer cells to express a chimeric antigen receptor having the binding protein of the present disclosure as an antigen-binding domain.

[0023] The object of the present disclosure is to provide a novel and enhanced binding protein specific for CD44v6 that can be used in treatment, diagnosis, medical imaging, and cell engineering.

[0024] Aspects of the present disclosure will be more fully described below with reference to the accompanying drawings. However, the binding proteins, composite binding proteins / ADCs, and methods disclosed herein can be implemented in many different forms and should not be construed as limited to the aspects described herein. Like numbers in the drawings refer to like elements throughout.

[0025] The terms used in this specification are for the purpose of describing particular aspects of the present disclosure only and are not intended to limit the present disclosure. As used in this specification, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0026] In some embodiments, the non-limiting term "binding protein" is used. As used herein, the term "binding protein" is used to refer to a binding protein that includes a binding domain of an antibody (i.e., a binding domain obtained from, derived from, or based on the binding domain of an antibody). Thus, a binding protein is an antibody-based or antibody-like molecule that includes a binding site of an antibody or a binding site derived from an antibody. Thus, it is an immunoconjugate.

[0027] In some embodiments, the non-limiting terms "antibody" or "antigen-binding fragment thereof" are used. As used herein, the term "antibody" is used in the broadest sense to include both monoclonal and polyclonal antibodies. As is well known, an antibody is an immunoglobulin molecule that can specifically bind to a target (antigen) such as a protein, carbohydrate, polynucleotide, lipid, polypeptide, etc. via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" or "antigen-binding fragment thereof" includes not only full-length or intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof such as Fab, Fab’, F(ab’)2, Fab3, Fv and variants thereof, fusion proteins containing one or more antibody moieties, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetra-bodies, linear antibodies, single-chain antibodies, multispecific antibodies (such as bispecific antibodies), and other modified constructs of immunoglobulin molecules containing an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0028] As is known to those skilled in the art, an antibody is a protein that includes four polypeptide chains, namely, two heavy chains and two light chains. Usually, the heavy chains are identical to each other, and the light chains are identical to each other. The light chains are shorter (and thus lighter) than the heavy chains. The heavy chains contain four or five domains, with a variable (VH) domain at the N-terminus, followed by three or four constant domains (CH1, CH2, CH3, and CH4 if present, from the N-terminus to the C-terminus). The light chains contain two domains, with a variable (VL) domain at the N-terminus and a constant (CL) domain at the C-terminus. In the heavy chains, an unstructured hinge region is located between the CH1 domain and the CH2 domain. The two heavy chains of the antibody are linked by disulfide bonds formed between cysteine residues present in the hinge region, and each heavy chain is linked to one light chain by a disulfide bond between cysteine residues present in the CH1 and CL domains, respectively. In mammals, two types of light chains are produced, known as lambda (λ) and kappa (κ). In the case of kappa light chains, the variable and constant domains may be referred to as the Vκ domain and the Cκ domain, respectively. Whether the light chain is a λ light chain or a κ light chain is determined by its constant region, and the constant regions of λ and κ light chains are different, but the same for all light chains of the same type in any given species. Depending on the amino acid sequence of the constant domain of its heavy chain, immunoglobulins are assigned to different classes. There are six major classes of antibodies: IgA, IgD, IgE, IgG, IgM, and IgY, and some of these may be further classified into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. As used herein, the term "full-length antibody" refers to an antibody of any class, such as IgD, IgE, IgG, IgA, IgM, or IgY (or any of its subclasses). The term "antigen-binding fragment" refers to a part or region of an antibody molecule or its derivatives that retains all or an important part of the antigen-binding of the corresponding full-length antibody. In some embodiments, the heavy chain of the antibody may include VH+CH1+hinge+CH2+CH3, and the light chain VL+CL.In a preferred embodiment, the antibody has an IgG1 LALA format, where CH1 is defined by SEQ ID NO: 117, CH2 is defined by SEQ ID NO: 119, CH3 is defined by SEQ ID NO: 120, CL is defined by SEQ ID NO: 116, and the hinge is defined by SEQ ID NO: 118.

[0029] As briefly listed above, examples of antigen-binding fragments include: (1) a Fab fragment that is a monovalent fragment having a VL-CL chain and a VH-CH chain; (2) a Fab’ fragment that is a Fab fragment having a heavy-chain hinge region; (3) a dimer of Fab’ fragments linked by a heavy-chain hinge region, for example, linked by a disulfide bridge in the hinge region, which is an F(ab’)2 fragment; (4) an Fc fragment; (5) an Fv fragment that is the smallest antibody fragment having a VL domain and a VH domain of a single arm of an antibody; (6) a single-chain Fv (scFv) fragment in which the V H domain and the V L domain are linked by a peptide linker; (7) two V H(scFv)2, which comprises two VL domains that associate via two VH domains through disulfide bonds, and (8) antibody single variable domain (VH or VL) polypeptides that specifically bind to an antigen, although not limited thereto. Antigen-binding fragments can be prepared by conventional methods. For example, F(ab’)2 fragments can be generated by pepsin digestion of full-length antibody molecules, and Fab fragments can be generated by reducing the disulfide bonds of F(ab’)2 fragments. Alternatively, the fragments can be prepared by recombinant techniques by expressing the heavy chain fragment and the light chain fragment in a suitable host cell (e.g., E. coli, yeast, mammalian, plant, or insect cells), assembling them, and forming the desired antigen-binding fragment in vivo or in vitro. Single-chain antibodies can be prepared by recombinant techniques by linking the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region. For example, a flexible linker can be incorporated between the two variable regions. Thus, throughout the description, the general terms "binding protein" or "antibody" are used. These terms are used in the broadest sense and thus also incorporate all variants and fragments described above and below. In some embodiments, the binding protein is a monoclonal antibody or antigen-binding fragment selected from the group consisting of Fv fragments (e.g., single-chain Fv and disulfide-bonded Fv), Fab-like fragments (e.g., Fab fragments, Fab’ fragments, and F(ab)2 fragments), and domain antibodies (e.g., single VH variable domain or VL variable domain).

[0030] Thus, the constant region of the heavy chain is the same for all antibodies of any given isotype of a species, but differs between isotypes. The specificity of an antibody is determined by the sequence of its variable region. The sequence of the variable region differs between antibodies of the same type in any individual. In particular, both the light and heavy chains of an antibody contain three hypervariable complementarity-determining regions (CDRs). In a pair of light and heavy chains, the CDRs of the two chains form the antigen-binding site. The CDR sequences determine the specificity of the antibody. The pair of the light-chain variable region and the heavy-chain variable region containing the (antigen) binding site is known as the (antigen) binding domain. The three CDRs of the heavy chain are known as VHCDR1, VHCDR2, and VHCDR3 from the N-terminus to the C-terminus, and the three CDRs of the light chain are known as VLCDR1, VLCDR2, and VLCDR3 from the N-terminus to the C-terminus.

[0031] In antibodies, as described above, the CDR sequences are located in the variable domains of the heavy and light chains. The CDR sequences are located within the polypeptide framework and properly position the CDRs for antigen binding. Thus, the remaining portion of the variable domain (i.e., the portion of the variable domain sequence that does not form part of any CDR) constitutes the framework region. The N-terminus of the mature variable domain forms framework region 1 (FR1), the polypeptide sequence between CDR1 and CDR2 forms FR2, the polypeptide sequence between CDR2 and CDR3 forms FR3, and the polypeptide sequence linking CDR3 to the constant domain forms FR4. In the binding protein of the present invention, the variable region framework region can have any suitable amino acid sequence such that the binding protein binds to CD44v6 via its CDRs.

[0032] When the binding protein is an antibody, the antibody can be of any isotype and subtype. Thus, it can be an IgA, IgD, IgE, IgG, or IgM antibody. The heavy chain constant domains corresponding to the different isotypes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of the different isotypes of immunoglobulins are well known. In some embodiments, the antibody is an IgG antibody. As described above, there are four subtypes of IgG antibodies: IgG1, IgG2, IgG3, and IgG4. The IgG anti-CD44v6 antibody of the present invention can be of any IgG subtype, i.e., it can be an IgG1, IgG2, IgG3, or IgG4 antibody. In a preferred embodiment, the antibody is an IgG1 or IgG4 antibody such as an IgG1 LALA antibody and / or an IgG1 IAHA antibody. In IgG1 LALA, leucine (L) is replaced by alanine (A) at amino acid positions 234 and 235 in the Fc region. The LALA mutation removes Fc-mediated binding to Fcγ receptors on immune cells and reduces effector function. Thus, removing the binding avoids the immune response, i.e., reduces the immunogenicity mediated by Fc effector functions such as ADCC and CDC, and reduces the risk of the biopharmaceutical causing undesirable off-target and on-target side effects. In the IAHA mutation, alanine (A) is replaced by isoleucine (I) and alanine (A) is replaced by histidine (H) at amino acid positions 253 and 310 in the Fc region of the antibody. Thus, in this embodiment, the binding protein can be an antibody designed as a "silent Fc" antibody without Fc-gamma receptor interaction via the LALA mutation to remove ADCC / CDC activity. Furthermore, these antibodies are characterized by a low risk of immunogenicity, as evaluated by in-silico T cell epitope prediction analysis. The IAHA double mutation in the Fc region reduces the interaction with FcRn (FcRn binding), and as a result, shortens the circulation time in the blood (DOI: 10.1080 / 19420862.2016.1156285).

[0033] As detailed above, the antibody light chain belongs to either the kappa (κ) type or the lambda (λ) type. The binding protein of the present invention may include a κ light chain or a λ light chain. In certain embodiments, the binding protein of the present invention includes a κ light chain.

[0034] Alternatively, the binding protein may be a binding fragment of an antibody (i.e., an antibody fragment), i.e., a fragment that retains the ability of the antibody to specifically bind to CD44v6. Such fragments are well known and may include, for example, Fab’, Fab, F(ab’)2, Fv, Fd, or dAb fragments, which can be prepared according to techniques well known in the art.

[0035] The Fab fragment consists of the antigen-binding domain of the antibody, i.e., it can be seen that each antibody contains two Fab fragments, each consisting of a light chain and the N-terminal section of the heavy chain linked thereto. Thus, the Fab fragment includes the entire light chain and the V H and C H 1 domain of the heavy chain to which it binds. The Fab fragment can be obtained by digesting the antibody with papain.

[0036] The F(ab’)2 fragment consists of two Fab fragments of the antibody and the hinge region of the heavy domain containing a disulfide bond that links the two heavy chains together. In other words, the F(ab’)2 fragment can be regarded as two covalently linked Fab fragments. The F(ab’)2 fragment can be obtained by digesting the antibody with pepsin. Reduction of the F(ab’)2 fragment generates two Fab’ fragments that can be regarded as Fab fragments containing additional sulfhydryl groups useful for binding the fragments to other molecules.

[0037] Alternatively, the binding protein may be a synthetic construct or an artificial construct, i.e., an antibody-like molecule that contains a binding domain but is genetically engineered or artificially constructed. This includes, in addition to chimeric antibodies or CDR-grafted antibodies, single-chain antibodies and scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, single-domain antibodies (DAB), TandAbs dimers, and V HIt includes other constructs such as heavy chain antibodies such as H. In certain embodiments, the artificial construct is a single-chain variable fragment (scFv). The scFv is a fusion protein in which a single polypeptide contains both the V H domain and the V L domain. The scFv fragment generally contains a peptide linker that covalently links the V H region and the V L region, contributing to the stability of the molecule. The linker can contain from 1 to 20 amino acids, for example, 1, 2, 3, or 4 amino acids, 5, 10, or 15 amino acids, or other intermediate numbers in the range of 1 to 20 as convenient. The peptide linker can be formed from any commonly convenient amino acid residue such as glycine and / or serine, as is well known to those skilled in the art. However, the presence of a linker is not essential, and the V L domain can be linked to the V H domain by a peptide bond. The scFv typically contains a V L region linked to the V H region by a linker sequence from the N-terminus to the C-terminus. The preparation of scFv molecules is well known in the art.

[0038] The binding domain of an antibody is composed of a light chain variable domain and a heavy chain variable domain (a classical bivalent antibody has two binding domains). Thus, the binding protein can be a natural antibody or a fragment thereof, or an artificial or synthetic antibody, or an antibody construct, or a derivative (e.g., a single-chain antibody as further discussed below). In summary, the binding protein of the present invention contains the binding domain of an antibody, and the binding domain of the antibody contains a light chain variable domain and a heavy chain variable domain.

[0039] As used herein, the term "capable of binding to X" (where X is an antigen) refers to the property of an antibody or a binding fragment thereof that can be tested, for example, by ELISA, by the use of surface plasmon resonance (SPR) technology, by the use of kinetic exclusion assay (KinExA®) or by the use of biolayer interferometry (BLI). Those skilled in the art know the said methods and other methods.

[0040] The term "specificity" of a binding protein for a target, also called "selectivity", refers to a binding protein that binds to the target with high affinity but usually does not bind to other antigens. A selective or specific binding protein / antibody does not cross-react or cross-reacts to a low degree with targets other than the antigen of interest. Thus, "specifically" binding means that the binding protein binds to its target (i.e., CD44v6) in a way that distinguishes it from binding to non-target molecules, more specifically, that the binding protein binds to its target (CD44v6) with a higher binding affinity than when it binds to other molecules. That is, the binding protein does not bind to other non-target molecules, or binds to them to a much lesser or insignificant extent, or binds to such other molecules with a lower affinity than when it binds to CD44v6. Alternatively, a binding protein that "specifically binds" to CD44v6 may be said to "target" or "recognize" CD44v6. In other words, CD44v6 is the antigen of the binding protein of the present invention, and thus this binding protein is an "antigen-binding protein" in the sense that it binds to CD44v6 as an antigen.

[0041] The binding protein of the present invention can bind to a drug to form a conjugate binding protein. In one aspect, the drug may be a detectable drug such as a certain label, and the conjugate binding protein is used for imaging. In other aspects, the conjugate binding protein can be formed by linking the binding protein to a therapeutic agent. In this case, the conjugate binding protein can be formed as an antibody-drug conjugate (ADC) or can function similarly to an antibody-drug conjugate (ADC) and can be used for treatment.

[0042] By linking to monoclonal antibodies via biodegradable and stable linkers, ADCs can deliver very potent cytotoxic anti-tumor agents to cancer cells and distinguish cancer from normal tissues. Thus, ADCs can combine a monoclonal antibody specific for a surface antigen present on specific tumor cells with a very potent anti-cancer agent linked via a chemical linker. ADCs typically consist of an antibody specific for a target-related antigen (selective for tumor-related antigens that are restricted or not expressed in normal healthy cells), a payload designed to kill the target cancer cells (a potent cytotoxic drug designed to induce target cell death after being taken up and released by tumor cells), and a chemical linker for conjugating the payload to the antibody (a linker that is stable in circulation but releases the cytotoxic drug inside target cells and can be either cleavable or non-cleavable). ADCs are usually monoclonal antibodies covalently conjugated to small molecule drugs that target specific cancer cells, reducing systemic toxicity, enhancing the cell-killing ability of the monoclonal antibody, conferring higher tumor selectivity, and as a result, providing higher tumor selectivity and limited systemic exposure, and thus, higher drug resistance. ADCs deliver therapeutic agents via a linker conjugated to a monoclonal antibody that binds to a specific target expressed on cancer cells. After binding to the target (cancer protein or receptor), the ADC releases the cytotoxic drug into the cancer cells. The chemical "linker" that conjugates both the antibody and the cytotoxic drug is highly stable and prevents cleavage (fission) before the ADC enters the tumor. The anti-cancer agent penetrates the tumor and causes cell death by damaging the DNA of cancer cells or preventing the formation and spread of new cancer cells. Thus, ADCs bind to and internalize proteins on the surface of cancer cells and release drugs during internalization to kill cancer cells. A schematic diagram of an ADC is shown in Figure 1, showing an mAb carrying a payload via a linker, where the drug may be present in multiple copies as indicated by the letter n (the number of individual drugs conjugated to the mAb).

[0043] As used herein, "therapy" means the treatment of any medical condition. Such treatment can be prophylactic (i.e., preventive), curative (or treatment aimed at cure), or palliative (i.e., treatment aimed at simply limiting, alleviating, or improving the symptoms of a condition). Thus, "therapy" or "treatment" of a disorder such as cancer / cancerous tumor by a binding protein or a complex binding protein as used herein refers to preventing or improving a particular disorder or medical condition, or curing it. In the case of cancer and tumors, treatment can cause the current tumor to shrink or disappear, or can stop or prevent further spread of the tumor. The amount sufficient to achieve this is defined as a "therapeutically effective amount". The effective amount for a given purpose varies depending on the disease or condition being treated, its severity, the size / weight and general condition of the subject. Thus, the binding proteins or complex binding proteins described herein can be used to improve any condition in which the target antigen is expressed / overexpressed in a subject, and can be administered systemically or locally by any suitable method known in the art. As used herein, a subject refers to any mammal, such as livestock animals such as cows, horses, sheep, pigs or goats, pet animals such as rabbits, cats or dogs, or primates such as monkeys, chimpanzees, gorillas, humans, etc. Preferably, the subject is a human.

[0044] Prophylactic treatment can include prevention of a condition, or delay in the progression or onset of a condition. For example, a complex binding protein can be used to prevent an infection, or to reduce the degree to which an infection may develop, or to prevent, delay or reduce the degree of cancer development or recurrence, or to prevent or reduce the degree of metastasis, for example.

[0045] As used herein, the term "diagnosis" or "diagnosing" means the process of determining whether a disease or condition, such as cancer, is present in a test subject. A diagnosis can be regarded, in the sense of a diagnostic procedure, as an attempt to classify an individual's condition into distinct different categories that enables medical decisions regarding treatment and prognosis. Thereafter, a diagnostic opinion is often explained with respect to the disease or other condition. The first task is to detect medical signs for performing a diagnostic procedure, such as detecting any deviation from a state known to be normal. The diagnostic procedure can be performed in vitro using the binding protein or complex binding protein of the present specification in, for example, an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry, or Western blotting, or can be performed in vivo, in which case the binding protein can carry a detectable agent such as a label for in vivo imaging of a tumor. The binding protein can be combined with a radioisotope for imaging such as immunoscintigraphy.

[0046] Accordingly, the binding protein or complex binding protein can be used in medical imaging that can be used for diagnosis or prognosis. As used herein, the term "prognosis" or "prognostic prediction" means the prediction or estimation of the likelihood of recovery or survival from a disease when treated. The prognosis of cancer can vary depending on several factors, such as the stage of the disease at diagnosis, the type and subtype of cancer, the molecular profile of the tumor, and even gender. Diagnosis / prognosis can also be used to distinguish patients into different subgroups based on the nature or aggressiveness of a disorder such as cancer. Further, it can be used to determine a treatment plan, i.e., a dosing plan. If the complex binding protein contains a radionuclide, dosimetry may be used, in which case the diagnosis / prognosis may involve determining the radiation dose by measurement, calculation, or a combination of measurement and calculation of the absorbed dose (the radiation energy accumulated in the tissue divided by the mass of the tissue) due to binding / uptake / internalization of the complex binding protein.

[0047] The CD44 cell surface glycoprotein plays a role in promoting cell-cell and cell-matrix interactions through its affinity for hyaluronic acid. Furthermore, it is known to confer adhesion and is also involved in the assembly of growth factors at the cell surface such as EGFR and HER4. Protein dysfunction and / or changes in expression cause various pathogenic phenotypes. The term "CD44" refers to CD44 derived from any species. Thus, it can be the human CD44 or its equivalent or corresponding molecule in other species, most notably other mammals. Human CD44 is assigned the UniProt accession number P16070. CD44 transcripts undergo complex alternative splicing to generate functionally distinct isoforms. As shown in Figure 2, CD44s is the standard isoform and CD44v is the variant. The CD44 protein is encoded by a single highly conserved gene consisting of 20 exons, and exons 1-5, 16-18, and 20 encode the minimal isoform CD44, the standard isoform CD44, and the hematopoietic isoform CD44. The exons lacking in CD44 are called CD44 exon isoform variants (referred to as CD44v1-10). Thus, the 10 variant exons 6-15 (v1-v10) in the middle of the CD44 gene can be selectively spliced to generate various CD44 variant (CD44v) isoforms, one of which is CD44 variant 6, isoform CD44v6. Furthermore, 19 different splice variants generated by alternative splicing of CD44 mRNA have been discovered, and they are all expressed at various levels in different tissues, but the roles of these variants are not fully understood. Preferably, CD44 is human CD44v6 such that the binding protein of the present invention specifically binds to human CD44v6.

[0048] CD44v6 is a non-internalizing cancer-related splice variant of CD44 (hyaluronic acid receptor). High expression of CD44v6 has been found in several cancers and is associated with poor prognosis and rapidly progressive disease. The expression of CD44v6 in normal tissues is restricted to the spinous epithelial layer above the basal layer, more specifically keratinocytes. Thus, while CD44 is widely expressed in most vertebrate cells, the expression of CD44v6 is restricted to only a few tissues and is thought to be associated with tumor progression and metastasis. As a result, the low expression levels in healthy tissues, combined with overexpression in various different cancer types, make CD44v6 a promising target for molecular radiotherapy.

[0049] The binding proteins herein can be linked, for example, by gene fusion, complexed or chemically linked to a "drug", i.e., a moiety having specific properties, or form a complex or fusion binding protein, where the binding protein and the drug may be directly linked to each other, as in the case of binding an iodine (I) radioisotope to the binding protein, or may be linked via a linker, as in the case of binding a lutetium (Lu) to the binding protein (i.e., indirectly linked to each other). The drug may be bound using a chelating agent (in the form of indirect binding), where the chelating agent is linked to the binding protein and chelates the drug. The drug may be a radioisotope, a photoactivatable compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytotoxic drug, a prodrug, a binding molecule with different specificities, a cytokine or other immunomodulatory or cytotoxic polypeptide.

[0050] The agent may be a therapeutic agent or a detectable contrast agent. The therapeutic agent or active pharmaceutical ingredient (API) linked, conjugated, or linked to the binding protein may be a cytotoxic agent comprising or consisting of one or more radioisotopes and / or one or more cytotoxic drugs. The term "radioisotope" may also be referred to as a "radionuclide" and refers to a nuclide that has excess nuclear energy, is unstable, and is prone to radioactive decay. The one or more radioisotopes are each independently selected from the group consisting of beta emitters, Auger emitters, conversion electron emitters, alpha emitters, and low photon energy emitters, and each independently can have a pattern of local absorption energy release that produces a high dose absorption in the vicinity of the agent. The one or more radioisotopes are 90 Y, 32 P, 186 Re / 188 Re, 166 Ho, 76 As / 77 As, 153 long-range beta emitters such as Sm, 131 I, 177 Lu, 67 Cu, 161 Tb, 47 medium-range beta emitters such as Sc, 45 Ca, 35 S, 14 low-energy beta emitters such as C, 51 Cr, 67 Ga, 99 TC m , 111 In, 123 I, 125 I, 201 conversion or Auger emitters such as TI, and 212 Bi, 212 Pb, 213 Bi, 223 Ac, 225 Ac, 227 Th, 149 Tb, and 211 each independently selected from the group consisting of alpha emitters such as At.

[0051] The binding protein or conjugate binding protein comprising the therapeutic agent of the present disclosure can be used in the medicine and therapy of any condition or disorder showing CD44v6 expression. In some embodiments, the condition or disorder is cancer such as advanced thyroid cancer, head and neck cancer, pancreatic cancer, squamous cell carcinoma, Hodgkin's lymphoma, colorectal cancer, liver cancer, cervical cancer, gastric cancer, ovarian cancer, lung cancer, bladder cancer, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, breast cancer, hepatocellular carcinoma, and esophageal cancer, and metastatic cancer of the brain including metastatic forms of the other cancers even when metastases have already formed. Since CD44v6 is associated with angiogenesis, i.e., the formation of new blood vessels from existing blood vessels, which is essential for tumor growth and other conditions, this binding protein may also act as an angiogenesis inhibitor in the treatment of cancer and other angiogenesis-related disorders.

[0052] In other aspects, the binding protein may be linked to a contrast agent. Molecular imaging combining the contrast agent and the targeting moiety of the binding protein form can be used to specifically image the affected site in the body. The binding protein can be used in molecular imaging for targeting contrast agents such as radionuclides to target cells in vivo. This enables the diagnosis and prediction of response of any tissue and disease in which the antigen is expressed / overexpressed, since the progression of the disease can be monitored and the response to a specific therapeutic agent can be predicted. The imaging / detectable agent can be, inter alia, a radioisotope, an enzyme, a fluorescent molecule, a dye, digoxigenin, and biotin. The detectable agent can be detectable by imaging techniques such as SPECT, PET, MRI, optical or ultrasonic imaging. When the detectable agent is a radioisotope, they are 111 In, 99m Tc, 67 Ga, 68 Ga, 72 As, 89 Zr, 123 I, 125 I, 124 I, 47 Sc and 201 TI and can be selected from. The conjugate binding protein is 86 γ / 90 γ, 111 In / 177 Lu, or 125 I / 211 It may include pairs of detectable and cytotoxic radioisotopes such as At, which can act simultaneously in a multimodal manner as a detectable agent and as a cytotoxic agent.

[0053] The binding protein has been shown to bind to CD44v6 with high affinity and can thus be used as a single cancer therapeutic agent or as a conjugate binding protein, as described above. The binding proteins of the present disclosure can also be used to design chimeric antigen receptors (CARs) against CD44v6 to obtain CD44v6-targeted CAR cells such as CAR-T cells. These CD44v6-targeted CAR T cells can then be used in therapies to eliminate CD44v6-expressing cells such as cancer cells. For example, CAR T cells are produced by isolating T cells from a subject and inserting the gene for the CAR into the T cells to produce CAR T cells that express the CAR protein. The CAR is a hybrid of an antibody receptor that includes a T cell and four different regions, an extracellular domain that recognizes an antigen (usually the scFv fragment of an antibody) connected to a transmembrane domain by a hinge (spacer), the transmembrane domain has a hydrophobic alpha-helical structure, the transmembrane domain is connected to an endodomain (intracellular domain), which undergoes a conformational change after antigen recognition and triggers downstream signaling pathways to induce an immune response. The endodomain may also include one or more co-stimulatory domains to enhance anti-tumor activity. Accordingly, the present disclosure provides cells engineered to express a chimeric antigen receptor (CAR), the CAR comprising an antigen-binding domain, a transmembrane domain connected to the antigen-binding domain by a hinge region, and an intracellular domain optionally connected to one or more co-stimulatory domains, the antigen-binding domain comprising the scFv fragment of the binding protein. The cells may be immune effector cells such as human cells, T cells, NK cells or macrophages.

[0054] The present invention provides novel and enhanced binding proteins that bind to human CD44v6. The binding proteins of the present invention have been evaluated for binding affinity and therapeutic effect and have been found to provide improved performance compared to other binding proteins in the art. One object of the present disclosure is to provide novel and enhanced tumor targeting agents for improving therapies such as radiotherapy for cancer that can discover and characterize tumor / cancer cells and also destroy them. The binding protein can be linked / linked to one or more agents as a payload as shown in FIG. 2, and by recognizing and binding to a target present / overexpressed on the tumor cell surface, when the agent is, for example, a contrast agent or a detectable agent, it can identify the location of the tumor cell, or carry a therapeutic agent such as one or more radioisotopes to kill the target cells. The radiation is an effector as shown in FIG. 3, and the radioisotope-conjugated binding protein may be called a radiopharmaceutical. The binding protein is introduced into the body by various means (such as injection or oral ingestion), localizes a specific antigen (CD44v6), binds to the receptor, stays on the tumor cell surface or is taken up into the tumor cell, exposes the radioisotope payload, where the radiation emitted from the radioisotope destroys the cancer cells, which is called molecular radiotherapy (MRT) or radionuclide therapy (RNT).

[0055] As discussed above and below, the binding proteins of the present disclosure can be used to treat many disorders associated with the expression / overexpression of the target antigen CD44v6. In particular, subjects suffering from advanced thyroid cancer (TC), including thyroid carcinoma, especially anaplastic (ATC) and radioiodine - refractory TC, are rare diseases that are resistant to standard cancer treatments and have no effective therapies, so they may benefit greatly from these treatments. Thyroid cancer is in most cases a curable disease by surgical treatment followed by adjuvant treatment with radioactive iodine. However, there are also many cases that do not respond to current treatments, and the prognosis of these patients is very poor. The median survival period after diagnosis of ATC is only 5 months, so the unmet clinical needs are very large. CD44v6 is highly expressed on tumor cells in a significant proportion of these patients and has thus been established as a relevant target. Since tumor cells express CD44v6, advanced thyroid cancer may be treatable with CD44v6 - targeted radiopharmaceuticals effective for molecular radiotherapy, as shown in Figure 4. Binding proteins of the present disclosure, such as CD44v6 - specific mAbs, are labeled with radionuclides to deliver a therapeutic dose of radioactivity to CD44v6 - expressing tumor cells while sparing normal non - CD44v6 - expressing tissues. In some embodiments, the radiolabeled binding protein conjugate contains a DOTA chelate with a therapeutic radionuclide 177 Lu or an imaging 111 In. 111 In / 177 Lu - DOTA is a well - characterized theranostic pair complex, and Lutathera ( 177 Lu - DOTATATE) is approved in both the United States and Europe for the treatment of neuroendocrine cancer.

[0056] This target has already been clinically validated for radioimmunotherapy, is specifically taken up by tumors, and has good tolerability. The experimental data summarized in this specification are further described in detail in the Examples section below. PK / PD studies have been conducted in mice, suggesting an appropriate half-life, normal tissue distribution, and tumor targeting ability of the binding protein. The experimental data also confirm, as shown in Figure 5a, that there is a dramatic effect without toxicity being observed in ATC-bearing mice, and Figure 5b shows the tumor volume of mice up to 40 days of treatment with a single administration of the radiopharmaceutical (having 177 Lu) of the present disclosure as a therapeutic agent, and the survival rate after such a single administration. Clear and thoroughly characterized antigen-specific binding of this binding protein, showing no signs of off-target binding or cross-reactivity, has been demonstrated. The radiolabeled conjugate has been evaluated in three species (mice, rabbits, cynomolgus monkeys), and in the dosimetry evaluation, good dosimetry has been demonstrated with the bone marrow being the dose-limiting organ as expected. These studies have also verified low uptake in CD44v6-specific normal tissues in rabbit and cynomolgus monkey studies, with no accumulation of radioactivity in normal tissues or active uptake in the bone marrow being observed, although the data are not shown.

[0057] The experimental data of the target binding specificity test shows binding of the binding protein and the conjugate (radio-labeled) binding protein that has high specificity and affinity for CD44v6 and a low risk of off-target binding, as will be described in more detail in the sections of the following examples, where such occurrences were not shown in in-silico and in-vitro evaluations of off-target binding and selectivity. In-silico, in-vitro, and in-vivo toxicology studies using three different animal models have demonstrated no toxic effects by the corresponding therapeutic levels of the unlabeled antibody, a low risk of antibody-mediated effects such as T cell activation, and a low risk of off-target binding. Pharmacokinetic evaluations in three different animal models also show feasible dosimetry and pharmacokinetic characteristics of the conjugate. SPR measurements have demonstrated specific binding to CD44v6 with low nanomolar affinity (regardless of the presence of the DOTA conjugate), and mapping of the binding epitope verified by SPR measurements indicates that the binding protein binds to the epitope defined by SEQ ID NO: 7. Evaluation of species specificity shows binding to the target of rabbit, cynomolgus monkey, and human CD44v6-peptides, verified by SPR measurements.

[0058] Accordingly, the binding protein of the present invention can be linked / conjugated to an agent for imaging and / or therapy. As an example, 177 The Lu conjugate binding protein may provide effective treatment of CD44v6-expressing radioiodine-refractory thyroid cancer. Such conjugate binding protein has been shown to bind to CD44v6 with high affinity, as demonstrated by radioimmunoassay against cultured thyroid cancer cells and squamous cell carcinoma cells. Real-time kinetic measurements against cultured thyroid cancer cells have demonstrated that the conjugate binding protein specifically binds to CD44v6 with high affinity to antigen-positive cells and does not bind to antigen-negative cells. In xenograft mouse models with 5 and 3 different tumor target expressions respectively 125 I / 177In the in vivo biodistribution evaluation of the Lu conjugate protein, antigen-dependent tumor uptake was shown, and no off-target binding was shown.

[0059] In xenografts of thyroid cancer mice 177 In the pharmacodynamic investigation and treatment data of the Lu conjugate antibody, no toxicity was observed with a single administration of 16.5 MBq / 50 μg of the radiolabeled antibody. 177 A Lu-dependent and antigen-specific therapeutic effect (decrease in tumor growth and even complete remission) was demonstrated. Data from two thyroid cancer mouse models show a dose-dependent and antigen-specific therapeutic effect without signs of toxicity (i.e., no weight loss or change in behavior of the mice). The experimental data further show a radiation delivery capacity equivalent to or greater than that of the clinically tested monoclonal antibody BIWA-4 with respect to in vitro and in vivo cell uptake. In addition to improved antigen affinity, more appropriate radionuclide labeling, and a fully human format, the LALA mutation introduced into the conjugate protein ensures the lack of ADCC / CDC function and a decrease in in vivo off-target uptake in the liver and the like. SPR measurements of the conjugate proteins of the present disclosure demonstrate a significant decrease in FcγR1 binding to the silenced ADCC / CDC of the LALA construct. These improvements result in clear advantages compared to previous humanized antibody-based CD44v6-targeted radiopharmaceuticals. In this experiment, 177 Lu is used as the therapeutic radionuclide, but any other radionuclide suitable for use in such treatments can also be used, and it is expected that similar results will be obtained.

[0060] Accordingly, the present invention provides a novel and enhanced binding protein that binds to human CD44v6. Exon 6 of the human CD44 gene has the amino acid sequence QATPSSTTEETATQKEQWFGNRWHEGYRQTPREDSHSTTGTAA (SEQ ID NO: 115). The binding protein is specific for an epitope encoded by exon v6 of CD44, particularly an epitope of the amino acid sequence WFGNRW (SEQ ID NO: 7). The binding protein of the present invention comprises an antibody binding domain, which contains a heavy chain variable domain (VH) and a light chain variable domain (VL), or derivatives thereof, and VH and VL each contain three complementarity determining regions (CDRs), i.e., a total of six CDRs, and the binding protein specifically binds to CD44v6, and more specifically, recognizes an epitope of CD44v6 defined by SEQ ID NO: (7). In some embodiments, the present invention relates to 21 binding protein variants having similar sequences and all recognizing the same epitope. The epitope to which the present binding protein binds is shown in FIG. 6 as compared to the BIWA-4 epitope WFGNRWHEGY (SEQ ID NO: 5). Accordingly, the binding protein may comprise an antibody binding domain containing a heavy chain variable domain (VH) and a light chain variable domain (VL), or any derivative thereof, and the derivative may be a single domain antibody containing a single monomeric variable antibody domain. In some embodiments, the binding protein is an IgG antibody lacking a light chain and consists of two heavy chains bound to the variable domain (V H H).

[0061] The light chain and heavy chain variable domains each contain three CDRs. The light chain variable domain contains VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable domain contains VHCDR1, VHCDR2 and VHCDR3. The six CDRs are VHCDR1 defined by SEQ ID NO: 1, VHCDR2 defined by SEQ ID NO: 2, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 4, A VLCDR2 defined by X1AS, where X1 can be T, A, or S It has an amino acid sequence of VLCDR3 defined by SEQ ID NO: 6. Here, VHCDR3 and VLCDR3 are identical in all 21 binding protein variants, but VHCDR1, VHCDR2, VLCDR1, and VLCDR2 contain some mutations as shown in Table 1 below. In addition to the CDRs according to SEQ ID NOs: 1 to 6, the present invention also encompasses CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity that also bind to the same epitope of CD44v6.

[0062] [Table 1]

[0063] The sequences of the CDRs of the binding protein are shown in Table 1 in one-letter amino acid code, where VHCDR1, VHCDR2, VLCDR1, and VLCDR2 contain sequence variations among the 21 binding protein variants as indicated by X. In VHCDR1, X3 can be S or T, X5 can be S, R, or G, X6 can be S or N, and X7 can be Y or F. In VHCDR2, X3 can be A or G, X4 can be S or G, and X6 can be T, S, Y, R, or G. In VLCDR1, X2 can be S, N, or T, X4 can be A, S, or G, and X5 can be S or N. In VLCDR2, X1 can be A, S, or T such that VLCDR2 is AAS, SAS, or TAS.

[0064] In some embodiments, some CDRs are surrounded by specific amino acids called framework amino acids (faa), and these amino acids are conserved among different binding proteins with some minor variations. Thus, VHCDR1, VHCDR2, and VLCDR2 of the binding protein can be present adjacent to specific framework amino acids, and the CDR and framework amino acid sequences are VHCDR1 and faa defined by SEQ ID NO: 8, and VHCDR2 and faa defined by SEQ ID NO: 9, and VLCDR2 and faa defined by SEQ ID NO: 10, and are selected from the group consisting of, as shown in Table 2 below. In addition to the CDR + faa according to SEQ ID NOS: 8 to 10, the present invention also encompasses CDR sequences having 95% or more, for example 96%, 97%, 98%, 99% or more identity, which also bind to the same epitope of CD44v6.

[0065]

Table 2

[0066] In the case of VHCDR1 and faa, the sequence is the same as VHCDR1, but two framework amino acids (MS) are added at the end. In VHCDR2 and faa, one additional faa is added at each of the beginning and end of the sequence, X1 is A or T, X 10 can be Y or F. The remaining unknowns in the middle correspond to the VHCDR2 sequence in Table 1 above. In VLCDR2 and faa, X4 of the additional faa can be S, T, N or I, and the remaining unknowns correspond to the sequences in Table 1 above.

[0067] In some embodiments, 21 different binding proteins having the combinations of individual CDRs shown in Tables 3 - 4 below are presented, where U - MN114 - 19 is the parent clone and the other 20 binding proteins are its affinity - matured versions.

[0068]

Table 3

[0069] Table 3 shows the CDR H1 and H2 of each of the 21 different binding proteins. The CDR (VHCDR3) of H3 for all 21 binding proteins is defined by the sequence ARHYYSDSDYRSSAAMDY (SEQ ID NO: 3).

[0070]

Table 4

[0071] Table 4 shows the CDR L1, L2, and L3 of each of the 21 different binding proteins.

[0072] In some embodiments, combinations of the above individual CDRs and 21 different binding proteins having the framework amino acids around VHCDR1, VHCDR2, and VLCDR2 shown in Tables 5 and 6 below are presented.

[0073]

Table 5-1

[0074]

Table 5-2

[0075]

Table 6

[0076] The binding proteins of the present invention can be synthesized by any method known in the art. Preferably, the binding protein is synthesized using a protein expression system such as a cell expression system using prokaryotic (e.g., bacterial) cells or eukaryotic (e.g., yeast, fungus, insect, or mammalian) cells. An alternative protein expression system is a cell-free in vitro expression system in which the nucleotide sequence encoding the binding protein is transcribed into mRNA and the mRNA is translated into protein in vitro. Cell-free expression system kits are widely available and can be purchased, for example, from Thermo Fisher Scientific. Alternatively, the binding protein can be chemically synthesized in a non-biological system. Liquid phase synthesis or solid phase synthesis can be used to form the binding proteins of the present invention or to generate the polypeptides contained within the binding proteins of the present invention.

[0077] One skilled in the art can easily generate the binding protein using appropriate methodologies common in the art. In particular, the binding protein can be recombinantly expressed in mammalian cells such as CHO cells. The binding protein synthesized in a protein expression system can be purified using standard techniques in the art. For example, it can be synthesized using an affinity tag and purified by affinity chromatography. When the binding protein is an antibody, it can be purified using affinity chromatography with one or more antibody-binding proteins such as Protein G, Protein A, Protein A / G, Protein L, etc.

[0078] As described above, the binding protein is an antibody-based or antibody-like molecule. Thus, the binding protein can be a natural antibody or a fragment thereof, or an artificial or synthetic antibody, or an antibody construct or derivative (e.g., single-chain antibody). In a preferred embodiment, the binding protein is a human protein (derived from human rather than humanized), particularly a human monoclonal antibody, antibody fragment or scFv. A human binding protein can include a VH region and a VL region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences, and can also include a human constant region if the protein includes a constant region. However, such a protein may contain amino acids not encoded by human germline Ig sequences, such as mutations introduced by random or site-directed mutagenesis.

[0079] As detailed above, the binding protein of the present invention includes a binding domain that includes the binding domain of an antibody in which the heavy chain variable domain (or variable region) and the light chain variable domain are included. Thus, in certain embodiments, the binding protein of the present invention is (i) a heavy chain variable domain (VH) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 35 to 54 and 147, or a variant thereof (or consisting thereof), and (ii) It includes (or consists of) a light chain variable domain (VL) containing the amino acid sequence described in any one of SEQ ID NOs: 55 to 74 and 148 or a variant thereof.

[0080] In some embodiments, the binding protein includes a heavy chain and a light chain containing a variable region and a constant region, and the binding protein of the present invention (i) a heavy chain containing the amino acid sequence described in any one of SEQ ID NOs: 34, 75 to 93, and 149, or a variant thereof (consisting of the same); (ii) a light chain containing the amino acid sequence described in any one of SEQ ID NOs: 94 to 113 and 150, or a variant thereof (consisting of the same).

[0081] The variant is defined as a sequence having at least 80% identity thereto, such as 85%, 90%, 95% or more. This means that the CDR sequence of the variant has not changed in consideration of the antibody variant defined by the VH or VL domain, i.e., it does not contain sequence variations, or the sequence variations of the CDR amino acid sequence are at most 5%, such as 4%, 3%, 2%, 1% or less, or the CDR sequence variation has at least 95% sequence identity to the defined sequence, such as 96%, 97%, 98%, 99% or more. Binding proteins having variants of the variable domain and / or constant domain sequences are functional variants having the above activities (i.e., they specifically bind to the defined epitope of CD44v6). The variant sequence may be modified from the native sequence by substitution, insertion and / or deletion of one or more amino acids.

[0082] Sequence identity can be evaluated by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs that create pairwise or multiple alignments of the sequences are useful. For example, EMBOSS Needle or EMBOSS Stretcher (both Rice, P. et al., Trends Genet., 16, (6) pp276-277, 2000) can be used for pairwise alignment, while Clustal Omega (Sievers F et al., Mol. Syst. Biol. 7:539, 2011) or MUSCLE (Edgar, R.C., Nucleic Acids Res. 32(5):1792-1797, 2004) can be used for multiple sequence alignment, although other suitable programs can be used. Whether the alignment is pairwise or multiple, it needs to be performed globally (i.e., over the entire reference sequence) rather than locally. The sequence alignment and % identity calculation may be determined using, for example, standard Clustal Omega parameters, i.e., matrix Gonnet, gap start penalty 6, gap extension penalty 1. Alternatively, standard EMBOSS Needle parameters, i.e., matrix BLOSUM62, gap start penalty 10, gap extension penalty 0.5, may be used. Alternatively, any other suitable parameters may be used.

[0083] In some embodiments, the disclosure encompasses conjugate proteins such as antibody-drug conjugates. Accordingly, the disclosure provides a conjugate protein comprising (i) at least one binding protein described above and below, and (ii) at least one agent, wherein at least one agent is linked to the binding protein and the binding protein and the agent are directly or indirectly linked.

[0084] In a further aspect, the present invention provides a pharmaceutical composition comprising the binding protein of the present invention as described above, or the composite binding protein as described above, or the engineered CAR cell as described above. Further, the pharmaceutical composition also comprises at least one pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable carrier or excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc. that are physiologically compatible.

[0085] Preferably, the carrier or excipient is suitable for parenteral administration, such as intradermal administration, intravenous administration, intramuscular administration, or subcutaneous administration (e.g., by injection or infusion). Depending on the route of administration, the binding protein or composite binding protein may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate or denature it.

[0086] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in pharmaceutical compositions, kits, and products include water, buffered water, and physiological saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferred to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, etc., sodium chloride, etc.

[0087] A pharmaceutical composition comprising a binding protein, a complex binding protein, or a binding protein or complex binding protein can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other parenteral routes of administration such as direct injection or infusion into the tumor site. As used herein, the term "parenteral administration" means a mode of administration other than enteral administration and topical administration, usually by injection. Alternatively, a parenteral route such as topical, epidermal, or mucosal administration may be used. Local administration is preferred, including administration around the tumor, near the tumor, within the tumor, within the lesion, around the lesion, intracavitary injection, intracellular administration, and inhalation. However, the antigen-binding protein, complex of engineered cells, may be administered systemically.

[0088] An appropriate dosage of the specific binding protein, complex binding protein, or pharmaceutical composition of the present invention can be determined by a skilled physician. The actual dosage level of the active ingredient in the pharmaceutical compositions and products of the present invention can be varied so as to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular subject, i.e., the patient, without causing toxicity to the patient. The dosage level selected will depend on various pharmacokinetic factors including the activity of the specific protein / complex used, the route of administration, the time of administration, the rate of excretion of the protein, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the pharmaceutical art.

[0089] Suitable dosages of the polypeptide or composition of the present invention can be, for example, in the range of about 0.1 μg / kg to about 100 mg / kg of the body weight of the patient to be treated. For example, appropriate dosages can be about 1 μg / kg to about 20 mg / kg of body weight per single administration, or about 10 μg / kg to about 10 mg / kg of body weight per single administration. In the case of a conjugate protein carrying a radioisotope, administration can be given at regular intervals such as every other week (bi-weekly). For other purposes, shorter intervals, such as daily administration, may be more suitable. The intervals suitable for different types of treatment will be apparent to those skilled in the art.

[0090] The dosing schedule may be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, doses may be administered over time in several divided doses, or the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as a unit dose for the subject to be treated. Each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0091] The conjugate protein or complex / composition can be administered in a single volume or multiple volumes. Multiple doses can be administered at the same or different locations via the same or different routes. Alternatively, they can be administered as sustained-release formulations, in which case less frequent administration is required. The dosage and frequency can vary depending on the half-life of the species administered to the patient and the desired treatment period. The dosage and frequency of administration may also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, relatively low dosages may be administered at relatively irregular intervals over a long period of time. In therapeutic use, relatively high dosages may be administered, for example, until the patient shows partial or complete improvement of the symptoms of the disease. In an exemplary dosing schedule, the conjugate protein is administered to the subject once a week, once every two weeks, or once every three weeks, in a cycle repeated 2 to 10 times.

[0092] The splice variant CD44v6 is mainly expressed in tumor cells, shows uniform expression of CD44v6 in many cancers, and is expressed only in a subset of epithelial tissues (keratinocytes) among normal tissues, but a pre-targeted therapy scheme may be used. In the pre-targeted therapy scheme, the binding protein itself does not carry the payload (drug), but is carried by a second molecule. In this embodiment, a binding protein containing a molecular binding tag is administered to the subject and becomes capable of binding to the target (CD44v6). Thereafter, the unbound binding protein is removed from the system naturally or by injection of a removing agent. After the unbound binding protein has exited the circulation, a second molecule that carries the payload, i.e., binds to the therapeutic agent, is administered. The second binding molecule binds to the molecular binding tag of the binding protein bound to the target, thereby delivering the payload to the target. Accordingly, a method of treating a subject in need thereof is provided, the method comprising administering a first binding protein comprising a molecular binding tag, enabling any unbound binding protein to leave the subject's circulation, and administering a therapeutically effective amount of a second molecule, wherein the second molecule is linked to a therapeutic agent, the second molecule binds to the first binding protein, thereby delivering the therapeutic agent to the CD44v6 epitope to which the first binding protein binds.

[0093] Thus, the content of the present disclosure enables the treatment, imaging and diagnosis of disorders associated with CD44v6 expression, such as cancer, by administering the composite binding protein of the present invention. Exemplary embodiments of the present disclosure are disclosed in the drawings and the specification. However, many variations and modifications can be made to these embodiments without substantially departing from the principles of the present disclosure. Accordingly, the present disclosure should be regarded as illustrative rather than limiting, and should not be regarded as limited to the specific embodiments described above. Thus, specific terms are used, but they are used only in a general and descriptive sense and not for purposes of limitation.

[0094] The description of the exemplary embodiments provided herein is presented for illustrative purposes. This description is not intended to be exhaustive or to limit the exemplary embodiments to the exact forms disclosed, and modifications and variations are possible in light of the above teachings or may be obtained from various alternative practices of the provided embodiments. The examples discussed herein are chosen and described in order to explain the principles and properties of the various exemplary embodiments and their practical application, so that those skilled in the art can utilize the various exemplary embodiments with various methods and various modifications suitable for the particular purposes contemplated. The features of the embodiments described herein can be combined in any possible combination of methods, products, and systems. It should be understood that the exemplary embodiments presented herein can be implemented in any arbitrary combination with each other. Also, the word "comprising" does not necessarily exclude the presence of other elements or steps other than those listed, and it should also be noted that the words "a" or "an" preceding an element do not exclude the presence of a plurality of such elements. Furthermore, it should be noted that any reference signs do not limit the scope of the claims, and the exemplary embodiments can be realized in the broadest sense of the claims.

Example

[0095] Briefly, CD44v6-binding antibodies were selected and affinity matured. Candidates were epitope mapped, extensively evaluated both in vitro and in vivo, radionuclide labeling, in vivo kinetics, and dosimetry were verified, and molecular radiotherapy experiments were also successfully conducted in several mouse xenografts of thyroid cancer.

[0096] The following Tables 7-9 summarize the related in-silico, in-vitro, and in-vivo experiments performed on the selected MN114 antibodies of the present disclosure after selection and format conversion. Selected exemplary experimental examples are outlined in more detail below. Many of these experiments related to pharmacology, toxicology, and pharmacokinetics are also described in detail in the following sections. Unless otherwise specified below, the antibody format is IgG1 LALA.

[0097]

Table 7-1

[0098]

Table 7-2

[0099]

Table 8

[0100]

Table 9-1

[0101]

Table 9-2

[0102]

Table 10

[0103] Example 1, Phage Display Selection on Human CD44v6 Using a Human scFv Library Phage display selection was performed to enable the isolation of scFv fragments with specificity for human CD44v6.

[0104] Phage Display Selection Biopanning was performed using four rounds of enrichment with a naive human synthetic scFv phage library, SciLifeLib2 (SciLifeLab, Stockholm, Sweden), with a design and construction similar to that previously reported (Sall et al., Protein Eng Des Sel (2016) 29:427-437). Briefly, the human germline genes IGHV3-23 and IGKV1-39 were used as scaffolds for the library, and Kunkel mutagenesis was used to introduce diversity into four out of six CDRs, namely VHCDR1, VHCDR2, VHCDR3, and VLCDR3. Selection was performed using a chemically biotinylated human CD44v6, called biotin-CD44v6, and magnetic beads (Dynabeads M-280, ThermoFisher Scientific, #11206D) coated with streptavidin. The selection pressure was increased by gradually reducing the amount of antigen (from 200 nM to 10 nM) and increasing the intensity and number of washes between different rounds. To remove non-specific or streptavidin binders and enhance the likelihood of selecting v6-specific binders, pre-selection was performed by incubating the phage stock on streptavidin beads with biotinylated human CD44 (isoform 6), called biotin-CD44, prior to selection rounds 1, 2, and 3. See Table 10 for the specifications of the various antigens used. Also, 2% bovine serum albumin (BSA) was included as a blocking agent throughout the selection procedure. Phage bound to the antigen was eluted using a trypsin-aprotinin approach. The entire selection process was automated and performed using a Kingfisher Flex robot. The recovered phage was grown in XL1 Blue Escherichia coli overnight at 37 °C on agar plates (round 1), or overnight at 30 °C in solution (rounds 2, 3, and 4). Phage stocks were prepared by infecting with an excess of M13K07 helper phage (New England Biolabs, #N0315S), and scFv expression was induced by the addition of IPTG. The overnight cultures were PEG / NaCl precipitated, resuspended in selection buffer, and used for the next round of selection.

[0105] Recloning and Expression of scFv To enable the production of soluble scFv fragments, phagemid DNA from selection rounds 3 and 4 was isolated. In the pool, the gene encoding the scFv fragment was digested with a restriction enzyme and subcloned into a screening vector that provided a secretion signal for the scFv fragment along with a C-terminal triple FLAG tag and a hexahistidine (His) tag. Subsequently, the construct was transformed into TOP10 Escherichia coli. Single colonies were picked, cultured, and induced with IPTG for soluble scFv expression in a 96-well format. A total of 189 scFv clones present in the bacterial supernatant were prepared for ELISA screening.

[0106] ELISA Screening CD44v6 and two negative control proteins, namely CD44 and streptavidin, or streptavidin at a concentration of 1 μg / ml were immobilized directly or indirectly on 384-well ELISA plates. ScFv clones present in the bacterial supernatant were diluted 1:10 with a blocking buffer (PBS supplemented with 0.5% BSA + 0.05% Tween20) and bound to the coated proteins. Detection of the binding was made possible by using an HRP-conjugated α-FLAG M2 antibody (Sigma-Aldrich#A8592) and then incubating with a TMB-ELISA substrate (ThermoFisher Scientific#34029). The generation of the colorimetric signal was stopped by adding 1 M sulfuric acid, and the plates were analyzed at a wavelength of 450 nm. All samples were analyzed in duplicate.

[0107] DNA Sequencing Ninety-five scFv clones showing binding to CD44v6 and / or CD44 were sent for Sanger DNA sequencing by Eurofins / GATC Biotech (Cologne, Germany).

[0108] Results Using the scFv phage library SciLifeLib2, phage display selection was performed against the antigen human CD44v6. After recloning the selected scFv fragments into the screening vector, a total of 189 scFv clones were selected from rounds 3 and 4 of the selection. By ELISA screening, 95 potential scFv positive hits were obtained. DNA sequencing of these hits identified 17 sequence-unique scFv clones.

[0109] Example 2, Kinetic Screening of 17 Sequence-Unique scFvs by SPR An approach based on kinetic screening by surface plasmon resonance (SPR) was performed on the 17 sequence-unique scFv clones from Example 1 to enable ranking of the different clones.

[0110] Materials and Methods Kinetic screening was performed on a Biacore T200 instrument (Cytiva). The α-FLAG M2 antibody (Sigma-Aldrich #F1804), which functions as a capture ligand, was immobilized on all four surfaces of a CM5-S amine sensor chip according to the manufacturer's recommendations. The scFv clones present in the bacterial supernatant were injected to capture on the chip surface, followed by injection of 50 nM CD44v6 or 50 nM CD44 (negative control). The surface was regenerated with 10 mM glycine-HCl, pH 2.1. All experiments were performed at 25 °C in running buffer (HBS, pH 7.5 supplemented with 0.05% Tween20). Response curve sensorgrams were obtained by subtracting the response curve of the reference surface (α-FLAG M2 antibody-immobilized surface) from that of the blank run (running buffer injected instead of antigen). The data were analyzed using Biacore T200 Evaluation 3.1 software and a 1:1 Langmuir binding model.

[0111] Results Of the 17 clones analyzed, 11 scFv clones were considered promising based on their binding to CD44v6. More specifically, high binding responses and favorable slow off-rates were considered.

[0112] Example 3, Conversion to the full antibody format Eleven scFv clones that appeared to specifically bind to the v6 region of CD44v6 were selected for conversion to the full-length human IgG4 S228P (EU numbering) antibody format. The rationale for including these specific clones was based on their performance in a panel of binding assays (see Examples 1 and 2). Additionally, the positive control BIWA-4 and the isotype control antibody were similarly converted.

[0113] In-Fusion cloning, transfection into HEK293, expression, and purification The VH and VL regions of the selected scFv clones were PCR amplified and inserted into the vector pHAT-hIgG4-S241P constructed in-house using the In-Fusion HD Plus Cloning Kit (Clontech #638909). Transfection of the plasmid DNA into expiHEK293 cells was performed using the ExpiFectamineTM 293 Transfection Kit (ThermoFisher Scientific #A14525) in a 4 ml culture. After 5 days of culture (37 °C, 6% CO2, 80% rH, 400 rpm), the culture was harvested and the antibody was purified on Protein A conjugated magnetic beads (ThermoFisher Scientific #88846) using a Kingfisher Flex instrument. The buffer was exchanged to PBS at pH 7.5 using a 96-well spin desalting plate (ThermoFisher Scientific #87775). SDS-PAGE was performed to determine the purity and integrity of the purified antibody, and the concentration was determined using an Implen NP80 UV-Vis spectrophotometer.

[0114] ELISA CD44v6, human (hm) v6 - peptide, and negative control CD44 along with streptavidin were immobilized directly or indirectly via streptavidin at a concentration of 1 μg / ml onto 384 - well ELISA plates. Purified IgG4 S228P clones were diluted to 1, 0.2, or 0.04 μg / ml with blocking buffer (PBS supplemented with 0.5% BSA + 0.05% Tween20) and allowed to bind to the coated proteins. Detection of binding was made possible by using HRP - conjugated α - human kappa antibody (Southern Biotech #9230) and then incubating with TMB - ELISA substrate (ThermoFisher Scientific #34029). The generation of the colorimetric signal was stopped by adding 1 M sulfuric acid, and the plates were analyzed at a wavelength of 450 nm. All samples were analyzed in duplicate.

[0115] Results A total of 11 scFv clones and BIWA - 4 were recloned into the human IgG4 S228P format, expressed in expiHEK293 cells, and purified by protein A - conjugated magnetic beads on a Kingfisher Flex instrument. When analyzed by SDS - PAGE, all antibodies were of the expected molecular weight, at an acceptable level of purity, and it was confirmed by ELISA that binding to the v6 region of CD44v6 was retained, i.e., positive signals were obtained for CD44v6 and hm v6 - peptide, while no binding was detected for CD44 and streptavidin. No binding was detected with the isotype control.

[0116] Example 4, Kinetic Measurement of a Novel Anti - CD44v6 IgG4 Antibody The rate constants of the purified IgG4 clones (Example 3) for CD44 and v6 - peptide were determined by surface plasmon resonance (SPR) using a single - cycle kinetics (SCK) approach.

[0117] Materials and Methods Kinetic measurements were performed on a BIAcore T200 instrument (Cytiva) using the SCK approach. The α-human kappa antibody (GE Healthcare #28958325), which functions as a capture ligand, was immobilized on all four surfaces of a CM5-S amine sensor chip according to the manufacturer's recommendations. The antibody was injected and captured at equal response units (RU). A four-fold dilution series of CD44v6 consisting of five concentrations in the range of 80 nM to 0.3 nM was prepared in running buffer and sequentially injected onto the chip surface. A single injection of 100 nM CD44 (negative control) was also performed.

[0118] For the kinetic measurement of the human v6-peptide, a streptavidin (SA) sensor chip was used and the v6-peptide was immobilized at approximately 20 RU. A five-fold dilution series of each antibody consisting of five concentrations in the range of 50 nM to 0.08 nM was prepared in running buffer and sequentially injected onto the chip surface.

[0119] All experiments were performed at 25 °C in running buffer (HBS supplemented with 0.05% Tween20, pH 7.5), and the chip surface was regenerated with 10 mM glycine-HCl, pH 2.1. Response curve sensorgrams were obtained by subtracting the response curve of the reference surface (α-human kappa antibody-immobilized surface or streptavidin-immobilized surface) and the response curve of the blank run (running buffer injected instead of antigen or antibody). The data were analyzed using Biacore T200 Evaluation 3.1 software and a 1:1 Langmuir binding model.

[0120] Results Converted U-MN114-19 showed retained binding to CD44v6 and hm v6-peptide. The reference antibody BIWA-4 hIgG4 also showed binding to CD44v6 and hm v6-peptide as expected. The apparent affinity (expressed as appKD) was in the range from low to sub-nanomolar. Compared to the reference BIWA4 hIgG4, U-MN114-19 bound with higher affinity to both CD44v6 and hm v6-peptide (Table 11).

[0121]

Table 11

[0122] Example 5, Cell Binding of IgG4 U-MN114-19 For all clones that were successfully converted and produced as IgG4, cell binding after radiolabeling was evaluated. For simplicity, in this example, data from U-MN114-19 and BIWA4 are shown.

[0123] Materials and Methods Radioiodination was performed using Pierce iodination tubes according to the manufacturer's protocol. Briefly, 2 - 5 MBq of 125 I (PerkinElmer) was added to a washed (1 mL of PBS) Pierce iodination tube (ThermoFisher) containing 50 μL of PBS. The iodine was incubated for 6 minutes with gentle swirling every 30 seconds inside the tube, and then transferred to an Eppendorf tube containing 10 μg of the MN114 antibody (hIgG4). The antibody / iodine reaction was incubated at 37 °C and 350 rpm for 15 minutes. The labeling yield was determined by ITLC. 177 For Lu labeling, first U-MN114-19 and BIWA-4 were complexed with DOTA using p-SCN-Bn-DOTA: According to the manufacturer's instructions, the buffer was switched from PBS to Na2HPO4 (0.1 M, pH 7.5 - 7.9, metal-free H2O) using an Amicon® Ultra 0.5 mL centrifugal filter (Sigma). A 10-fold molar excess of p-SCN-Bn-DOTA dissolved in Na2HPO4 (0.1 M, pH 7.5 - 7.9, metal-free H2O) was added to the antibody and incubated at 37 °C and 350 rpm for 4 hours. After incubation, the antibody was purified from the excess p-SCN-Bn-DOTA using an Amicon® Ultra filter. 177 Radiolabeling with 177This was carried out by adding Lu and incubating at 42 °C and 350 rpm for 2 hours. The labeling yield was determined by ITLC.

[0124] All ligand tracer experiments were carried out according to the manufacturer's standard protocol. Briefly, 3 - 5×10 5 BHT-101 or ACT-1 cells were seeded into 10 cm Petri dishes and incubated at 37 °C and 5% CO2 at least 24 hours before the start of the experiment. The dishes were placed in the ligand tracer instrument (gray or yellow) and a baseline was established for about 30 minutes before adding the first concentration. Each concentration (1 nM and 3 nM, or 1 nM, 3 nM and 10 nM for U-MN114-19, 1 nM, 3 nM and 10 nM for BIWA-4) was run at room temperature for about 90 minutes. All media containing the radioactive antibody were removed and retention was started with 3 mL of fresh media. For kinetic measurements, the total run time of the experiment was 12 hours.

[0125] Results U-MN114-19, 125 labeled with both 177 I and D Lu, showed low affinity binding to CD44v6 positive cell lines with various antigen expression levels. A representative example of the iodinated antibody on BHT-101 cells is shown in Figure 7. In all cell lines, U-MN114-19 showed lower or comparable K

[0126] Example 6. Comparison of the in vivo distribution of U-MN114-19 and BIWA-4 The main candidate (U-MN114-19) from the U-MN114 antibody selection was evaluated in vivo in balb / c nu / nu mice in direct comparison with BIWA-4.

[0127] Materials and methods Animal experiments were conducted in accordance with Swedish laws and regulations using ethical approvals C33 / 16, C9 / 16, and 10966 / 20. For the inoculation of tumor cells (ACT-1), after harvesting the cells with trypsin, approximately 10 7 cells per mouse were injected into the right hind flank of the mouse in 100 μL of serum-free medium. Radio-labeling was performed as described in Example 5. On days 8 - 10 after tumor cell inoculation, the radio-labeled U-MN114-19 or BIWA4 (both in IgG4 format) was injected intravenously (i.v.) into the tail vein. A total of 15 μg of U-MN114-19 or BIWA4 was injected, which consisted of 1 - 2 μg of radio-labeled antibody and 13 - 14 μg of non-radio-labeled antibody, resulting in a total of 15 μg / 50 μL per mouse. The injection activity was 100 - 300 kBq per mouse. The animals were euthanized and dissected at 24 hours, 48 hours, and 192 hours (8 days) post-injection (p.i.). Organs were analyzed on a Wizard 1460 well counter (PerkinElmer), and the %ID relative to the organ weight (g) was calculated.

[0128] Results 125 The biodistribution of I-U-MN114-19 was repeatedly evaluated and reproducible results were obtained. 125 In a direct comparison with I-BIWA-4 (Figures 8a and 8b), 125 the tumor peak uptake of I-U-MN114-19 was 125 significantly higher than that of I-BIWA4, and the total area under the curve of the tumor was 125 significantly larger for I-U-MN114-19 compared to 125 I-BIWA4.

[0129] Conclusion 125 I-U-MN114-19, in ACT-1 tumor-bearing mice, 125It showed a better tumor-to-blood ratio and higher peak tumor uptake than I-BIWA4. The in vivo distribution results showed no off-target binding or accumulation of the antibody, indicating that it is a stable and specific compound. The results suggest that U-MN114-19 has higher therapeutic utility than BIWA4.

[0130] Example 7, biodistribution of binuclear species of U-MN114-19 125 I and 177 The binuclear species study using Lu was used to determine whether the antibody is more effective than halogen or radioactive metal in terms of tumor targeting, total tumor dose, and safety. The data were later used for the calculation of dosimetry (data not shown).

[0131] Materials and Methods Animal experiments were carried out as described in Example 6 using the ACT-1 xenograft model. Radiolabeling was performed as in Example 5. A total of 15 μg of antibody per mouse was injected, which contained 1 μg of 125 I-U-MN114-19 (100 kBq) and 1 μg of 177 Lu-U-MN114-19 (100 kBq) in the same injection and was diluted with 13 μg of unlabeled U-MN114-19. The animals were euthanized and dissected at 1 hour, 24 hours, 48 hours, and 168 hours post-injection (p.i.). Organs were analyzed with a Wizard 1460 well counter (PerkinElmer), and %ID relative to organ weight (g) was calculated.

[0132] Results 177 The peak tumor uptake of Lu-U-MN114-19 was significantly greater than that of 125 I-U-MN114-19 at all time points, resulting in a better tumor-to-blood ratio (Figure 9). Based on the dosimetry evaluation of the in vivo distribution data, 177 it was confirmed that Lu will be a more suitable therapeutic radionuclide in the future. Figure 9 shows the following, upper left: 125In vivo distribution of I-U-MN114-19 (IgG4), lower left: in ACT-1 xenografts 177 Tumor-to-organ ratios from the in vivo distribution of Lu-U-MN114-19 (IgG4). Upper right: 177 In vivo distribution of Lu-U-MN114-19 (IgG4), lower right: 177 Tumor-to-organ ratios of Lu-U-MN114-19 (IgG4). Error bars represent SD, N = 15.

[0133] Conclusion 177 Lu-U-MN114-19 was superior to I-U-MN114-19 with respect to peak tumor uptake and tumor-to-blood ratio. In the dual-isotope study, 125 tumor uptake was significantly greater compared to I, and the cross-fire dose to healthy tissue was low, 131 suggesting that Lu is highly likely to be the most effective therapeutic radionuclide in future studies. 177

[0134] Example 8, Affinity Maturation of U-MN114-19 Clone U-MN114-19 was matured for the purpose of generating clones with improved affinity for the v6 region within CD44v6.

[0135] Library Design and Construction ​Since U-MN114-19 originally derived from a scFv library (Example 1), the scFv format was chosen as a scaffold for library generation. An affinity matured library called MN114-19-Lib1 (Lib1) was diversified in four out of six CDR loops, namely VHCDR1, VHCDR2, VLCDR1 and VLCDR2. CDRH3 of VH and VL are generally considered the most important regions for antigen binding. These loops were inferred to be kept constant because they are likely to be important for the target interaction of U-MN114-19. Next-generation sequencing of the natural repertoire revealed that antibody evolution by somatic hypermutation occurs through pathways defined based on the germline gene origin of the antibody (DOI: 10.3389 / fimmu.2018.01391). The spatial and chemical diversities introduced into Lib1 were inspired by these in vivo evolution patterns. Thirteen positions most substituted in VHCDR1 and VHCDR2 of the natural IGHV3-23 repertoire were targeted for mutation, and similarly, five positions with the most mutations in VLCDR1 and VLCDR2 of IGKV1-39 were targeted. Also, the amino acid composition at each of these positions was motivated by the natural repertoire. Overall, this procedure targeted 18 positions in MN114-19 Lib1, resulting in a theoretical diversity of combinations of approximately 3.2×10 8 combinations of mutants.

[0136] Library diversity was introduced into the scaffold gene using Kunkel mutagenesis essentially as described (Fellouse FA, Sidhu, S.S. (2007)). To assess whether the intended diversity was incorporated, a small aliquot of the generated Kunkel DNA was used to chemically transform TOP10 E. coli cells, and 96 clones were selected and sent for sequencing (GATC, Germany). Subsequently, the remaining DNA was electroporated into SS320 cells (Lucigen, Middleton, Wisconsin, USA), and when measured by the number of bacterial colonies obtained after transformation, approximately 1.5×10 9A very diverse library containing individual clones was generated. Transformed SS320 cells were harvested and stored at -80 °C with 15% glycerol. Using the bacterial glycerol stock, a total of 600 ml of 2×YT containing antibiotics selective for both phagemid and F’ episome was inoculated. After growing the bacteria to the logarithmic phase, they were infected with M13KO7 helper phage (New England Biolabs, Ipswich, Massachusetts, USA) using a multiplicity of infection of 5. The culture was grown overnight, and scFv-display phage were recovered by standard polyethylene glycol (PEG) / NaCl precipitation.

[0137] Phage display selection Phage display selection was performed using 3 or 4 rounds of enrichment with MN114-19 Lib1. For biotinylated targets (CD44v6 and hm v6-peptide), selection was carried out using magnetic beads coated with streptavidin (as described in Example 1). Similarly, magnetic beads coated with Protein G (Thermofisher Scientific #10004D) were used to capture Fc-fusion CD44v6 (non-biotinylated). The selection tracks were designed such that either CD44v6 or hm v6-peptide alone was used throughout the selection rounds, or the two antigens were used alternately between different selection rounds, resulting in a scheme covering a total of 4 different selection tracks. The selection pressure was increased between different selection rounds by reducing the amount of antigen (50 - 1 or 0.05 nM) and increasing the intensity and number of washes (5 - 8). Prior to round 1, negative selection (pre-selection) was performed using Bio-CD44. Elution of antigen-binding phage was carried out using a trypsin-aprotinin approach. The entire phage display selection process, excluding the phage-target antigen incubation step, was automated and carried out using a Kingfisher Flex robot (ThermoFisher Scientific).

[0138] Recloning and Expression of AL-MN114 scFv Clone Phagemid DNA from the second and third, or second, third, and fourth selection rounds of each selection track was isolated, digested enzymatically, and subcloned into the in-house screening vector pHAT-6 within the pool to enable soluble expression of the AL-MN114 scFv clone fused with a triple FLAG tag and a hexahistidine tag (Hisx6) at the C-terminus. Subsequently, the vector construct was transformed into Escherichia coli TOP10 cells. Single colony clones were picked, cultured, and induced with IPTG for soluble scFv expression in 96-well format. A total of 468 scFv clones were prepared to be analyzed in the primary ELISA screening.

[0139] ELISA Screening The antigens Bio-CD44v6 and hm v6-peptide, together with the negative control Bio-CD44, were indirectly coated onto 384-well ELISA plates via streptavidin at 1 μg / ml in PBS. The 3×FLAG-tagged scFv clones present in the bacterial supernatants were diluted 1:7 with blocking buffer (PBS with 0.5% BSA + 0.05% Tween20) and bound to the coated antigens. Detection of binding was made possible by using the HRP-conjugated a-FLAG M2 antibody (Sigma-Aldrich#A8592) or HRP-conjugated a-human kappa antibody (Southern biotech#2060-05), followed by incubation with the TMB ELISA substrate (ThermoFisher Scientific#34029). The generation of the colorimetric signal was stopped by adding 1M sulfuric acid, and the plates were analyzed on a Spectramax plus instrument (Molecular Devices) at a wavelength of 450 mm (absorbance 450 nm). All clones were assayed in duplicate, and the mean absorbance 450 nm values were calculated from them, subtracting the background with the mean absorbance 450 nm value of the blank sample (blocking buffer added instead of the scFv clone).

[0140] DNA Sequencing Clones showing binding to CD44v6 and hm v6-peptide were sent to Eurofins genomics (Ebersberg, Germany) for Sanger DNA sequencing.

[0141] Results To generate MN114-Lib1, a total of 18 positions within the gene encoding U-MN114-19scFv were targeted. Sequencing of 96 randomly selected clones confirmed the introduction of the intended diversity.

[0142] After phage display selection and ELISA screening of 468 scFv clones, 354 positive hits were identified, showing binding only to the v6 region within CD44v6 and no binding to the negative control antigen CD44. DNA sequencing of the 354 positive hits identified 247 sequence-unique clones. These 247 clones were named AL-MN114 and subsequently assigned unique numbers.

[0143] Example 9, Kinetic measurements of newly selected AL-MN114 scFv clones The 247 sequence-unique AL-MN114 scFv clones (Example 8) were further analyzed by SPR in an initial kinetic screening for binding to hm v6-peptide. The 95 most promising clones ranked by apparent off-rate were further subjected to more precise kinetic measurements using the SCK approach against CD44v6, human v6-peptide, and cynomolgus v6-peptide.

[0144] Materials and methods Kinetic screening and SCK measurement were performed on a BIAcore T200 instrument (Cytiva). The α-FLAG M2 antibody (Merck #F1804), which functions as a capture ligand, was immobilized on all four surfaces of a CM5 series S sensor chip using EDC / NHS amine coupling chemistry according to the manufacturer's recommendations. All experiments were performed at 25 °C in running buffer (HBS supplemented with 0.05% Tween20, pH 7.5). The 3×FLAG-tagged AL-MN114 scFv clones present in the bacterial supernatant were diluted with running buffer to obtain equal capture RU levels. For kinetic screening, 100 nM hm v6-peptide (Table 10, Example 13) was prepared in running buffer and injected onto the surface captured by AL-MN114 scFv and allowed to bind. For SCK measurement, three-fold dilution series of hm v6-peptide and cm v6-peptide at five concentrations ranging from 200 to 2.5 nM (Table 10, Example 13) were prepared in running buffer. Each dilution series was injected sequentially from the lowest antigen concentration to the highest antigen concentration. All chip surfaces were regenerated with 10 mM glycine-HCl, pH 2.1.

[0145] Response curve sensorgrams were obtained by subtracting the response curve of the reference surface (α-FLAG M2 antibody-immobilized surface) from the response curve of the blank run (running buffer injected instead of antigen). The data were analyzed using Biacore T200 Evaluation 3.1 software and a 1:1 Langmuir binding model.

[0146] Results The SCK measurements of the clones were in good correlation with the apparent affinity obtained during kinetic screening. The most promising clones showed approximately a 5-fold improvement in off-rate compared to the parental clone U-MN114-19. The 20 most promising AL-MN114 scFv clones were converted to the hIgG4 format as described above (Example 3) and further characterized.

[0147] Example 10. Characterization of the AL-MN114 hIgG4 Clone in Cancer Cells In this experiment, the time-resolved interaction analysis (ligand tracer) and kinetic properties of the radiolabeled U-MN114-19, AL-MN114 antibody, and BIWA4 that were evaluated will be described.

[0148] Materials and Methods The radiolabeling, ligand tracer cell seeding, and experiments were conducted as described in Example 5.

[0149] Results The undifferentiated thyroid cancer cell line BHT-101 was used for the ligand tracer evaluation of the affinity-matured AL-MN114 antibody (hIgG4). To better detect differences in affinity, the cell line was selected based on its CD44v6 antigen level (in the medium). All 125 I-MN114 antibodies were superior in affinity 125 to I-BIWA4 (Figure 10). 125 Most of the I-AL-MN114 clones showed better retention than the parental clone 125 I-U-MN114-19 (Table 12). Measured with the ligand tracer on A431 cells (a high antigen-expressing cell line), 177 for the Lu-labeled antibodies, both U-MN114-19 and AL-MN114-465 were superior to BIWA4 in affinity (Table 13). Furthermore, 125 I / 177 Lu-AL-MN114-465 both showed better retention compared to BIWA4 and U-MN114-19.

[0150] [Table 12]

[0151] [Table 13]

[0152] Conclusion Affinity maturation improved the affinity, exceeding that of the parental clone and the comparative antibody BIWA4. Four AL-MN114 clones, namely AL-MN114-71, AL-MN114-132, AL-MN114-444, and AL-MN114-465, were selected for hIgG1 conversion and small-scale production.

[0153] Example 11, Conversion to the full antibody IgG1 format AL-MN114-71, AL-MN114-132, AL-MN114-444, AL-MN114-465, U-MN114-19, and BIWA4 were converted into the human IgG1 LALA and / or human IgG1 LALA IAHA formats; refer to Table 14. For simplicity, the conversion to the IgG1 LALA format is described below.

[0154] InFusion cloning, transfection into HEK293, expression, and purification The VH and VL regions of AL-MN114-132, AL-MN114-465, and BIWA4 were PCR amplified and inserted into the vector pHAT-hIgG1-LALA constructed in-house using the In-Fusion HD Plus Cloning Kit (Clontech #638909). Transfection of plasmid DNA into expiHEK293 cells was performed using the ExpiFectamineTM 293 Transfection Kit (ThermoFisher Scientific #A14525) in a 230 mL culture. After culturing for 5 days (37 °C, 7% CO2, 70% rH, 105 rpm), the culture was harvested, and the antibody was purified by affinity chromatography using a HiTrap PrismA column (Cytiva), followed by buffer exchange to PBS, pH 7.4 using a HiTrap desalting column. The endotoxin level measured by the LALA chromogenic endotoxin assay was less than 0.25 EU / mg. SDS-PAGE was performed to determine the purity and integrity of the purified antibody, and the concentration was determined using an Implen NP80 UV-Vis spectrophotometer. Furthermore, size exclusion chromatography was performed on each purified antibody using an Agilent Bio SEC-3.

[0155]

Table 14

[0156] Results The selected clones were converted to the IgG1 LALA and / or IgG1 LALA IAHA format and produced and purified.

[0157] Example 12, Epitope Mapping Antibody clones from Example 12 were epitope mapped within the human v6 region by ELISA using a peptide-based approach. A peptide array consisting of 29 synthetic peptides covering the 43-amino acid-long human v6 region was ordered from JPT Peptides (Germany). Each peptide was 15 amino acids long and had a 1-amino acid shift with a biotin moiety at the N-terminus.

[0158] Materials and Methods The peptide array and full-length human v6-peptide (43aa) were coated onto 384-well ELISA plates via streptavidin (1 μg / mL). All purified antibodies (Example 11) were diluted to 1 μg / mL in blocking buffer (PBS supplemented with 0.5% BSA + 0.05% Tween20) and bound to the coated peptides. Detection of binding was made possible by using HRP-conjugated α-human kappa antibody (Southern Biotech #9230) and then incubating with TMB-ELISA substrate (ThermoFisher Scientific #34029). The generation of the colorimetric signal was stopped by adding 1 M sulfuric acid, and the plates were analyzed at a wavelength of 450 nm. Each sample was assayed in duplicate, from which the average absorbance value was calculated, and the background was subtracted by the average absorbance value of the blank well (blocking buffer added instead of antibody).

[0159] Results The results showed that all MN114 clones exhibited the same 6-amino acid-long epitope, i.e., the amino acids WFGNRW (SEQ ID NO: 7) located at positions 18 - 23 within the human v6 region. BIWA-4 showed a 10-amino acid-long partially overlapping epitope, i.e., the amino acids WFGNRWHEGY (SEQ ID NO: 5) located at positions 18 - 27 within the human v6 region.

[0160] Conclusion The MN114 antibody clones evaluated shared the same 6-amino acid-long epitope and were partially overlapping with the 10-amino acid-long epitope of BIWA-4.

[0161] Example 13, Kinetic Measurement of IgG1 Clones Kinetic measurements of AL-MN114-71, -132, -444, -465 and BIWA4 after conversion to the IgG1 format were performed by SPR using the SCK approach. The parental clone U-MN114-19 was also included as IgG1.

[0162] Materials and Methods SPR measurements were performed on a BIAcore T200 instrument (Cytiva) using the SCK approach. Each antibody was directly immobilized onto a separate surface of a CM5 series S sensor chip using EDC / NHS amine coupling chemistry according to the manufacturer's recommendations. The immobilization level was set to 1500 RU. All experiments were performed at 25 °C in running buffer (HBS, 0.05% Tween20, pH 7.5). Three-fold dilution series of CD44v6 and negative control CD44, consisting of five concentrations in the range of 10 - 0.12 nM, were prepared in running buffer. Four-fold dilution series of human, cynomolgus monkey, rabbit v6-peptides, consisting of five concentrations in the range of 100 - 0.39 nM, were also prepared in running buffer. Each dilution series was injected sequentially onto the immobilized antibody clone, starting with the lowest concentration first, and the chip surface was regenerated with 10 mM glycine-HCl, pH 2.1. See Table 10 for the specifications of the antigens used in the SPR measurements.

[0163] Response curve sensorgrams were obtained by subtracting the response curve of the reference surface (activated and inactivated chip surface) and the response curve of the blank run (running buffer injected instead of antigen). The data were analyzed using Biacore T200 Evaluation 3.1 software and a 1:1 Langmuir binding model.

[0164] Results All antibody clones showed binding to CD44v6 but no binding to CD44 (control antigen). The apparent affinity for CD44v6 obtained app K D was in the sub-nanomolar range,[[]]app K D was 1 - 2 nM (Table 15). In contrast to BIWA4, which did not show binding to the rb-v6-peptide, binding of the antibody clones was also detected against the human (hm) v6-peptide, cynomolgus monkey (cm) v6-peptide, and rabbit (rb) v6-peptide. This data correlates well with the epitope mapping performed (Example 12), demonstrating that all novel antibodies share a 6 amino acid-long epitope present in the v6 regions of cynomolgus monkey and rabbit. The epitope found with BIWA4 is not present in the rabbit v6 region and is present only in the cynomolgus monkey v6 region. The introduced LALA or LALA IAHA Fc modifications are not expected to affect the antigen-binding portion of the antibody.

[0165]

Table 15-1

[0166]

Table 15-2

[0167] Conclusion AL-MN114-71, -132, -444, -465 and the parental clone U-MN114-19 showed binding to hm v6-peptide, cm v6-peptide, rb v6-peptide, and CD44v6 iso4.

[0168] Example 14, Specificity after radiolabeling The specificity of the radiolabeled MN114 antibodies was evaluated by a specificity assay in which the radiolabeled antibody competes with a molar excess of unlabeled antibody with respect to antigen binding, and was evaluated using two ATC cell lines.

[0169] Materials and Methods 125 Radioiodination with I and 177 radiolabeling with Lu were performed as described in Example 5. ACT-1 and BHT-101 (3 - 5*10 per well4 Cells were seeded into 48-well plates at least 24 hours prior to the start of the experiment and incubated at 37 °C and 5% CO2. 30 nM of a radiolabeled antibody, or 30 nM of a radiolabeled antibody in a solution containing 3 μM of an excess of unlabeled antibody, was added per well (100 μL) and incubated at 37 °C and 5% CO2 for 24 hours. After incubation, the cells were washed three times with PBS and harvested using 100 μL of trypsin per well. The cells were counted and the CPM measured with a Wizard 1460 well counter (PerkinElmer) was adjusted according to the number of cells, and the data was presented as CPM / 100,000 cells. As described in Example 7, they were seeded into ligand tracer dishes. In the competitive assay, 10 nM of 125 I-U-MN114-19 and 125 I-AL-MN114-444 were incubated for 2 hours, after which 30 nM of an unlabeled antibody was added.

[0170] Results All radiolabeled antibodies retained specificity after labeling with both 125 I and 177 Lu (Figure 11), and the specificity of AL-MN114-465 was carried out in the presence of a 100-fold molar excess of the parental antibody U-MN114-19. The total CPM / 100,000 cells of AL-MN114-465 was higher than that of both BIWA4 and U-MN114-19, indicating that the uptake of AL-MN114-465 into both cell lines was greater, regardless of the radiolabeling method. Similarly, the affinity-matured AL-MN114-444 had a slower dissociation rate than U-MN114-19, which also indicates the retention of the affinity-matured clone and its excellent affinity (Figure 12).

[0171] Conclusion Both U-MN114-19 and the affinity-matured variant (Al-MN114-465) bind antigen-dependently with higher specificity and excellent affinity than BIWA4. Furthermore, AL-MN114-444 had improved retention compared to U-MN114-19 in the presence of a three-fold molar excess of unlabeled antibody.

[0172] Example 15, In Vivo Distribution of IgG1 LALA and LALA / IAHA Formats in Mice Four selected AL-MN114 clones were 125 both labeled with 177 both I and

[0173] Materials and Methods Animal experiments were performed as described in Example 6 using the ACT-1 or A431 xenograft models. Radiolabeling was performed as in Example 5. For the I comparison of LALA and LALA / IAHA, a total of 15 μg per mouse was injected, consisting of 1 μg of 125 I-U-MN114-19 (100 kBq) and 14 μg of unlabeled U-MN114-19 (IgG1 LALA or IgG1 LALA / IAHA). Animals were euthanized and dissected at 1 hour, 24 hours, 48 hours, and 168 hours post-infection (IgG1 LALA), and at 1 hour, 4 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 168 hours post-infection (IgG1 LALA / IAHA). Organs were analyzed on a Wizard 1460 well counter (PerkinElmer), and %ID relative to organ weight (g) was calculated. For the dual-isotope studies of U-MN114-19 with AL-MN114-71, 132, 444, and 465, a total of 15 μg per mouse was injected, consisting of 1 μg of 125 I-U-MN114-19 (100 kBq) and 1 μg of 125 Lu-U-MN114-19 (100 kBq) diluted with 13 μg of unlabeled U-MN114-19. 177 For the Lu-labeled IgG1 LALA antibody, 1 μg of the labeled antibody and 14 μg of the unlabeled antibody were injected per animal in 50 μL. Animals were euthanized and dissected at 24 hours, 48 hours, 96 hours, and 168 hours post-injection (p.i.). Organs were analyzed on a Wizard 1460 well counter (PerkinElmer), and %ID relative to organ weight (g) was calculated. 177 ​

[0174] Results The IgG1 LALA / IAHA format showed a higher tumor-to-blood ratio compared to the IgG1 LALA format, although the absolute tumor dose was higher for the LALA format (Figure 13). All affinity matured IgG1 clones and U-MN114-19 evaluated in vivo showed good tumor uptake and retention. 177 The Lu-labeled antibody 125 showed higher peak tumor uptake and improved retention compared to the I-labeled antibody (Figure 14).

[0175] Two AL-MN114 clones 177 were evaluated as IgG1 LALA in vivo using Lu and compared to U-MN114-19 IgG1 LALA, both showing good biodistribution profiles with high tumor uptake compared to blood. The affinity matured variants showed better tumor-to-blood ratios compared to the parental versions (Figure 15).

[0176] Conclusion All evaluated affinity matured IgG1 clones showed good tumor uptake and retention in vivo. 177 The Lu-labeled antibody 125 was demonstrated to have improved tumor uptake and retention compared to the I-labeled antibody.

[0177] Example 16, Therapeutic Studies in Mice In two separate studies on two different xenograft models, ACT-1 and BHT-101, 177 the therapeutic ability of U-MN114-19 labeled with Lu was evaluated.

[0178] Materials and Methods Animal studies were conducted as described in Example 6 using the ACT-1 and BHT-101 xenograft models. The xenograft models were selected based on antigen expression levels. ACT-1 was estimated to have a 10-fold higher expression level of CD44v6 than BHT-101. For ACT-1 xenografts,177 Radiolabeling was performed in the same manner as in Example 5, except for the injection activity of Lu and the amount of antibody injected per antibody. For the treatment study, approximately 15 MBq of 177 Lu-U-MN114-19 (50 μg, IgG4) was injected. In the control animals, 50 μg of unlabeled U-MN114-19 (IgG4) was injected. For the BHT-101 xenograft, as described in Example 6, 10 7 cells were inoculated into the right posterior abdomen. For the treatment of BHT-101 tumors, approximately 7 MBq of 177 Lu-U-MN114-19 (50 μg, IgG4) was injected. In the control, approximately 7 MBq of 177 Lu-isotope control antibody (IgG4) was injected. The tumors were measured and the tumor volume was calculated as (H × L × W) * 0.52. The body weights of the animals were monitored for general health status.

[0179] Results In the ACT-1 study, complete remission was achieved in all treated animals with a single administration (15 MBq), and no signs of tumor regrowth were seen until the end of the study. In the BHT-101 study, at a dose of 7 MBq, tumor growth was delayed compared to the isotope control, and the median tumor survival time was almost doubled (Figure 16).

[0180] Conclusions In the treatment study, the potential for treatment has been demonstrated in both a high-tumor model (ACT-1) and a medium-tumor model (BHT-101).

[0181] Example 17, Comparison with BIWA4 To establish the efficiency of the current antibody compared to the prior art, a series of experiments were conducted. For example, affinity and treatment efficiency were tested.

[0182] A. Affinity Regarding affinity, the approximate dissociation constant K D ( app K D ) was evaluated, and K DSince the value can be considered to be related to the concentration of the antibody (the amount of antibody required for a specific experiment), K D The lower the value (the lower the concentration), the higher the affinity of the antibody.

[0183] The parent clone MN19 (also called U-MN114-19) has an approximate K of about 0.16 nM for BHT-101 cells (i.e., the BHT-101 cell line derived from thyroid cancer, the cell with DSMZ number ACC279). D , app K D was confirmed to have. All affinity matured variants of the parent MN19 (defined in Tables 3 - 6) show a K of less than 0.2 nM when measured on BHT-101. app K D On the other hand, BIWA4 shows a K of 9 nM on the BHT-101 cell line, which is significantly higher. In this regard, for comparison, it should be noted that the BIWA4 tested in these experiments was carried out using the BIWA4 antibody in the same format / construct as the antibodies of the present disclosure. In SPR, the affinity is higher than that for live cells. app K D

[0184] B. Biodistribution Regarding the therapeutic efficiency using the antibody, the biodistribution of the injection dose (ID) was determined as %ID / g of the bound antibody. As shown in Figure 17, the biodistribution of iodinated parent clone U-MN114-19, 125 I-U-MN114-19 and iodinated BIWA4, 125 I-BIWA4 revealed that 125 I-U-MN114-19 had significantly superior tumor uptake compared to 125 I-BIWA4. Figure 17 shows a comparative plot of the tumor uptake of 125 I-U-MN114-19 and 125 I-BIWA4 in ACT-1 xenografts. The calculated area under the curve AUC was significantly larger for 125 I-U-MN114-19 than for 125 I-BIWA4, assuming that 0% of the injected activity was in the tumor at t = 0.

[0185] C. Influence on Reaction Time and Tumor Growth In animal studies using xenograft-bearing mice, 177 Lu-BIWA4 and 177 Lu-AL-MN114-465 were tested in parallel with BHT-101 xenografts. A single dose (10 MBq / 50 μg) was injected into the tail vein of the mice, the growth of the xenografts was tracked by caliper measurement, and the health status of the animals was monitored using the body weight and behavior of the animals. The results are shown in Figures 18 and 19.

[0186] Figure 18 shows the tumor growth after treatment with 10 MBq of 177 Lu-AL-MN114-465 or 177 Lu-BIWA4 in BHT-101 xenografts. 177 The survival rate of the animals treated with either 177 Lu-AL-MN114-465 or 177 Lu-BIWA4 was 100%, whereas it can be seen that all controls were euthanized by day 12 after injection. Figure 19 shows the time to complete response, and Figure 19 (top) shows the time to complete response of 177 Lu-AL-MN114-465 or 177 Lu-BIWA4 in BHT-101 xenografts, and Figure 19 (bottom) shows the time to partial response of 177 Lu-AL-MN114-465 or 177 Lu-BIWA4 in BHT-101 xenografts. In BHT-101 xenografts, 177 it can be seen that animals treated with

[0187] In a 3D multicellular tumor spheroid assay using BHT-101 cells, treatment with 60 kBq of 177 Lu-AL-MN114-465 resulted in faster complete and partial responses compared to untreated controls and 60 kBq of 177Both spheroids treated with Lu-isotope control antibody became significantly smaller on day 10 after treatment as compared to both spheroids treated with Lu-isotope control antibody. Similarly, in the treatment with 60 kBq of 177 Lu-BIWA4, the spheroids became significantly smaller on day 10 after treatment as compared to the untreated control, but were not smaller as compared to the spheroids treated with 60 kBq of 177 Lu-isotope control antibody. Therefore, treatment with both 60 kBq of 177 Lu-AL-MN114-465 and 177 Lu-BIWA4 had a significant effect on the growth of the spheroids as compared to the untreated control, but BIWA4 could not have a significantly greater effect than the isotope control. This indicates that AL-MN114-465 is superior to BIWA4 in this 3D setting (Figure 20).

[0188] Figure 20 shows the tumor size / growth rate. Figure 20 (top) shows the growth rate of 3D multicellular tumor spheroids of BHT-101 cells treated with either 60 kBq of 177 Lu-AL-MN114-465, BIWA4, or isotope control (ISO-c) antibody. The spheroids were measured over time, and the growth rate was defined as the relative size compared to the size on day 0 (start of treatment). Figure 20 (bottom) shows the one-way ANOVA of the size ratios on day 10 after treatment, which demonstrated that the spheroids treated with AL-MN114-465 were not significantly different from the spheroids treated with BIWA4, but were different from the isotope control (**), and no such difference was seen in the spheroids treated with BIWA4.

[0189] Conclusion All of the binding proteins, parent clones, and mature clones of the present disclosure defined in Tables 3 to 6 show high affinity as compared to BIWA4. Typically, the dissociation constant K measured in cells derived from the BHT-101 cell line Dis less than 9 nM, such as less than 8, 7, 6, 5, 4, 3, 2, or 1 nM, preferably less than 1 nM, such as less than 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 nM. Preferably, the CD44v6 binding protein binds to BHT-101 cells such that the KD value of the interaction is at most 0.2 nM for the disclosed binding protein. D <binds to BHT-101 cells such that the KD value of the interaction is at most 0.2 nM.

[0190] Furthermore, the binding proteins of the present disclosure may be seen to provide a faster response and a greater therapeutic effect in treatment. Thus, the binding proteins of the present invention have a significant technical effect compared to the prior art BIWA4 antibody.

[0191] Embodiments by item Item 1 A binding protein that specifically binds to CD44v6 and contains an antibody binding domain, wherein the binding domain contains a heavy chain variable domain (VH) and a light chain variable domain (VL) each containing three complementarity determining regions (CDRs) or derivatives thereof, and the amino acid sequences of the CDRs are VHCDR1 defined by SEQ ID NO: 1, VHCDR2 defined by SEQ ID NO: 2, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 4, VLCDR2 defined by X1AS, where X1 is T, A, or S, VLCDR3 defined by SEQ ID NO: 6, a CDR sequence and a CDR sequence having at least 95%, such as 96%, 97%, 98%, 99% or more identity thereto, and is selected from the group consisting of a binding protein that recognizes the epitope of CD44v6 defined by SEQ ID NO: 7. Item 2 The VHCDR1 of the binding protein according to item 1 is defined by the amino acid sequence GFX3FX5X6X7A, where X3 is S, X5 is G, X6 is S, and / or X7 is Y. Item 3 The VHCDR2 of the binding protein according to item 1 or 2 is defined by the amino acid sequence ISX3X4GX6ST, where X3 is A, X4 is G, and / or X6 is S. Item 4 The VLCDR1 of the binding protein according to any one of items 1 to 3 is defined by the amino acid sequence QX2IX4X5Y, where X2 is S, X4 is S, and / or X5 is S. Item 5 The VLCDR2 of the binding protein according to any one of items 1 to 4 is defined by the amino acid sequence X1AS, where X1 is T or S. Item 6 VHCDR1, VHCDR2, and VLCDR2 are present adjacent to specific framework amino acids, and the CDR sequences and framework amino acid (faa) sequences are VHCDR1 and faa defined by SEQ ID NO: 8, VHCDR2 and faa defined by SEQ ID NO: 9, VLCDR2 and faa defined by SEQ ID NO: 10, CDR sequences that have at least 95% identity, such as 96%, 97%, 98%, 99% or more, to the CDR sequences, and are selected from the group consisting of: the binding protein according to item 1. Item 7 The CDR is VHCDR1 selected from SEQ ID NOs: 11 - 18, VHCDR2 selected from SEQ ID NOs: 19 - 25, 32, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 selected from SEQ ID NOs: 26 - 31, VLCDR2 selected from TAS, SAS, and AAS, VLCDR3 defined by SEQ ID NO: 6, A CDR sequence, and a CDR sequence having an identity of 95% or more, such as 96%, 97%, 98%, 99% or more, thereto, a binding protein according to any one of items 1 to 5, which is individually selected from the group consisting of. Item 8 The CDR is VHCDR1 defined by SEQ ID NO: 11, and VHCDR2 selected from SEQ ID NOs: 19 to 25, and VHCDR3 defined by SEQ ID NO: 3, and VLCDR1 defined by SEQ ID NO: 26, and VLCDR2 selected from TAS, SAS and AAS, and VLCDR3 defined by SEQ ID NO: 6, and A CDR sequence, and a CDR sequence having an identity of 95% or more, such as 96%, 97%, 98%, 99% or more, thereto, a binding protein according to any one of items 1 to 5 and 7, which is selected from the group consisting of. Item 9 The amino acid sequence of the CDR is i) VHCDR1 defined by SEQ ID NO: 11, and VHCDR2 defined by SEQ ID NO: 19, and VHCDR3 defined by SEQ ID NO: 3, and VLCDR1 defined by SEQ ID NO: 26, and VLCDR2 defined by TAS, and VLCDR3 defined by SEQ ID NO: 6, a binding protein having ii) VHCDR1 defined by SEQ ID NO: 11, and VHCDR2 defined by SEQ ID NO: 20, and VHCDR3 defined by SEQ ID NO: 3, and VLCDR1 defined by SEQ ID NO: 26, and VLCDR2 defined by SAS, and VLCDR3 defined by SEQ ID NO: 6, a binding protein having iii) VHCDR1 defined by SEQ ID NO: 12, VHCDR2 defined by SEQ ID NO: 20, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 26, VLCDR2 defined by AAS, VLCDR3 defined by SEQ ID NO: 6, and a binding protein having the same; iv) VHCDR1 defined by SEQ ID NO: 16, VHCDR2 defined by SEQ ID NO: 20, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 26, VLCDR2 defined by AAS, VLCDR3 defined by SEQ ID NO: 6, and a binding protein having the same; v) VHCDR1 defined by SEQ ID NO: 17, VHCDR2 defined by SEQ ID NO: 20, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 27, VLCDR2 defined by SAS, VLCDR3 defined by SEQ ID NO: 6, and a binding protein having the same; vi) VHCDR1 defined by SEQ ID NO: 12, VHCDR2 defined by SEQ ID NO: 21, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 26, VLCDR2 defined by AAS, VLCDR3 defined by SEQ ID NO: 6, and a binding protein having the same; vii) VHCDR1 defined by SEQ ID NO: 12, a VHCDR2 defined by SEQ ID NO: 22, a VHCDR3 defined by SEQ ID NO: 3, a VLCDR1 defined by SEQ ID NO: 27, a VLCDR2 defined by TAS, and a VLCDR3 defined by SEQ ID NO: 6; and a binding protein having the same, viii) a VHCDR1 defined by SEQ ID NO: 13, a VHCDR2 defined by SEQ ID NO: 19, a VHCDR3 defined by SEQ ID NO: 3, a VLCDR1 defined by SEQ ID NO: 27, a VLCDR2 defined by SAS, and a VLCDR3 defined by SEQ ID NO: 6; and a binding protein having the same, ix) a VHCDR1 defined by SEQ ID NO: 11, a VHCDR2 defined by SEQ ID NO: 19, a VHCDR3 defined by SEQ ID NO: 3, a VLCDR1 defined by SEQ ID NO: 27, a VLCDR2 defined by SAS, and a VLCDR3 defined by SEQ ID NO: 6; and a binding protein having the same, x) a VHCDR1 defined by SEQ ID NO: 14, a VHCDR2 defined by SEQ ID NO: 19, a VHCDR3 defined by SEQ ID NO: 3, a VLCDR1 defined by SEQ ID NO: 27, a VLCDR2 defined by SAS, and a VLCDR3 defined by SEQ ID NO: 6; and a binding protein having the same, xi) a VHCDR1 defined by SEQ ID NO: 14, a VHCDR2 defined by SEQ ID NO: 19, a VHCDR3 defined by SEQ ID NO: 3, and a VLCDR1 defined by SEQ ID NO: 26, and a VLCDR2 defined by AAS, and a VLCDR3 defined by SEQ ID NO: 6, and a binding protein having the same; xii) a VHCDR1 defined by SEQ ID NO: 12, and a VHCDR2 defined by SEQ ID NO: 19, and a VHCDR3 defined by SEQ ID NO: 3, and a VLCDR1 defined by SEQ ID NO: 26, and a VLCDR2 defined by AAS, and a VLCDR3 defined by SEQ ID NO: 6, and a binding protein having the same; xiii) a VHCDR1 defined by SEQ ID NO: 15, and a VHCDR2 defined by SEQ ID NO: 19, and a VHCDR3 defined by SEQ ID NO: 3, and a VLCDR1 defined by SEQ ID NO: 26, and a VLCDR2 defined by AAS, and a VLCDR3 defined by SEQ ID NO: 6, and a binding protein having the same; xiv) a VHCDR1 defined by SEQ ID NO: 17, and a VHCDR2 defined by SEQ ID NO: 19, and a VHCDR3 defined by SEQ ID NO: 3, and a VLCDR1 defined by SEQ ID NO: 28, and a VLCDR2 defined by AAS, and a VLCDR3 defined by SEQ ID NO: 6, and a binding protein having the same; xv) a VHCDR1 defined by SEQ ID NO: 15, and a VHCDR2 defined by SEQ ID NO: 19, and a VHCDR3 defined by SEQ ID NO: 3, and VLCDR1 defined by SEQ ID NO: 26, VLCDR2 defined by AAS, a binding protein having VLCDR3 defined by SEQ ID NO: 6, xvi) VHCDR1 defined by SEQ ID NO: 12, VHCDR2 defined by SEQ ID NO: 19, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 26, VLCDR2 defined by AAS, a binding protein having VLCDR3 defined by SEQ ID NO: 6, xvii) VHCDR1 defined by SEQ ID NO: 12, VHCDR2 defined by SEQ ID NO: 23, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 29, VLCDR2 defined by SAS, a binding protein having VLCDR3 defined by SEQ ID NO: 6, xviii) VHCDR1 defined by SEQ ID NO: 15, VHCDR2 defined by SEQ ID NO: 20, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 30, VLCDR2 defined by TAS, a binding protein having VLCDR3 defined by SEQ ID NO: 6, xix) VHCDR1 defined by SEQ ID NO: 18, VHCDR2 defined by SEQ ID NO: 24, VHCDR3 defined by SEQ ID NO: 3, VLCDR1 defined by SEQ ID NO: 31, a binding protein having VLCDR2 defined by SAS and VLCDR3 defined by SEQ ID NO: 6; and xx) VHCDR1 defined by SEQ ID NO: 12 and VHCDR2 defined by SEQ ID NO: 25 and VHCDR3 defined by SEQ ID NO: 3; and VLCDR1 defined by SEQ ID NO: 26 and VLCDR2 defined by AAS and VLCDR3 defined by SEQ ID NO: 6; and xxi) VHCDR1 defined by SEQ ID NO: 12 and VHCDR2 defined by SEQ ID NO: 32 and VHCDR3 defined by SEQ ID NO: 3; and VLCDR1 defined by SEQ ID NO: 26 and VLCDR2 defined by AAS and VLCDR3 defined by SEQ ID NO: 6; and a CDR sequence having at least 95%, such as 96%, 97%, 98%, 99% or more identity thereto; a binding protein according to any one of items 1 to 5 and 7 to 8 selected from the group comprising Item 10 The sequence of the CDR containing the framework amino acid (faa) is i) a binding protein having VHCDR1 and faa defined by SEQ ID NO: 121 and VHCDR2 and faa defined by SEQ ID NO: 129 and VLCDR2 and faa defined by SEQ ID NO: 139; and ii) a binding protein having VHCDR1 and faa defined by SEQ ID NO: 121 and VHCDR2 and faa defined by SEQ ID NO: 130 and A binding protein having VLCDR2 and faa defined by SEQ ID NO: 140, iii) VHCDR1 and faa defined by SEQ ID NO: 122, VHCDR2 and faa defined by SEQ ID NO: 131, A binding protein having VLCDR2 and faa defined by SEQ ID NO: 141, iv) VHCDR1 and faa defined by SEQ ID NO: 126, VHCDR2 and faa defined by SEQ ID NO: 130, A binding protein having VLCDR2 and faa defined by SEQ ID NO: 144, v) VHCDR1 and faa defined by SEQ ID NO: 127, VHCDR2 and faa defined by SEQ ID NO: 130, A binding protein having VLCDR2 and faa defined by SEQ ID NO: 140, vi) VHCDR1 and faa defined by SEQ ID NO: 122, VHCDR2 and faa defined by SEQ ID NO: 133, A binding protein having VLCDR2 and faa defined by SEQ ID NO: 141, vii) VHCDR1 and faa defined by SEQ ID NO: 122, VHCDR2 and faa defined by SEQ ID NO: 134, A binding protein having VLCDR2 and faa defined by SEQ ID NO: 142, viii) VHCDR1 and faa defined by SEQ ID NO: 123, VHCDR2 and faa defined by SEQ ID NO: 129, A binding protein having VLCDR2 and faa defined by SEQ ID NO: 140, ix) A binding protein having VHCDR1 and faa defined by SEQ ID NO: 121, and VHCDR2 and faa defined by SEQ ID NO: 132, and VLCDR2 and faa defined by SEQ ID NO: 140; and x) A binding protein having VHCDR1 and faa defined by SEQ ID NO: 124, and VHCDR2 and faa defined by SEQ ID NO: 129, and VLCDR2 and faa defined by SEQ ID NO: 143; and xi) A binding protein having VHCDR1 and faa defined by SEQ ID NO: 124, and VHCDR2 and faa defined by SEQ ID NO: 132, and VLCDR2 and faa defined by SEQ ID NO: 141; and xii) A binding protein having VHCDR1 and faa defined by SEQ ID NO: 122, and VHCDR2 and faa defined by SEQ ID NO: 129, and VLCDR2 and faa defined by SEQ ID NO: 141; and xiii) A binding protein having VHCDR1 and faa defined by SEQ ID NO: 125, and VHCDR2 and faa defined by SEQ ID NO: 129, and VLCDR2 and faa defined by SEQ ID NO: 141; and xiv) A binding protein having VHCDR1 and faa defined by SEQ ID NO: 127, and VHCDR2 and faa defined by SEQ ID NO: 135, and VLCDR2 and faa defined by SEQ ID NO: 144; and xv) A binding protein having VHCDR1 and faa defined by SEQ ID NO: 125, and The VHCDR2 and faa defined by SEQ ID NO: 132, and The VLCDR2 and faa defined by SEQ ID NO: 141, and a binding protein having the same; xvi) The VHCDR1 and faa defined by SEQ ID NO: 122, and The VHCDR2 and faa defined by SEQ ID NO: 132, and The VLCDR2 and faa defined by SEQ ID NO: 141, and a binding protein having the same; xvii) The VHCDR1 and faa defined by SEQ ID NO: 122, and The VHCDR2 and faa defined by SEQ ID NO: 136, and The VLCDR2 and faa defined by SEQ ID NO: 145, and a binding protein having the same; xviii) The VHCDR1 and faa defined by SEQ ID NO: 125, and The VHCDR2 and faa defined by SEQ ID NO: 131, and The VLCDR2 and faa defined by SEQ ID NO: 146, and a binding protein having the same; xix) The VHCDR1 and faa defined by SEQ ID NO: 128, and The VHCDR2 and faa defined by SEQ ID NO: 137, and The VLCDR2 and faa defined by SEQ ID NO: 145, and a binding protein having the same; xx) The VHCDR1 and faa defined by SEQ ID NO: 122, and The VHCDR2 and faa defined by SEQ ID NO: 138, and The VLCDR2 and faa defined by SEQ ID NO: 141, and a binding protein having the same; xxi) The VHCDR1 and faa defined by SEQ ID NO: 122, and The VHCDR2 and faa defined by SEQ ID NO: 33, and A binding protein having VLCDR2 and faa defined by SEQ ID NO: 141 The binding protein according to item 6, selected from the group consisting of a CDR sequence and a CDR sequence having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto. Item 11 The VH sequence includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 54 and a sequence having 80% or more, such as 85%, 90%, 95% or more identity thereto, and the VL sequence includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 to 74 and a sequence having 80% or more, such as 85%, 90%, 95% or more identity thereto. The binding protein according to any one of items 1 to 5 and 7 to 10. Item 12 The CDR sequence does not contain mutations in the amino acid sequence, or the sequence mutations in the CDR amino acid sequence are at most 5%, such as 94%, 3%, 2%, 1% or less. The binding protein according to item 11. Item 13 The amino acid sequences of the VH and VL are i) A binding protein having VH defined by SEQ ID NO: 35 And VL defined by SEQ ID NO: 55 ii) A binding protein having VH defined by SEQ ID NO: 36 And VL defined by SEQ ID NO: 56 iii) A binding protein having VH defined by SEQ ID NO: 37 And VL defined by SEQ ID NO: 57 iv) A binding protein having VH defined by SEQ ID NO: 46 And VL defined by SEQ ID NO: 66 v) A binding protein having VH defined by SEQ ID NO: 54 A binding protein having VL defined by SEQ ID NO: 74, and vi) A binding protein having VH defined by SEQ ID NO: 38, and A binding protein having VL defined by SEQ ID NO: 58, and vii) A binding protein having VH defined by SEQ ID NO: 39, and A binding protein having VL defined by SEQ ID NO: 59, and viii) A binding protein having VH defined by SEQ ID NO: 40, and A binding protein having VL defined by SEQ ID NO: 60, and ix) A binding protein having VH defined by SEQ ID NO: 41, and A binding protein having VL defined by SEQ ID NO: 61, and x) A binding protein having VH defined by SEQ ID NO: 42, and A binding protein having VL defined by SEQ ID NO: 62, and xi) A binding protein having VH defined by SEQ ID NO: 43, and A binding protein having VL defined by SEQ ID NO: 63, and xii) A binding protein having VH defined by SEQ ID NO: 44, and A binding protein having VL defined by SEQ ID NO: 64, and xiii) A binding protein having VH defined by SEQ ID NO: 45, and A binding protein having VL defined by SEQ ID NO: 65, and xiv) A binding protein having VH defined by SEQ ID NO: 47, and A binding protein having VL defined by SEQ ID NO: 67, and xv) A binding protein having VH defined by SEQ ID NO: 48, and A binding protein having VL defined by SEQ ID NO: 68, and xvi) A VH defined by SEQ ID NO: 49 and a VL defined by SEQ ID NO: 69, and a binding protein having the same; and xvii) A VH defined by SEQ ID NO: 50 and a VL defined by SEQ ID NO: 70, and a binding protein having the same; and xviii) A VH defined by SEQ ID NO: 51 and a VL defined by SEQ ID NO: 71, and a binding protein having the same; and xix) A VH defined by SEQ ID NO: 52 and a VL defined by SEQ ID NO: 72, and a binding protein having the same; and xx) A VH defined by SEQ ID NO: 53 and a VL defined by SEQ ID NO: 73, and a binding protein having the same; and xxi) A VH defined by SEQ ID NO: 147 and a VL defined by SEQ ID NO: 148, and a sequence having 80% or more, such as 85%, 90%, 95% or more identity thereto, and a binding protein according to any one of items 11 - 12 selected from the group comprising the same. Item 15 The binding protein according to any one of items 1 - 13, wherein the binding protein is a monoclonal antibody or an antigen - binding fragment selected from the group consisting of Fv fragments, Fab - like fragments, and domain antibodies. Item 16 The binding protein according to item 14, wherein the Fv fragment is a scFv fragment. Item 17 The binding protein according to item 14, wherein the Fab - like fragment is a Fab or F(ab’)2 fragment. Item 18 Item 19 The binding molecule is a monoclonal antibody of the IgG1 isotype, such as an IgG1 LALA antibody or an IgG1 LALA IAHA antibody, or a monoclonal antibody of the IgG4 isotype, which is a binding protein according to any one of items 1 to 14. Item 18 The binding protein according to any one of items 1 to 17, wherein the binding protein is human or derived from a human. Item 19 The antibody includes a heavy chain and a light chain. The heavy chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 75 to 93, and 149, and a sequence having 80% or more, for example, 85%, 90%, 95% or more identity thereto. The light chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 94 to 113 and 150, and a sequence having 80% or more, for example, 85%, 90%, 95% or more identity thereto. The binding protein according to any one of items 17 to 18. Item 20 The amino acid sequences of the heavy chain and the light chain are i) A binding protein having a heavy chain defined by SEQ ID NO: 75 and a light chain defined by SEQ ID NO: 94, ii) A binding protein having a heavy chain defined by SEQ ID NO: 76 and a light chain defined by SEQ ID NO: 95, iii) A binding protein having a heavy chain defined by SEQ ID NO: 77 and a light chain defined by SEQ ID NO: 96, iv) A binding protein having a heavy chain defined by SEQ ID NO: 34 and a light chain defined by SEQ ID NO: 105, v) A binding protein having a heavy chain defined by SEQ ID NO: 93 and a light chain defined by SEQ ID NO: 113, vi) a binding protein having a heavy chain defined by SEQ ID NO: 78 and, a light chain defined by SEQ ID NO: 97; and, vii) a binding protein having a heavy chain defined by SEQ ID NO: 79 and, a light chain defined by SEQ ID NO: 98; and, viii) a binding protein having a heavy chain defined by SEQ ID NO: 80 and, a light chain defined by SEQ ID NO: 99; and, ix) a binding protein having a heavy chain defined by SEQ ID NO: 81 and, a light chain defined by SEQ ID NO: 100; and, x) a binding protein having a heavy chain defined by SEQ ID NO: 82 and, a light chain defined by SEQ ID NO: 101; and, xi) a binding protein having a heavy chain defined by SEQ ID NO: 83 and, a light chain defined by SEQ ID NO: 102; and, xii) a binding protein having a heavy chain defined by SEQ ID NO: 84 and, a light chain defined by SEQ ID NO: 103; and, xiii) a binding protein having a heavy chain defined by SEQ ID NO: 85 and, a light chain defined by SEQ ID NO: 104; and, xiv) a binding protein having a heavy chain defined by SEQ ID NO: 86 and, a light chain defined by SEQ ID NO: 106; and, xv) a binding protein having a heavy chain defined by SEQ ID NO: 87 and, a light chain defined by SEQ ID NO: 107; and, xvi) a binding protein having a heavy chain defined by SEQ ID NO: 88 and, A binding protein having a light chain defined by SEQ ID NO: 108, xviii) A binding protein having a heavy chain defined by SEQ ID NO: 89, A binding protein having a light chain defined by SEQ ID NO: 109, xviii) A binding protein having a heavy chain defined by SEQ ID NO: 90, A binding protein having a light chain defined by SEQ ID NO: 110, xix) A binding protein having a heavy chain defined by SEQ ID NO: 91, A binding protein having a light chain defined by SEQ ID NO: 111, xx) A binding protein having a heavy chain defined by SEQ ID NO: 92, A binding protein having a light chain defined by SEQ ID NO: 112, xxi) A binding protein having a heavy chain defined by SEQ ID NO: 149, A binding protein having a light chain defined by SEQ ID NO: 150, The binding protein according to item 19, selected from the group consisting of a sequence having 80% or more, such as 85%, 90%, 95% or more identity thereto. Item 21 The binding protein according to any one of items 20 to 21, wherein the CDR sequence does not contain a mutation in the amino acid sequence, or the sequence mutation of the CDR amino acid sequence is at most 5%, such as 4%, 3%, 2%, 1% or less. Item 22 (i) At least one binding protein according to any one of items 1 to 21, (ii) A composite binding protein comprising at least one agent. Item 23 The composite binding protein according to item 22, wherein the at least one agent is linked to the binding protein, and the binding protein and the agent are directly or indirectly linked. Item 24 The agent is a radioisotope, a photoactivatable compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytotoxic drug, a prodrug, a binding molecule having different specificities, a cytokine, or another immunomodulatory polypeptide, the conjugate protein according to item 22 or 23. Item 25 The conjugate protein according to any one of items 22 to 24, wherein the agent is a therapeutic agent. Item 26 The conjugate protein according to item 25, wherein the therapeutic agent is a cytotoxic drug containing or consisting of one or more radioisotopes. Item 27 The conjugate protein according to item 24 or 26, wherein the one or more radioisotopes are each independently selected from the group consisting of a beta emitter, an Auger emitter, a conversion electron emitter, an alpha emitter, and a low photon energy emitter. Item 28 The conjugate protein according to item 27, wherein the one or more radioisotopes each independently have a pattern of local absorption energy release that produces a high dose absorption in the vicinity of the agent. Item 29 The one or more radioisotopes are 90 Y, 32 P, 186 Re / 188 Re, 166 Ho, 76 As / 77 As, 153 Long-range beta emitters such as Sm, 131 I, 177 Lu, 67 Cu, 161 Tb, 47 Medium-range beta emitters such as Sc, 45 Ca, 35 S or 14 Low-energy beta emitters such as C, 51 Cr, 67 Ga, 99 TC m , 111 In, 123 I, 125 I, 201 Conversion or Auger emitters such as TI, and212 Bi, 212 Pb, 213 Bi, 223 Ac, 225 Ac, 227 Th, 149 Tb, 211 The conjugate protein according to item 27 or 28, independently selected from the group consisting of alpha emitters such as At. Item 30 The radioactive isotope is 177 Lu, the conjugate protein according to any one of items 24 or 26 - 29. Item 31 The therapeutic agent is a cytotoxic agent containing or consisting of one or more cytotoxic drugs, the conjugate protein according to item 25. Item 32 The one or more cytotoxic drugs are the conjugate protein according to item 31, selected from cell growth inhibitors, toxins, and chemotherapeutic drugs. Item 33 The agent is a detectable agent, the conjugate protein according to any one of items 22 - 24. Item 34 The detectable agent is, inter alia, the conjugate protein according to item 33, selected from the group consisting of radioactive isotopes, enzymes, fluorescent molecules, dyes, digoxigenin, and biotin. Item 35 The detectable agent contains or consists of a radioactive isotope, the conjugate protein according to item 33 or 34. Item 36 The radioactive isotope is 111 In, 99m Tc, 67 Ga, 68 Ga, 72 As, 89 Zr, 123 I, 125 I, 124 I, 47 Sc and 201 TI, the conjugate protein according to item 34 or 35, selected from the group consisting of. Item 37 The composite binding protein is 86 γ / 90 γ, 111 In / 177 Lu, 125 I / 211 a pair of detectable and cytotoxic radioisotopes such as At, and the composite binding protein according to any one of items 24, 26 to 28, or 34 to 35. Item 38 The radioisotope can act in a multimodal manner simultaneously as a detectable agent and as a cytotoxic agent, and the composite binding protein according to item 37. Item 39 The detectable agent is detectable by imaging techniques such as SPECT, PET, MRI, optical or ultrasonic imaging, and the composite binding protein according to items 33 to 38. Item 40 The agent is indirectly linked to the binding protein via a linker, and the composite binding protein according to item 22 or 23. Item 41 The linker is in the form of a chelating agent, and the composite binding protein according to item 40. Item 42 The chelating agent is a derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a derivative of deferoxamine (DFO), a derivative of diethylenetriaminepentaacetic acid (DTPA), a derivative of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), a derivative of (tBu)4(1-(1-carboxy-3-tert-butoxypropyl)-4,7,10-(carboxy tert-butoxymethyl)-1,4,7,10-tetraazacyclododecane) (DOTAGA), a derivative of 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), a derivative of 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), a derivative of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and is selected from the group consisting of, and the composite binding protein according to item 41. Item 43 The linker is a molecular binding tag for pre-targeting treatment using a secondary binding molecule, the secondary binding molecule is bound to the agent, the secondary binding molecule is linked to the binding tag of the binding protein, thereby linking the agent to the binding protein to form a composite binding protein, the composite binding protein according to item 40. Item 44 A cell engineered to express a chimeric antigen receptor (CAR), the CAR comprising an antigen-binding domain, a transmembrane domain connected to the antigen-binding domain by a hinge region, and an intracellular domain optionally connected to one or more co-stimulatory domains, the antigen-binding domain comprising the binding protein according to any one of items 1 to 15. Item 45 The cell according to item 44, wherein the cell is a human cell. Item 46 The cell according to item 44 or 45, wherein the cell is an immune effector cell such as a T cell, NK cell or macrophage. Item 47 A pharmaceutical composition comprising the binding protein according to any one of items 1 to 21, the composite binding protein according to any one of items 22 to 32 or 37 to 43, the cell according to any one of items 44 to 46, and a pharmaceutically acceptable carrier or excipient. Item 48 The binding protein according to any one of items 1 to 21, the composite binding protein according to any one of items 22 to 32 or 37 to 43, the cell according to any one of items 44 to 46, or the pharmaceutical composition according to item 47 for use in therapy. Item 49 The binding protein, composite binding protein, cell, or pharmaceutical composition according to item 48 for use in cancer treatment. Item 50 A method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of the binding protein according to any one of items 1 to 21, the composite binding protein according to any one of items 22 to 32 or 37 to 43, the cell according to any one of items 44 to 46, or the pharmaceutical composition according to item 47. Item 51 A method of treating a subject in need thereof, said method comprising: administering a therapeutically effective amount of a first binding protein according to any one of Items 1 to 21, said binding protein further comprising a molecular binding tag; allowing any unbound binding protein to leave the circulation of said subject; administering a therapeutically effective amount of a second molecule, said second molecule being linked to a therapeutic agent, said second molecule binding to said first binding protein, thereby delivering said therapeutic agent to a CD44v6 epitope to which said first binding protein binds. Item 52 The method according to Item 50 or 51, wherein said subject has a disorder characterized by the expression of CD44 variant CD44v6. Item 53 The method according to Item 52, wherein said disorder is cancer or another angiogenesis-related disorder. Item 54 Use of a binding protein according to any one of Items 1 to 21, a conjugate binding protein according to any one of Items 22 to 32 or 37 to 43, a cell according to any one of Items 44 to 46, or a pharmaceutical composition according to Item 47 for the treatment, prevention or diagnosis of cancer. Item 55 The binding protein, conjugate binding protein, cell, or pharmaceutical composition according to Item 49, the method according to Item 53, or the use according to Item 54, wherein said cancer is selected from the group consisting of advanced thyroid cancer, head and neck cancer, pancreatic cancer, squamous cell carcinoma, Hodgkin's lymphoma, colorectal cancer, liver cancer, cervical cancer, gastric cancer, ovarian cancer, lung cancer, bladder cancer, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, breast cancer, hepatocellular carcinoma, and esophageal cancer, and metastatic cancer of the brain. Item 56 An in vitro method for detecting the expression of said CD44 variant CD44v6, said method comprising: (i) Contacting the binding protein according to items 1 to 21, or the composite binding protein according to items 22 to 24 or 33 to 43, with a biological sample such as a tissue sample or a liquid obtained from a subject, and if the binding protein or the composite binding protein is present in the biological sample, binding it to the epitope of CD44v6 defined by SEQ ID NO: 7; (ii) Washing the biological sample to remove unbound binding protein or composite binding protein; (iii) Detecting any binding protein or composite binding protein bound to the epitope in the biological sample, an in vitro method comprising. Item 57 An in vivo method for detecting the expression of the CD44 variant CD44v6, the method comprising: (i) Administering to a subject the composite binding protein according to items 22 to 24 or 33 to 43, wherein the composite binding protein binds to the epitope of CD44v6 defined by SEQ ID NO: 7; (ii) Detecting that the conjugate binding protein has bound to cells expressing the epitope, an in vivo method comprising. Item 58 The composite binding protein comprises one or more 111 In radioisotope atoms, the in vivo method according to item 57. Item 59 (iii) At least one binding protein according to any one of items 1 to 14, and (iv) At least one 177 Lu radioisotope atom linked to the binding protein, and A composite binding protein used for the treatment of advanced thyroid cancer. Item 60 The dissociation constant K measured in the cells derived from the BHT-101 cell line DThe binding protein according to any one of items 1 to 21, which is less than 9 nM, for example, less than 8, 7, 6, 5, 4, 3, 2, or 1 nM, preferably less than 1 nM, for example, less than 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 nM. Item 61 The dissociation constant K measured with the cells derived from the BHT-101 cell line D is K D The binding protein according to item 60, which is less than 0.2 nM such that it is <0.2 nM.

Claims

1. A binding protein that specifically binds to CD44v6 and contains an antibody binding domain, The binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), or derivatives thereof, each containing three complementarity-determining regions (CDRs), and the amino acid sequence of the CDRs is: VHCDR1 as defined by sequence number 1, VHCDR2, defined by sequence number 2, VHCDR3, defined by Sequence ID 3, VLCDR1 as defined by Sequence ID 4, A VLCDR2 defined by X1AS, where X1 may be T, A, or S, VLCDR3 as defined by sequence number 6, A binding protein selected from the group comprising a CDR sequence having 95% or more identity thereto, for example, 96%, 97%, 98%, 99%, or more, wherein the binding protein recognizes the epitope of CD44v6 as defined by Sequence ID No.

7.

2. VHCDR1, VHCDR2, and VLCDR2 are located next to specific framework amino acids, and the CDR and framework amino acid (faa) sequences are, VHCDR1 and faa as defined by Sequence ID 8, VHCDR2 and faa as defined by Sequence ID No. 9, VLCDR2 and faa as defined by sequence number 10, The binding protein according to claim 1, selected from the group comprising a CDR sequence having 95% or more identity thereto, for example, 96%, 97%, 98%, 99%, or more.

3. The aforementioned CD-R is VHCDR1 selected from sequence numbers 11 to 18, VHCDR2 selected from sequence numbers 19-25 and 32, VHCDR3, defined by Sequence ID 3, VLCDR1 selected from sequence numbers 26 to 31, VLCDR2 selected from TAS, SAS, and AAS, VLCDR3 as defined by sequence number 6, The binding protein according to claim 1, individually selected from the group comprising a CDR sequence having 95% or more identity thereto, for example, 96%, 97%, 98%, 99%, or more.

4. The binding protein according to claim 1, wherein the VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 54 and 147, and a sequence having 80% or more identity with respect to it, for example, 85%, 90%, 95%, or more, and the VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 to 74 and 148, and a sequence having 80% or more identity with respect to it, for example, 85%, 90%, 95%, or more.

5. The binding protein is a monoclonal antibody, or an Fv fragment such as an scFv fragment, Fab or F(ab') ２ The binding protein according to claim 1, which is an antigen-binding fragment selected from the group consisting of Fab-like fragments such as fragments and domain antibodies.

6. The binding protein according to claim 1, wherein the binding molecule is an IgG1 isotype monoclonal antibody such as an IgG1 LALA antibody or an IgG1 LALA IAHA antibody.

7. The binding protein according to claim 1, wherein the binding protein is human or of human origin.

8. The binding protein according to claim 5, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 75-93 and 149, and a sequence having 80% or more identity with respect to it, for example, 85%, 90%, 95%, or more, and the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-113 and 150, and a sequence having 80% or more identity with respect to it, for example, 85%, 90%, 95%, or more.

9. The binding protein according to claim 4, wherein the CDR sequence does not contain amino acid sequence mutations, or the sequence mutations in the CDR amino acid sequence are at most 5%, for example, 4%, 3%, 2%, 1%, or less.

10. The binding protein according to claim 1, wherein the binding protein binds to BHT-101 cells such that the interaction KD value is a maximum of 1 nM, for example, preferably a maximum of 0.2 nM.

11. At least one binding protein according to Claim 1, A complex binding protein containing at least one drug.

12. The complex-binding protein according to claim 11, wherein the at least one drug is a therapeutic agent or a detectable drug.

13. The complex-binding protein according to claim 12, wherein the at least one therapeutic agent is one or more cytotoxic agents such as radioisotopes, cell proliferation inhibitors, toxins, or chemotherapeutic agents, and the at least one detectable agent is one or more radioisotopes, enzymes, fluorescent molecules, dyes, digoxigenin, or biotin.

14. The radioactive isotope used as a therapeutic agent is, １７７ Lu, １３１ I, ６７ Cu, １６１ Tb, ４７ medium-range beta emitters such as Sc, ９０ Y, ３２ P, １８６ Re / １８８ Re, １６６ Ho, ７６ As / ７７ As, １５３ long-range beta emitters such as Sm, ４５ Ca, ３５ S or １４ low-energy beta emitters such as C, ５１ Cr, ６７ Ga, ９９ Tc, １１１ In, １２３ I, １２５ I, ２０１ conversion or Auger emitters such as TI, and ２１２ Bi, ２１２ Pb, ２１３ Bi, ２２３ Ac, ２２５ Ac, ２２７ Th, １４９ Tb, and ２１１ alpha emitters such as At selected from the group consisting of, the radioactive isotope used as a detectable agent is, １１１ In, ９９ mTc, ６７ Ga, ６８ Ga, ７２ As, ８９ Zr, １２３ I, １２５ I, １２４ I, ４７ Sc and ２０１ TI selected from the group consisting of, or the composite binding protein contains a pair of detectable and cytotoxic radioactive isotopes, the pair of radioactive isotopes is, １１１ In / １７７ Lu, ８６ γ / ９０ γ, and １２５ I / ２１１ At selected from, the composite binding protein according to claim 13.

15. The aforementioned drug is indirectly linked to the binding protein via a linker such as a chelating agent, the chelating agent being a derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), a derivative of deferoxamine (DFO), a derivative of diethylenetriaminepentaacetic acid (DTPA), a derivative of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), (tBu)4(1-(1-carboxy-3-carbotert The complex-binding protein according to claim 11, selected from the group consisting of a derivative of (butoxypropyl)-4,7,10-(carbotert-butoxymethyl)-1,4,7,10-tetraazacyclododecane) (DOTAGA), a derivative of 1,4,8,11-tetraazacyclodosedane-1,4,8,11-tetraacetic acid (TETA), a derivative of 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (NODAGA), and a derivative of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

16. A cell manipulated to express a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain connected to the antigen-binding domain by a hinge region, and an intracellular domain optionally connected to one or more costimulatory domains, the antigen-binding domain comprising the binding protein described in claim 1.

17. A pharmaceutical composition comprising the binding protein described in claim 1, and a pharmaceutically acceptable carrier or excipient.

18. The binding protein according to claim 1, for use in therapeutic purposes.

19. The binding protein according to claim 18 for use in cancer treatment.

20. The complex binding protein according to claim 19, wherein the cancer is advanced thyroid cancer.

21. An in vitro method for detecting the expression of the CD44 mutant CD44v6, wherein the method is (i) The binding protein described in claim 1 is brought into contact with a biological sample such as a tissue sample or liquid obtained from a subject, and the binding protein, if present in the biological sample, is configured to bind to the CD44v6 epitope as defined by SEQ ID NO:

7. (ii) Wash the biological sample to remove any unbound proteins, (iii) an in vitro method comprising detecting an arbitrary binding protein bound to the epitope in the biological sample.

22. An in vivo method for detecting the expression of the CD44 variant CD44v6, wherein the method is: An in vivo method comprising detecting that the complex-binding protein described in claim 11 has bound to a cell expressing the CD44v6 epitope as defined by SEQ ID NO: 7.