Anti-CD44v6 antibodies and uses thereof for treating CD44v6 overlexing

Abstract

The present disclosure relates to binding proteins that bind to human CD44v6. The binding protein may be conjugated to an agent to form a conjugated binding, where the agent may be an imaging agent or a therapeutic agent, such as a radioisotope. The binding proteins or conjugated binding proteins or pharmaceutical compositions thereof may be used in medical treatment, such as cancer therapy, or in diagnosis and medical imaging. The binding proteins may also be used to engineer cells to express chimeric antigen receptors having binding proteins of the disclosure as antigen binding domains.

Description

Technical Field

[0001] The present disclosure relates to binding proteins that bind to epitopes of human CD44v6. In some aspects, the binding protein constitutes a CD44v6 binding antibody or fragment thereof, or a conjugated binding protein or monoclonal antibody carrying an imaging agent or therapeutic agent (such as an anti-tumor agent). In some embodiments, the binding protein, antibody, conjugated binding protein / antibody or its pharmaceutical composition is used for medical treatment, such as cancer therapy. In some embodiments, the binding protein, antibody or conjugated 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 a binding protein of the present disclosure as an antigen binding domain. Background Art

[0002] Cancer is one of the most prevalent lethal diseases and, despite recent advances in diagnosis and treatment, still causes a large number of deaths each year. Monoclonal antibodies (mAbs) that modulate immune responses or conjugated mAbs carrying anti-tumor agents are providing highly effective and promising treatments for a variety of cancers.

[0003] CD44 is a cell surface glycoprotein involved in cell-cell interactions, cell proliferation, differentiation, adhesion and migration. The standard isoform CD44s contains exons 1-5 and 16-20, while the CD44 splice variant containing the alternative exon is named CD44v. The alternative splice variant of CD44 containing exon 6 is called CD44v6. Exon v6 is associated with cancer and is thought to be involved in metastatic spread, and therefore CD44v6 has been identified as a potential target for cancer therapy.

[0004] A mAb against CD44v6 (BIWA-1 or VFF-18) has been previously developed for the treatment of squamous cell carcinoma (WO97 / 21104), as well as a humanized mAb against CD44v6 (BIWA-4 or Bivatuzumab) used as a conjugated mAb for the treatment of inoperable recurrent or metastatic head and neck cancer (Postema EJ, et al. Journal of Nuclear Medicine 2003, 44(10):1690–9).

[0005] Despite previous progress, there remains a need for new and better binding proteins that target CD44v6 that can be used in diagnostics and medical treatments, such as cancer therapy. Summary of the invention

[0006] The object of the present disclosure is to provide novel and enhanced binding molecules that can be used for medical treatment, diagnosis and medical imaging. This object is achieved by a binding protein that specifically binds to CD44v6 and comprises a binding domain of an antibody, the binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), each comprising three complementary determining regions (CDRs), wherein the amino acid sequence of the CDRs is selected from the group comprising: VHCDR1 as defined in SEQ ID NO: 1, VHCDR2 as defined in SEQ ID NO: 2; VHCDR3 as defined in SEQ ID NO: 3; VLCDR1 as defined in SEQ ID NO: 4; VHCDR2 as defined in SEQ ID NO: 5; VHCDR3 as defined in SEQ ID NO: 6; VHCDR4 as defined in SEQ ID NO: 7; VHCDR5 as defined in SEQ ID NO: 8; VHCDR6 as defined in SEQ ID NO: 9; VHCDR7 as defined in SEQ ID NO: 10; VHCDR8 as defined in SEQ ID NO: 11; VHCDR9 as defined in SEQ ID NO: 12; VHCDR10 as defined in SEQ ID NO: 13; VHCDR11 as defined in SEQ ID NO: 14; VHCDR12 as defined in SEQ ID NO: 15; VHCDR13 as defined in SEQ ID NO: 16; VHCDR14 as defined in SEQ ID NO: 17; VHCDR15 as defined in SEQ ID NO: 18; VHCDR16 as defined in SEQ ID NO: 19; VHCDR17 as defined in SEQ ID NO: 20 1 VLCDR2 defined by AS, where X 1 It can be T, A or S; VLCDR3 as defined by SEQ ID NO:6; and a CDR sequence having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto, wherein the binding protein recognizes the epitope of CD44v6 as defined by SEQ ID NO:(7). In some embodiments, the amino acid sequence of the CDR of the binding protein is VHCDR1 as defined by SEQ ID NO:11, VHCDR2 as defined by SEQ ID NO:19, VHCDR3 as defined by SEQ ID NO:3, VLCDR1 as defined by SEQ ID NO:26, VLCDR2 as defined by sequence TAS, and VLCDR3 as defined by SEQ ID NO:6.

[0007] In some embodiments, the VHCDR1, VHCDR2 and VLCDR2 of the binding protein are present in close proximity to specific framework amino acids, wherein the CDR and framework amino acid (faa) sequence is selected from the group comprising: VHCDR1 and faa defined by SEQ ID NO: 8; VHCDR2 and faa as defined by SEQ ID NO: 9; VLCDR2 and faa as defined by SEQ ID NO: 10; and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto.

[0008] In some aspects, the binding protein is a monoclonal antibody, or an antigen binding fragment selected from the group consisting of an Fv fragment, a Fab-like fragment, and a domain antibody. 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 IgG1LALA antibody or an IgG1IAHA antibody. Typically, the binding protein is human or human-derived.

[0009] In some aspects, a conjugated binding protein is provided. The conjugated 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 a radioactive isotope, a cytostatic drug, a toxin, and a chemotherapeutic agent. In some embodiments, the therapeutic agent is 177 Lu radioactive isotope.

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

[0012] In some aspects, the agent is attached to the binding protein via a linker in the form of a chelating agent. The chelating agent can be selected from the group consisting of: 1,4,7,10-tetraazacyclododecane-1,4,7,10, tetraacetic acid (DOTA) derivatives, desferrioxamine (DFO) derivatives, diethylenetriaminepentaacetic acid (DTPA) derivatives, S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivatives, (tBu)4(1-(1-carboxyl-3-carbon tert-butyloxypropane) derivatives, The invention also includes derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NODAGA), and derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0013] In some aspects, an engineered cell is provided. The cell can be engineered 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, wherein the antigen binding domain comprises an scFv fragment of a protein-binding protein of the present disclosure.

[0014] According to some aspects, a pharmaceutical composition is provided, which comprises the binding protein, conjugated binding protein or engineered cell as described above, and a pharmaceutically acceptable carrier or excipient.

[0015] The binding proteins, conjugated binding proteins, engineered cells or pharmaceutical compositions as described above can be used for therapy. In some embodiments, the therapy is a cancer therapy, including 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 brain metastasis. In some embodiments, the cancer is advanced thyroid cancer.

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

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

[0018] Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description taken with reference to the following illustrative drawings and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and additional objects, features and advantages of the present inventive concept will be better understood through the following illustrative and non-limiting detailed description of different embodiments of the present inventive concept with reference to the accompanying drawings, in which:

[0020] Figure 1 The general concept of antibodies conjugated with agents as payloads is demonstrated.

[0021] Figure 2 is a schematic diagram of the GST-CD44 (v3-v10) fusion protein, in which the arrow marks the thrombin cleavage site.

[0022] Figure 3 The basic mechanism of molecular radiotherapy targeting cancer cells using monoclonal antibodies linked to radioisotopes is demonstrated.

[0023] Figure 4 Schematic representation of a CD44v6-targeted radiopharmaceutical for molecular radiotherapy of advanced thyroid cancer.

[0024] FIG5 shows the results of a treatment with a radiopharmaceutical of the present disclosure in mice with ATC, Figure 5a Tumor volumes in mice are shown, and Figure 5b Survival rates are shown.

[0025] Figure 6 is a graphical representation of the epitope of human CD44v6 bound by the binding proteins of the present invention compared to the previous BIWA-4 antibody.

[0026] Figure 7 Shown 125 IU-MN114-19 and 125 Binding of I-BIWA-4 to BHT-101 cells at 1 and 3 nM (U-MN114-19) and 1, 3 and 10 nM (BIWA-4).

[0027] Figure 8 shows 125 IU-MN114-19 and 125 Direct comparison of the biodistribution of I-BIWA-4. Figure 8a ACT-1 xenografts are shown 125 IU-MN114-19 and 125 Tumor and blood profiles of I-BIWA-4 (expressed as %ID / g as IgG4). Figure 8b The left picture shows: 125 IU-MN114-19 (IgG4) (N=12), right: 125 Tumor to organ ratios of biodistribution of I-BIWA-4 (IgG4) (N=11), error bars represent SD.

[0028] Fig. 9 Shows 125 IU-MN114-19 (IgG4) and 177 Biodistribution of Lu-U-MN114-19(IgG4) (upper panel), and tumor to organ ratios derived from the biodistribution (lower panel).

[0029] FIG. 10 shows a LigandTracer comparison of different antibodies. Fig.10a The results are shown for BHT-101 cells normalized to CPS at the end of association. 125 I-labeled MN114 antibody and 125 LigandTracer comparison of I-BIWA-4. Fig.10bComparisons of U-MN114-19 (dark grey), AL-MN114-465 (black), and BIWA-4 (light grey) are shown. Two concentrations of the MN114 clone (1 nM and 3 nM) and three concentrations of BIWA-4 (1 nM, 3 nM, and 10 nM) were each run for approximately 90 minutes before initiation of dissociation.

[0030] Fig.11 Specificity was demonstrated in the presence of a 50-100-fold molar excess of non-radiolabeled antibody (50-fold over BIWA-4 for radiolabeled BIWA-4, 100-fold over U-MN114-19 for radiolabeled U-MN114-19, and 100-fold over U-MN114-19 for radiolabeled AL-MN114-465).

[0031] Fig.12 showed a 3-fold molar excess 125 IU-MN114-19 (10 nM) and 125 LigandTracer assessment of antibody retention during competition with non-radiolabeled antibody I-AL-MN114-444 (10 nM) on BHT-101 cells.

[0032] Fig.13 The biodistribution is shown above: 125 Biodistribution of IU-MN114-19 (IgG1 LALA). Below: 125 Biodistribution of IU-MN114-19 (IgG1 LALA / IAHA) ACT-1 xenograft model, error bars represent SD, N=13 (LALA) and N=25 (LALA / IAHA).

[0033] Fig.14 Demonstrating tumor retention, left: 125 IU-MN114-19(IgG1 LALA / IAHA) and 125 Tumor retention of I-AL-MN114 variant (IgG1 LALA / IAHA) from 24hpi to 168hpi. Right: 177 Lu-U-MN114-19(IgG1 LALA / IAHA) and 177 Tumor retention of Lu-AL-MN114 variants (IgG1 LALA / IAHA) from 24hpi to 168hpi. Error bars represent SD, n≥14, N=61.

[0034] Fig.15 A431 xenografts are shown 177Blood and tumor uptake of Lu-labeled U-MN114-19 (left), AL-MN114-132 (middle), and AL-MN114-465 (right) expressed as % ID / g. Error bars represent SD, n=16, N=48.

[0035] Fig.16 Tumor growth compared to isotope control is shown. Left column, from top to bottom: ACT-1 tumor volume measurements, survival rate, and animal weight. Right column, from top to bottom: BHT-101 tumor volume measurements, survival rate, and animal weight. Error bars represent SD, N=10 (5+5) for ACT-1 study, N=8 (4+4) for BHT-101 study.

[0036] Fig.17 Demonstrated that ACT-1 xenografts 125 IU-MN114-19 and 125 Comparison of tumor uptake of I-BIWA4.

[0037] Fig.18 demonstrated the use of 10 MBq in BHT-101 xenografts. 177 Lu-AL-MN114-465 or 177 Tumor growth after Lu-BIWA4 treatment.

[0038] Fig.19 The time to complete reaction is shown, where Fig.19 (Top) shows the expression of BHT-101 xenografts 177 Lu-AL-MN114-465 or 177 The time it takes for Lu-BIWA4 to fully react, and Fig.19 (Bottom) shows the expression of BHT-101 xenografts in 177 Lu-AL-MN114-465 or 177 Partial reaction time of Lu-BIWA4

[0039] Fig. 20 The size / growth ratio of the tumor is shown, where Fig. 20 (Top) shows the use of 60kBq 177 Growth ratio of 3D multicellular tumor spheroids of BHT-101 cells treated with Lu-AL-MN114-465, BIWA4, or isotope control (ISO-c) antibodies, and Fig. 20 (Lower panel) shows one-way ANOVA of size ratios at day 10 after treatment.

[0040] The drawings are not necessarily drawn to scale and generally only show parts which are necessary in order to elucidate the inventive concept, wherein other parts may be omitted or merely suggested. DETAILED DESCRIPTION

[0041] The present disclosure relates to novel binding proteins, such as monoclonal antibodies or antigen binding fragments thereof, that selectively bind to CD44v6, and to conjugated binding proteins that carry therapeutic agents, such as antibody drug conjugates (ADCs). The binding proteins, antibodies, or conjugated antibodies can be used for medical treatments, such as cancer therapy, or for imaging applications, for in vitro and in vivo diagnosis. The binding proteins can also be used to engineer cells to express chimeric antigen receptors having the binding proteins of the present disclosure as antigen binding domains.

[0042] It is an object of the present disclosure to provide new and enhanced binding proteins specific for CD44v6 that can be used in therapy, diagnostics, medical imaging, and cell engineering.

[0043] Various aspects of the present disclosure will be described more fully below with reference to the accompanying drawings. However, the binding proteins, conjugated binding proteins / ADCs and methods disclosed herein can be implemented in many different forms and should not be construed as being limited to the aspects set forth herein. The same numbers in the accompanying drawings always refer to the same elements.

[0044] The terms used herein are only used for the purpose of describing specific aspects of the present disclosure and are not intended to limit the present disclosure. As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are also intended to include the plural forms.

[0045] In some embodiments, the non-limiting term "binding protein" is used. The term "binding protein" is used herein to refer to a binding protein that comprises a binding domain of an antibody (i.e., a binding domain obtained or derived from an antibody, or a binding domain based on an antibody). Thus, a binding protein is an antibody-based or antibody-like molecule that comprises a binding site of an antibody or a binding site derived from an antibody. Thus, it is an immunobinder.

[0046] In some embodiments, the non-limiting term "antibody" or "antigen-binding fragment thereof" is used. The term "antibody" is used in its broadest sense herein, including monoclonal antibodies 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 or other) through 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" not only covers full-length or complete polyclonal or monoclonal antibodies, but also covers antigen-binding fragments thereof, such as Fab, Fab', F(ab')2, Fab3, Fv and variants thereof, fusion proteins comprising one or more antibody portions, humanized antibodies, chimeric antibodies, mini antibodies, double antibodies, three antibodies, four antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0047] As known to the skilled person, antibodies are proteins comprising four polypeptide chains: two heavy chains and two light chains. Typically, the heavy chains are identical to each other and the light chains are identical to each other. The light chains are shorter (and therefore lighter) than the heavy chains. The heavy chain comprises four or five domains: the variable (VH) domain is located at the N-terminus, followed by three or four constant domains (CH1, CH2, CH3 and CH4 (if present) from N-terminus to C-terminus, respectively). The light chain comprises two domains: the variable (VL) domain is located at the N-terminus, and the constant (CL) domain is located at the C-terminus. In the heavy chain, the unstructured hinge region is located between the CH1 and CH2 domains. The two heavy chains of the antibody are connected by disulfide bonds formed between the cysteine ​​residues present in the hinge region, and each heavy chain is connected to one light chain by a disulfide bond between the cysteine ​​residues present in the CH1 and CL domains, respectively. Two types of light chains are produced in mammals, referred to as λ (λ) and κ (κ). For the κ light chain, the variable domain and the constant domain may be referred to as VL, ... K and C KDomain. Whether a light chain is a λ or κ light chain is determined by its constant region: the constant regions of λ and κ light chains are different, but are the same in all light chains of the same type in any given species. Antibodies are divided into different categories according to the amino acid sequence of their heavy chain constant domains. There are six main categories of antibodies: IgA, IgD, IgE, IgG, IgM and IgY, and several of these can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The term "full-length antibody" as used herein refers to antibodies of any class, such as IgD, IgE, IgG, IgA, IgM or IgY (or any subclass thereof). The term "antigen-binding fragment" refers to a portion or region of an antibody molecule or a derivative thereof that retains all or most of the antigen binding of the corresponding full-length antibody. In some embodiments, the heavy chain of an antibody may include VH+CH1+hinge+CH2+CH3, and the light chain may include VL+CL. In a preferred embodiment, the antibody has an IgG1 LALA format, and 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.

[0048] As briefly listed above, examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which are monovalent fragments having a VL-CL chain and a VH-CH chain; (2) Fab' fragments, which are Fab fragments having a heavy chain hinge region, (3) F(ab')2 fragments, which are dimers of Fab' fragments joined by the heavy chain hinge region (e.g., linked by a disulfide bridge in the hinge region); (4) Fc fragments; (5) Fv fragments, which are minimal antibody fragments having the VL and VH domains of a single arm of an antibody; (6) single-chain Fv (scFv) fragments, which are single polypeptide chains in which the VL and VH domains of the scFv are H and V L The domains are connected by a peptide linker; (7) (scFv) 2, which contains two V HA domain and two VL domains, which are associated through two VH domains via a disulfide bridge, and (8) domain antibodies, which can be antibody single variable domain (VH or VL) polypeptides that specifically bind to an antigen. Antigen binding fragments can be prepared via conventional methods. For example, F(ab')2 fragments can be produced by pepsin digestion of full-length antibody molecules, and Fab fragments can be produced by reducing the disulfide bridges of F(ab')2 fragments. Alternatively, fragments can be prepared via recombinant technology by expressing heavy chain and light chain fragments in suitable host cells (e.g., Escherichia coli, yeast, mammals, plants or insect cells) and assembling them in vivo or in vitro to form the desired antigen binding fragments. Single-chain antibodies can be prepared via recombinant technology by connecting a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region. For example, a flexible linker can be incorporated between the two variable regions. Therefore, the general terms "binding protein" or "antibody" are used throughout the specification. These terms are used in their broadest sense and therefore also include 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., a single VH variable domain or a VL variable domain).

[0049] The constant region of the heavy chain is therefore the same in all antibodies of any given isotype in a species, but is different between isotypes. The specificity of an antibody is determined by the sequence of its variable region. The sequence of the variable region is different between antibodies of the same type in any individual. In particular, both the light chain and the heavy chain of an antibody comprise three hypervariable complementary determining regions (CDRs). In a pair of light chain and heavy chain, the CDRs of the two chains form an antigen binding site. The CDR sequence determines the specificity of an antibody. A pair of light chain variable regions and heavy chain variable regions comprising (antigen) binding sites are referred to as (antigen) binding domains. The three CDRs of the heavy chain are referred to as VHCDR1, VHCDR2 and VHCDR3 from N-terminal to C-terminal, and the three CDRs of the light chain are referred to as VLCDR1, VLCDR2 and VLCDR3 from N-terminal to C-terminal.

[0050] In antibodies, as described above, CDR sequences are located in the variable domains of the heavy and light chains. The CDR sequences are located within a polypeptide framework that positions the CDRs in the appropriate position for antigen binding. Therefore, the remainder 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 CDR3 is connected to the polypeptide sequence of the constant domain to form FR4. In the binding protein of the present invention, the variable region framework region may have any suitable amino acid sequence so that the binding protein binds to CD44v6 via its CDR.

[0051] If the binding protein is an antibody, the antibody can be of any isotype and subtype. Therefore, it can be an IgA, IgD, IgE, IgG or IgM antibody. The heavy chain constant domains corresponding to different isotypes of immunoglobulins are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional configuration of different isotypes of immunoglobulins are well known. Preferably, the antibody is an IgG antibody. As mentioned above, IgG antibodies have four subtypes: IgG1, IgG2, IgG3 and IgG4. The IgG anti-CD44v6 antibody of the present invention can be any IgG subtype, that is, 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 or IgG1 IAHA antibody. In IgG1 LALA, the leucine (L) in amino acid positions 234 and 235 in the Fc region has been replaced by alanine (A). The LALA mutation eliminates Fc-mediated binding to the Fcγ receptors of immune cells, which reduces effector function. Remove binding to escape immune response, i.e. reduce the immunogenicity mediated by Fc effector functions (such as ADCC and CDC), and the risk of undesirable off-target and on-target side effects caused by biopharmaceuticals. In the IAHA mutation, at amino acid positions 253 and 310 in the Fc region of the antibody, isoleucine (I) has replaced alanine (A), and histidine (H) has replaced alanine (A). Therefore, in this embodiment, the binding protein can be an antibody designed as a "silent Fc" Fc-gamma receptor-free antibody by LALA mutation to eliminate ADCC / CDC activity. In addition, as evaluated in computer T cell epitope prediction analysis, these antibodies are characterized by a low risk of immunogenicity. The IAHA double mutation in the Fc region reduces the interaction with FcRn (FcRn binding), which in turn reduces its circulation time in the blood (DOI: 10.1080 / 19420862.2016.1156285).

[0052] As described in detail above, antibody light chains belong to the kappa (κ) and lambda (λ) types. The binding protein of the present invention may contain a kappa or lambda light chain. In a specific embodiment, the binding protein of the present invention comprises a kappa light chain.

[0053] 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 examples include Fab', Fab, F(ab'), 2 , Fv, Fd or dAb fragments, which can be prepared according to techniques well known in the art.

[0054] The Fab fragment consists of the antigen-binding domain of the antibody, that is, a single antibody can be considered to contain two Fab fragments, each of which consists of a light chain and the N-terminal segment of the heavy chain to which it is attached. H and C H 1 domain. Fab fragments can be obtained by digesting antibodies with papain.

[0055] F(ab') 2 The fragment consists of two Fab fragments of an antibody plus the hinge region of the heavy chain domain (including the disulfide bonds that link the two heavy chains together). In other words, F(ab') 2 The fragment can be viewed as two covalently joined Fab fragments. F(ab') 2 Fragments can be obtained by digesting antibodies with pepsin. F(ab') 2 Reduction of the fragments produces two Fab' fragments which can be considered as Fab fragments containing an additional thiol group which can be used to conjugate the fragment to other molecules.

[0056] Alternatively, the binding protein may be a synthetic or artificial construct, i.e., an antibody-like molecule that contains a binding domain but is genetically engineered or artificially constructed. This includes chimeric or CDR-grafted antibodies, as well as single-chain antibodies and other constructs, such as scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, single domain antibodies (DABs), TandAb dimers, and heavy chain antibodies such as V H In one embodiment, the artificial construct is a single chain variable fragment (scFv). ScFv is a fusion protein in which a single polypeptide comprises the V H and V L The scFv fragment generally comprises a covalently joined V H and V LThe peptide linker of the peptide region contributes to the stability of the molecule. The linker may contain 1 to 20 amino acids, such as, for example, 1, 2, 3 or 4 amino acids, 5, 10 or 15 amino acids, or other intermediate numbers within the range of 1 to 20 when convenient. The peptide linker may be formed by any commonly convenient amino acid residues, such as glycine and / or serine, as is familiar to those skilled in the art. However, the presence of the linker is not essential, and V L The domain can be linked to V by a peptide bond H The scFv usually contains a linker sequence from the N-terminus to the C-terminus to connect the V L Area connected to V H The preparation of scFv molecules is well known in the art.

[0057] The binding domain of an antibody consists of a light chain variable domain and a heavy chain variable domain (a classic 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 derivative (e.g., a single chain antibody, as further discussed below). In summary, the binding protein of the present invention comprises the binding domain of an antibody, the binding domain of the antibody comprising a light chain variable domain and a heavy chain variable domain.

[0058] As used herein, the term "capable of binding to X", wherein X is an antigen, refers to a property of an antibody or binding fragment thereof that can be determined, for example, by ELISA, by using surface plasmon resonance (SPR) technology, by using kinetic exclusion assays, Or bio-layer interferometry (BLI) to test. The skilled person is aware of these and other methods.

[0059] The term "specificity" of a binding protein for a target, sometimes referred to as "selectivity", refers to a binding protein that will bind to a target with high affinity but generally does not bind to other antigens. A selective or specific binding protein / antibody will not or to a low degree cross-react with other targets other than the intended antigen. Thus, "specific" binding refers to the binding protein binding to its target (i.e., CD44v6) in a manner that is distinguishable from binding to non-target molecules, and more specifically, the binding protein binds to its target (i.e., CD44v6) with a greater binding affinity than it binds to other molecules. That is, the binding protein does not bind to other non-target molecules, or does not bind to a significant or significant degree, or binds to such other molecules with a lower affinity than its affinity for binding to CD44v6. A binding protein that "specifically binds" to CD44v6 may alternatively be referred to as "directed to" or "recognizing" CD44v6. In other words, CD44v6 is an antigen for the binding protein of the present invention, and the binding protein is therefore an "antigen binding protein" in the sense that it binds to CD44v6 as its antigen.

[0060] The associated proteins of the present invention can be conjugated with reagents to form conjugated associated proteins. In one aspect, the reagent can be a detectable reagent, such as a certain label, and the conjugated associated proteins can be used for imaging. In other aspects, the conjugated associated proteins can be formed by connecting the associated proteins to a therapeutic agent. In the case described, the conjugated associated proteins can be formed as antibody drug conjugates (ADC) or function similar to antibody drug conjugates (ADC), and can be used for therapy.

[0061] ADC can deliver highly effective cytotoxic anticancer agents to monoclonal antibodies through biodegradable and stable joints, and distinguish cancer from normal tissues. Therefore, ADC can combine monoclonal antibodies with specific surface antigens present on specific tumor cells with highly effective anticancer agents connected via chemical joints. ADC is usually composed of three parts: an antibody with specificity for target-related antigens (selective for tumor-related antigens with limited or no expression on normal healthy cells), a payload intended to kill target cancer cells (an effective cytotoxic agent, intended to induce target cell death after internalization and release in tumor cells), and a chemical joint (a stable joint in the circulation, but releasing cytotoxic agents in target cells) attached to the payload to the antibody, wherein the joint is cleavable or non-cleavable. ADC is usually a monoclonal antibody covalently linked to a small molecule drug, which targets specific cancer cells to reduce systemic toxicity, increases the cell killing potential of monoclonal antibodies, and confers higher tumor selectivity, which leads to higher tumor selectivity and limited systemic exposure, and therefore has higher drug tolerance. ADCs deliver therapeutic agents via a linker attached 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 cell. The chemical "linker" that joins the antibody and cytotoxic drug together is highly stable to prevent the ADC from being cut (split) before it enters the tumor. Anticancer drugs penetrate the tumor and cause cell death by damaging the DNA of the cancer cells or by preventing new cancer cells from forming and spreading. Therefore, the ADC binds to proteins on the surface of the cancer cell, is internalized, and releases the drug while internalized, killing the cancer cell. A schematic diagram of an ADC is shown below. Figure 1 Shown, a mAb carrying a payload via a linker is shown, where the agent can be present in several copies, as indicated by the letter n (the number of individual agents attached to the mAb).

[0062] As used herein, "therapy" means the treatment of any medical condition. Such treatment can be preventive (i.e., preventive), curative (or intended to be a curative treatment) or palliative (i.e., a treatment intended only to limit, alleviate or improve the symptoms of a condition). Therefore, "therapy" or "treatment" of a condition (such as cancer / cancer tumor) by a binding protein or conjugated binding protein as used herein refers to preventing or improving certain conditions or medical conditions or curing it. In the case of cancer and tumors, treatment can shrink or eliminate existing tumors, or they can stop or prevent further spread of tumors. An amount sufficient to achieve this is defined as a "therapeutically effective amount". The effective amount for a given purpose will depend on the disease or condition to be treated, its severity, and the size / weight and overall condition of the subject. Therefore, the binding protein or conjugated binding protein as described herein can be used for the treatment / therapy of any condition in which the target antigen is expressed / overexpressed in a subject to improve the condition, and can be administered systemically or topically by any suitable method known in the art. A subject as defined herein refers to any mammal, e.g., a farm animal (such as a cow, horse, sheep, pig or goat), a pet animal (such as a rabbit, cat or dog) or a primate (such as a monkey, chimpanzee, gorilla or human). Most preferably, the subject is a human.

[0063] Prophylactic treatment may include preventing a condition, or delaying the development or onset of a condition. For example, the conjugated binding protein may be used to prevent infection, or reduce the extent to which an infection may develop, or to prevent, delay or reduce the extent to which a cancer develops or recurs, or, for example, to prevent or reduce the extent of metastasis.

[0064] As used herein, the term "diagnosis (diagnosis / diagnosing)" means the process of determining whether there is a disease or illness (such as cancer) in the tested subject. In terms of diagnostic procedures, diagnosis can be regarded as an attempt to classify individual illnesses into separate and different categories in order to make medical decisions about treatment and prognosis. Subsequently, diagnostic opinions are usually described according to diseases or other conditions. The initial task is to detect medical indications to perform diagnostic procedures, such as detecting any deviations from known normal conditions. Diagnostic procedures can be performed in vitro using the binding proteins or conjugated binding proteins of this article in, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemical method or Western blot, or it can be performed in vivo, wherein the binding proteins can carry detectable agents, such as labels, for in vivo tumor imaging. The binding proteins can be combined with radioisotopes for imaging, such as immunoscintigraphy.

[0065] Therefore, the associated protein or the associated protein of conjugation can be used for medical imaging, which can be used for diagnosis or prognosis. As used herein, the term "prognosis (prognosis / prognosing)" means the prediction or estimation of the chance of recovery or survival from the disease during treatment. The prognosis of cancer may depend on several factors, such as the disease stage during diagnosis, the type and subtype of cancer, the molecular spectrum of the tumor, and even gender. Diagnosis / prognosis can also be used to distinguish patients into different subgroups according to the nature or aggressiveness of illness such as cancer. It can be further used for treatment planning, i.e., for determining dosage regimens. In the case where the associated protein of conjugation includes a radionuclide, dosimetry can be used, wherein diagnosis / prognosis can include the combination / intake / internalization of the associated protein of conjugation, and the radiation dose is determined by the measurement, calculation, or measurement and calculation combination of absorbed dose (radiation energy deposited in tissue divided by tissue mass) of absorbed dose.

[0066] The CD44 cell surface glycoprotein plays a role in promoting cell-cell and cell-matrix interactions through its affinity for hyaluronic acid. In addition, it is known to impart adhesion and is also involved in the assembly of growth factors (such as EGFR and HER4) on the cell surface. Dysfunction and / or altered expression of the protein cause various pathogenic phenotypes. The term "CD44" refers to CD44 from any species. Therefore, it may be human CD44 or its equivalent or corresponding molecule in other species, most notably other mammals. Human CD44 has UniProt accession number P16070. CD44 transcripts undergo complex alternative splicing to produce functionally different subtypes, among which CD44s is the standard subtype and CD44v is a variant, such as Figure 2 As shown. The CD44 protein is encoded by a single and highly conserved gene, which consists of 20 exons, of which exons 1-5, 16-18 and 20 encode the smallest, standard and hematopoietic subtypes CD44s. The exons lacking in CD44s are referred to as CD44 exon subtype variants (called CD44v1-10). Therefore, the ten variant exons 6-15 (v1-v10) located in the middle of the CD44 gene can be selectively spliced ​​to produce a variety of CD44 variants (CD44v) subtypes, one of which is the CD44 variant 6 subtype CD44v6. In addition, 19 different splicing variants have been discovered, which are produced by selective splicing of CD44 mRNA, all of which are expressed at different levels in different tissues, and the effects of these variants are not yet fully understood. Preferably, CD44 is human CD44v6, so that the binding protein of the present invention specifically binds to human CD44v6.

[0067] CD44v6 is a non-internalizing cancer-associated splice variant of CD44 (hyaluronan receptor). High expression of CD44v6 has been found in a variety of cancers and is associated with poor prognosis and accelerated invasive disease. The expression of CD44v6 in normal tissues is restricted to the suprabasal spinous epithelial layer, more specifically in keratinocytes. Therefore, while CD44 is widely expressed in most vertebrate cells, the expression of CD44v6 is restricted to a few tissues and is thought to be associated with tumor progression and metastasis. Therefore, the low expression levels in healthy tissues, coupled with overexpression on a variety of different cancer types, make CD44v6 a promising target for molecular radiotherapy.

[0068] The binding protein herein can be joined, for example, by gene fusion, conjugation or chemical connection to "reagent" (i.e., part with a certain characteristic), to form a conjugated or fused binding protein, wherein the binding protein and the reagent can be directly joined to each other, such as when the iodine (I) radioisotope is coupled to the binding protein, or can be joined via a joint (i.e., indirectly joined to each other), such as when lutetium (Lu) is coupled to the binding protein. Chelating agents (a form of indirect joining) can be used to couple the reagent, wherein the chelating agent is joined to the binding protein and chelates the reagent. The reagent can be a radioisotope, a photoactivated compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytotoxic agent, a prodrug, a binding molecule with different specificities, a cytokine, or another immunomodulatory or cytotoxic polypeptide.

[0069] The agent can be a therapeutic agent or a detectable imaging agent. The therapeutic agent or active pharmaceutical ingredient (API) that is joined, conjugated or connected to the binding protein can be a cytotoxic agent that contains or consists of one or more radioactive isotopes and / or one or more cytotoxic drugs. The term "radioactive isotope" may also be referred to as a "radionuclide" and refers to a nuclide that has excess nuclear energy, making it unstable and susceptible to radioactive decay. One or more radioactive isotopes are each independently selected from the group consisting of beta-emitters, Auger emitters, conversion electron emitters, alpha-emitters, and low photon energy emitters, and may each independently have an emission mode that locally absorbs energy that produces high-dose absorbance near the agent. One or more radioactive isotopes are each independently selected from the group consisting of: long-range beta emitters, such as 90 Y. 32 P. 186 Re / 188 Re; 166 Ho, 76 As / 77 As, 153 Sm; medium-range-beta emitters such as 131 I. 177 Lu, 67Cu, 161 Tb, 47 Sc; low energy beta emitters, such as 45 Ca, 35 S or 14 C; conversion or Auger emitters, such as 51 Cr, 67 Ga, 99 TC m , 111 In, 123 I. 125 I. 201 TI, as well as α-emitters such as 212 Bi, 212 Pb, 213 Bi, 223 Ac, 225 Ac, 227 Th, 149 Tb and 211 At.

[0070] Binding proteins or conjugated binding proteins comprising therapeutic agents of the present disclosure can be used in medicine and therapy for 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 brain metastases, including metastatic forms of the other cancers, including cases where metastases have already formed. Since CD44v6 is associated with angiogenesis, which refers to the formation of new blood vessels from existing blood vessels, which is essential for tumor growth and other conditions, the binding proteins of the present invention can also be used as anti-angiogenic agents in the treatment of cancer and other angiogenesis-related disorders.

[0071] In other aspects, the binding protein can be connected to an imaging agent. Molecular imaging that combines an imaging agent with a targeting moiety in the form of a binding protein can be used to specifically image the diseased site in the body. Binding proteins can be used for molecular imaging to target imaging agents (such as radionuclides) to cells of interest in the body. This provides the ability to monitor disease progression and predict responses to specific therapeutic agents, thereby enabling diagnosis and response prediction of any tissue and disease expressing / overexpressing an antigen. Imaging agents / detectable agents can be radioisotopes, enzymes, fluorescent molecules, dyes, digoxin, and biotin, among others. Detectable agents can be detected by imaging techniques such as SPECT, PET, MRI, optical or ultrasound imaging. When detectable agents are radioisotopes, they can be selected from 111 In, 99m Tc, 67Ga, 68 Ga, 72 As, 89 Zr, 123 I. 125 I. 124 I. 47 Sc and 201 TI. The conjugated binding protein may contain a pair of detectable and cytotoxic radioisotopes, such as 86 γ / 90 γ, 111 In / 177 Lu or 125 I / 211 At, where the radioisotope is able to act both as a detectable agent and as a cytotoxic agent in a multimodal manner.

[0072] Binding proteins have been shown to bind CD44v6 with high affinity, and therefore can be used as independent cancer therapeutic agents or as conjugated binding proteins, as described above. The binding proteins disclosed herein can also be used to design chimeric antigen receptors (CARs) for CD44v6 to obtain CAR cells targeting CD44v6, such as CAR-T cells. Then, these CAR T cells targeting CD44v6 can be used to eliminate the therapy of cells (such as cancer cells) expressing CD44v6. For example, by isolating T cells from a subject, inserting CAR genes in T cells to produce CAR T cells expressing CAR proteins to prepare CAR T cells, wherein CAR is a hybrid of T cells and antibody receptors comprising 4 different regions; the extracellular domain of the recognition antigen (usually the scFv fragment of an antibody) is connected to a transmembrane domain by a hinge (spacer), wherein the transmembrane domain has a hydrophobic α-helical structure, and wherein the transmembrane domain is connected to an internal domain (intracellular domain), which undergoes conformational changes after antigen recognition, thereby triggering downstream signaling pathways to induce an immune response. The intracellular domain may also include one or more costimulatory domains to enhance anti-tumor activity. Therefore, the present disclosure provides cells engineered to express chimeric antigen receptors (CARs), wherein CARs include antigen binding domains, transmembrane domains connected to antigen binding domains by hinge regions, and intracellular domains optionally connected to one or more costimulatory domains, wherein the antigen binding domains include scFv fragments of binding proteins. Cells may be human cells, immune effector cells, such as T cells, NK cells, or macrophages.

[0073] The present invention provides novel enhanced binding proteins that bind to human CD44v6. The binding affinity and therapeutic efficacy of the binding proteins of the present invention have been evaluated and it has been determined that they provide enhanced performance compared to other binding proteins in the art. One object of the present disclosure is to provide novel enhanced tumor targeting agents for improved cancer therapies, such as radiation therapy, to be able to find and characterize tumor / cancer cells, and also to destroy them. Figure 2 As shown, the binding proteins can be conjugated / linked to one or more agents as a payload and can localize tumor cells by recognizing and binding to targets present / overexpressed on tumor cells if the agent is, for example, an imaging agent or a detectable agent, or they can kill target cells by carrying a therapeutic agent (such as one or more radioisotopes, where radiation is the effector), e.g. Figure 3 As shown in , the binding protein conjugated with a radioisotope can be referred to as a radiopharmaceutical. The binding protein is introduced into the body by various means (such as injection or ingestion), locates the specific antigen (CD44v6), binds to the receptor, stays on the surface of the tumor cell or is internalized into the tumor cell, and exposes its payload of radioisotope to the tissue, where the radiation emitted by the radioisotope destroys the cancer cells, which is called molecular radiation therapy (MRT) or radionuclide therapy (RNT).

[0074] The binding proteins disclosed herein can be used to treat a variety of conditions associated with the expression / overexpression of the target antigen CD44v6 of the present invention, as discussed above and below. In particular, subjects with thyroid cancer, especially advanced thyroid cancer (TC), including anaplastic (ATC) and iodine-refractory TC, can benefit greatly from these treatments because they are orphan diseases that are resistant to standard cancer treatments and therefore no effective treatments are available. In most cases, thyroid cancer is a disease that can be cured by surgical treatment followed by adjuvant therapy with radioactive iodine. However, there are many cases that are refractory to current treatments and these patients have a very poor prognosis. The median survival after diagnosis of ATC is only 5 months, and therefore the unmet clinical needs are huge. It has been determined that CD44v6 is highly expressed on a significant proportion of tumor cells in these patients and is therefore a relevant target. Because tumor cells express CD44v6, advanced thyroid cancer can be treated with molecular radiotherapy using effective CD44v6-targeted radiopharmaceuticals, such as Figure 4 As shown. The binding proteins of the present disclosure (such as CD44v6-specific mAbs) are radionuclide-labeled to bring therapeutic amounts of radioactivity to tumor cells expressing CD44v6 while leaving normal, non-CD44v6-expressing tissues unharmed. In some embodiments, the radiolabeled binding protein conjugate contains a radionuclide for therapy. 177 Lu or for imaging111 DOTA chelate of In. 111 In / 177 Lu-DOTA is a well-characterized theranostic pair complex and Lutathera ( 177 Lu-DOTATATE) is approved in the United States and the European Union for the treatment of neuroendocrine cancer.

[0075] This target has been previously clinically validated for radioimmunotherapy, has tumor-specific uptake, and is well tolerated. The experimental data as summarized herein are further described in more detail in the Examples section below. PK / PD studies have been performed in mice, and the results show that the binding protein has an appropriate half-life, normal tissue distribution, and tumor targeting ability. The experimental data also confirmed that no significant effects of toxicity were observed in mice carrying ATC, such as Figure 5a As shown, a dose of a radiopharmaceutical of the present disclosure (having 177 Lu as therapeutic agent) for up to 40 days, and Figure 5b Survival after one such dose is shown in . Clear and thoroughly characterized antigen-specific binding of the binding proteins of the present invention has been demonstrated, with no evidence of off-target binding or cross-reactivity. The radioconjugate has been evaluated in three species (mouse, rabbit, cynomolgus monkey), and dosimetry assessments have shown good dosimetry, with the bone marrow being the dose-limiting organ as expected. These studies also validated low uptake of 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, data not shown.

[0076] As described in more detail in the Examples section below, experimental data from target binding specificity testing showed binding of the binding protein and conjugated (radiolabeled) binding protein with high specificity and affinity for CD44v6, low risk of off-target binding, and in silico and in vitro assessments of off-target binding and selectivity showed no such occurrence. In silico, in vitro, and in vivo toxicology studies using three different animal models showed no toxic effects of the non-radioactive antibody at therapeutic levels, low risk of antibody-mediated effects (such as T cell activation), and low risk of off-target binding. Pharmacokinetic assessments in three different animal models also demonstrated feasible dosing and pharmacokinetic properties of the conjugate. SPR measurements demonstrated specific binding to CD44v6 with low nanomolar affinity (with or without DOTA conjugation), and mapping of the binding epitope verified by SPR measurements showed that the binding protein bound to the epitope defined as SEQ ID NO:7. Species specificity assessments demonstrated target binding in rabbit, cynomolgus monkey, and human CD44v6 peptides, verified by SPR measurements.

[0077] Thus, the binding proteins of the invention can be linked / conjugated to agents for imaging and / or therapy. As an example, 177 Lu-conjugated binding proteins can provide effective treatment for radioiodine-refractory thyroid cancers that express CD44v6. Such conjugated binding proteins have been shown to bind CD44v6 with high affinity, as demonstrated by radioimmunoassays on cultured thyroid cancer cells and squamous cell carcinoma cells. Real-time kinetic measurements on cultured thyroid cancer cells showed that the conjugated binding proteins specifically bind to CD44v6 with high affinity to antigen-positive cells and do not bind to antigen-negative cells. The conjugated binding proteins were tested in five and three xenograft mouse models with different tumor target expression, respectively. 125 I / 177 In vivo biodistribution assessment of the Lu-conjugated binding protein showed antigen-dependent tumor uptake and no off-target binding.

[0078] Thyroid cancer xenografts in mice 177 The efficacy study data of Lu-conjugated antibodies showed that at a single dose of 16.5 MBq / 50 μg radiolabeled antibody, 177 Lu-dependent and antigen-specific therapeutic effects (reduced tumor growth, even complete remission), with no observed toxicity. Data from two thyroid cancer mouse models showed dose-dependent and antigen-specific therapeutic effects, with no signs of toxicity (i.e., no mouse weight loss or behavioral changes). Experimental data further demonstrated that the radiation delivery efficacy was equivalent to and exceeded that of the clinically tested monoclonal antibody BIWA-4 in terms of cellular uptake in vitro and in vivo. In addition to improved antigen affinity, more suitable radionuclide labeling, and fully human form, the introduced LALA mutation of the binding protein ensures the lack of ADCC / CDC function and reduces off-target uptake in vivo, such as in the liver. SPR measurements of the binding protein disclosed herein showed that the FcγR1 binding of the silent ADCC / CDC of the LALA construct was severely reduced. These improvements provide significant advantages over previous humanized antibody-based radiopharmaceuticals targeting CD44v6. Although in this experiment 177 Lu has been used as a radioisotope for therapy, but any other radioisotope suitable for such treatment may be used with similar results expected.

[0079] Therefore, the present invention provides a novel 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, in particular for an epitope of the amino acid sequence WFGNRW (SEQ ID NO: 7). The binding protein of the present invention comprises an antibody binding domain, the binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) or a derivative thereof, VH and VL each comprising three complementary determining regions (CDRs), i.e. a total of six CDRs, wherein the binding protein specifically binds to CD44v6, and more specifically, they recognize an epitope of CD44v6 as defined in SEQ ID NO: (7). In some embodiments, the present invention relates to 21 binding protein variants that have similar sequences and all recognize the same epitope. Compared to the BIWA-4 epitope WFGNRWHEGY (SEQ ID NO: 5), the epitope bound by the binding protein of the present invention is as follows Figure 6 Thus, the binding protein may comprise a binding domain of an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) or any derivative thereof, wherein the derivative may be, for example, a single domain antibody comprising a single monomeric variable antibody domain. In some embodiments, the binding protein is an IgG antibody lacking a light chain, which consists of a heavy chain variable domain (VH) and a light chain variable domain (VL). H H) consists of two heavy chains.

[0080] The light chain variable domain and the heavy chain variable domain 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. These six CDRs have the following amino acid sequences:

[0081] VHCDR1 as defined by SEQ ID NO: 1;

[0082] VHCDR2 as defined by SEQ ID NO: 2;

[0083] VHCDR3 as defined by SEQ ID NO:3;

[0084] VLCDR1 as defined in SEQ ID NO:4;

[0085] Such as X 1 VLCDR2 defined by AS, where X 1 Can be T, A or S;

[0086] VLCDR3 as defined in SEQ ID NO:6.

[0087] Wherein VHCDR3 and VLCDR3 are identical in all 21 binding protein variants, while VHCDR1, VHCDR2, VLCDR1 and VLCDR2 contain some variations, as shown in the following Table 1. In addition to the CDRs according to SEQ ID NOs: 1-6, the present invention also encompasses CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto, which also bind to the same epitope of CD44v6.

[0088] Table 1

[0089] CDR SEQ ID NO sequence VHCDR1 SEQ ID NO:1 <![CDATA[GFX 3 FX 5 X 6 X 7 A]]> VHCDR2 SEQ ID NO:2 <![CDATA[ISX 3 X 4 GX 6 ST]]> VHCDR3 SEQ ID NO:3 ARHYYSDSDYRSSAAMDY VLCDR1 SEQ ID NO:4 <![CDATA[QX 2 IX 4 X 5 Y]]> VLCDR2 N / A <![CDATA[X 1 AS]]> VLCDR3 SEQ ID NO:6 QQDLYLLT

[0090] The sequences of the CDRs of the binding proteins are shown in Table 1 in single-letter amino acid codes, where VHCDR1, VHCDR2, VLCDR1 and VLCDR2 contain sequence variations between the 21 binding protein variants, as indicated by X. In VHCDR1, X 3 Can be S or T, X 5 Can be S, R or G, X 6 Can be S or N, and X 7 Can be Y or F. In VHCDR2, X 3 Can be A or G, X 4 Can be S or G, and X 6 Can be T, S, Y, R, or G. In VLCDR1, X 2 Can be S, N or T, X 4 Can be A, S or G, and X 5 Can be S or N. In VLCDR2, X 1 It can be A, S or T, so that VLCDR2 is AAS, SAS or TAS.

[0091] In some aspects, some CDRs are surrounded by specific amino acids called framework amino acids (faa), which are conserved between different binding proteins with some small variations. Thus, VHCDR1, VHCDR2 and VLCDR2 of the binding protein can be present next to specific framework amino acids, wherein the CDR and framework amino acid sequences are selected from the group comprising the following:

[0092] VHCDR1 and faa defined by SEQ ID NO:8;

[0093] VHCDR2 and faa as defined in SEQ ID NO:9;

[0094] VLCDR2 and faa as defined in SEQ ID NO: 10;

[0095] As shown in Table 2 below. In addition to the CDR+faa according to SEQ ID NO: 8-10, the present invention also encompasses CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto, which also bind to the same epitope of CD44v6.

[0096] Table 2

[0097] CDR SEQ ID NO sequence VHCDR1 and faa SEQ ID NO:8 <![CDATA[GFX 3 FX 5 X 6 X 7 AMS]]> VHCDR2 and faa SEQ ID NO:9 <![CDATA[X 1 ISX 4 X 5 GX 7 STX 10 ]]> VLCDR2 and faa SEQ ID NO:10 <![CDATA[X 1 ASX 4 ]]>

[0098] For VHCDR1 and faa, the sequence is the same as VHCDR1, but with two additional framework amino acids MS at the ends. In VHCDR2 and faa, an additional faa is added at the beginning and end of the sequence, where X 1 Can be A or T, and X 10 Can be Y or F. The remaining unknown sequence in the middle corresponds to the VHCDR2 sequence in Table 1 above. In VLCDR2 and faa, the additional faa X 4 It can be S, T, N or I, and the remaining unknown sequences correspond to the sequences in Table 1 above.

[0099] In some aspects, 21 different binding proteins are provided having various CDR combinations as shown in Tables 3-4 below, wherein U-MN114-19 is the parental clone and the other 20 binding proteins are affinity matured versions thereof.

[0100] Table 3

[0101] Clone Name VHCDR1 VHCDR2 U-MN114-19 GFTFSSYA (SEQ ID NO: 12) ISGSGGST (SEQ ID NO: 32) AL-MN114-465 GFSFGSYA (SEQ ID NO: 11) ISAGGSST (SEQ ID NO: (19) AL-MN114-451 GFTFSSYA (SEQ ID NO: 12) ISAGGYST (SEQ ID NO: 22) AL-MN114-452 GFSFSSYA (SEQ ID NO: 13) ISAGGSST (SEQ ID NO: 19) AL-MN114-460 GFSFGSYA (SEQ ID NO: 11) ISAGGSST (SEQ ID NO: 19) AL-MN114-423 GFTFSSYA (SEQ ID NO: 12) ISAGGTST (SEQ ID NO: 21) AL-MN114-145 GFSFSSFA (SEQ ID NO: 14) ISAGGSST (SEQ ID NO: 19) AL-MN114-429 GFSFSSFA (SEQ ID NO: 14) ISAGGSST (SEQ ID NO: 19) AL-MN114-435 GFTFSSYA (SEQ ID NO: 12) ISAGGSST (SEQ ID NO: 19) AL-MN114-439 GFSFGSFA (SEQ ID NO: 15) ISAGGSST (SEQ ID NO: 19) AL-MN114-71 GFSFRSFA (SEQ ID NO: 16) ISGGGSST (SEQ ID NO: 20) AL-MN114-79 GFTFRSFA (SEQ ID NO: 17) ISAGGSST (SEQ ID NO: 19) AL-MN114-93 GFSFGSFA (SEQ ID NO:15) ISAGGSST (SEQ ID NO:19) AL-MN114-126 GFTFSSYA (SEQ ID NO:12) ISAGGSST (SEQ ID NO:19) AL-MN114-42 GFTFSSYA (SEQ ID NO:12) ISGGGYST (SEQ ID NO:23) AL-MN114-18 GFSFGSFA (SEQ ID NO:15) ISGGGSST (SEQ ID NO:20) AL-MN114-19 GFSFGSYA (SEQ ID NO:11) ISGGGSST (SEQ ID NO:20) AL-MN114-132 GFTFSSYA (SEQ ID NO:12) ISGGGSST (SEQ ID NO:20) AL-MN114-23 GFSFRNFA (SEQ ID NO:18) ISGGGGST (SEQ ID NO:24) AL-MN114-14 GFTFSSYA (SEQ ID NO:12) ISGSGRST (SEQ ID NO:25) AL-MN114-444 GFTFRSFA (SEQ ID NO:17) ISGGGSST (SEQ ID NO:20)

[0102] The corresponding CDRs H1 and H2 of 21 different binding proteins are shown in Table 3. The CDRs of H3 (VHCDR3) of all 21 binding proteins are defined by the sequence ARHYYSDSDYRSSAAMDY (SEQ ID NO: 3).

[0103] Table 4

[0104]

[0105]

[0106] Table 4 shows the corresponding CDRs L1, L2 and L3 of 21 different binding proteins.

[0107] In some aspects, 21 different binding proteins are provided having various CDR combinations as described above, and the surrounding framework amino acids of VHCDR1, VHCDR2, and VLCDR2 as presented in Tables 5 and 6 below.

[0108] Table 5

[0109]

[0110]

[0111] Table 6

[0112] Clone Name VLCDR2 + faa U-MN114-19 AASS (SEQ ID NO:141) AL-MN114-465 TASS (SEQ ID NO:139) AL-MN114-451 TAST (SEQ ID NO:142) AL-MN114-452 SASS (SEQ ID NO:140) AL-MN114-460 SASS (SEQ ID NO:140) AL-MN114-423 AASS (SEQ ID NO:141) AL-MN114-145 SASN (SEQ ID NO:143) AL-MN114-429 AASS (SEQ ID NO:141) AL-MN114-435 AASS (SEQ ID NO:141) AL-MN114-439 AASS (SEQ ID NO:141) AL-MN114-71 AAST (SEQ ID NO:144) AL-MN114-79 AAST (SEQ ID NO:144) AL-MN114-93 AASS (SEQ ID NO:141) AL-MN114-126 AASS (SEQ ID NO:141) AL-MN114-42 SASI (SEQ ID NO:145) AL-MN114-18 TASN (SEQ ID NO: 146) AL-MN114-19 SASS (SEQ ID NO: 140) AL-MN114-132 AASS (SEQ ID NO: 141) AL-MN114-23 SASI (SEQ ID NO: 145) AL-MN114-14 AASS (SEQ ID NO: 141) AL-MN114-444 SASS (SEQ ID NO: 140)

[0113] The associated proteins of the present invention can be synthesized by any method known in the art. Preferably, a protein expression system is used, such as a cell expression system using a prokaryotic (e.g., bacterial) cell or a eukaryotic (e.g., yeast, fungi, insect or mammal) cell, to synthesize the associated proteins. Another protein expression system is a cell-free in vitro expression system, in which the nucleotide sequence encoding the associated proteins is transcribed into mRNA in vitro, and the mRNA is translated into protein in vitro. Cell-free expression system kits are widely available and can be purchased from, for example, Thermo Fisher Scientific. Alternatively, the associated proteins can be chemically synthesized in abiotic systems. Liquid phase synthesis or solid phase synthesis can be used to produce polypeptides that can form the associated proteins of the present invention or are included in the associated proteins of the present invention.

[0114] Technicians can easily produce associated proteins using appropriate methods commonly used in this area. Especially, associated proteins can be recombinantly expressed in mammalian cells such as CHO cells. Standard techniques in this area can be used to purify the associated proteins synthesized in protein expression systems, for example, it can be synthesized with affinity tags and purified by affinity chromatography. If the associated protein is an antibody, one or more antibody-associated proteins (such as protein G, protein A, protein A / G or protein L) can be used, and affinity chromatography is used to purify.

[0115] As mentioned above, the binding protein is a molecule or antibody-like molecule based on an antibody. Therefore, 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 (such as a single-chain antibody). In a preferred embodiment, the binding protein is a human protein (humanized compared to humanization), particularly a human monoclonal antibody, an antibody fragment or a scFv. People's binding proteins can include VH and VL regions, wherein both framework regions and CDR regions are derived from human germline immunoglobulin sequences, and if the constant region is included in the protein, human constant regions may also be included. However, such proteins may include amino acids not encoded by human germline Ig sequences, such as mutations introduced by random or site-specific mutagenesis.

[0116] As described in detail above, the binding protein of the present invention comprises a binding domain of an antibody, which comprises a heavy chain variable domain (or variable region) and a light chain variable domain. Therefore, in a specific embodiment, the binding protein of the present invention comprises:

[0117] (i) a heavy chain variable domain (VH) comprising (or consisting of) an amino acid sequence as shown in any one of SEQ ID NOs: 35-54 and 147 or a variant thereof; and

[0118] (ii) a light chain variable domain (VL) comprising (or consisting of) an amino acid sequence shown in any one of SEQ ID NOs: 55-74 and 148 or a variant thereof.

[0119] In some embodiments, the binding protein comprises a heavy chain and a light chain comprising a variable region and a constant region, wherein the binding protein of the invention comprises:

[0120] (i) a heavy chain comprising (or consisting of) an amino acid sequence shown in any one of SEQ ID NOs: 34, 75-93 and 149, or a variant thereof; and

[0121] (ii) a light chain comprising (or consisting of) the amino acid sequence shown in any one of SEQ ID NOs: 94-113 and 150 or a variant thereof.

[0122] Variants are defined as sequences with 80% or more, such as 85%, 90%, 95% or more identity thereto. The condition is that the CDR sequence of the variant is unchanged with respect to the antibody variant defined by the VH or VL domain, i.e., does not contain sequence variation, or wherein the sequence variation of the CDR amino acid sequence is at most 5%, such as 4%, 3%, 2%, 1% or less, or the CDR sequence variation is made to have at least 95%, such as 96%, 97%, 98%, 99% or more sequence identity with the defined sequence. The binding proteins with variants of the sequence of the variable and / or constant domains are functional variants with the above-mentioned activity (i.e., they specifically bind to the defined epitope of CD44v6). The variant sequence can be modified relative to the native sequence by substitution, insertion and / or deletion of one or more amino acids.

[0123] Sequence identity can be assessed by any convenient method. However, in order to determine the degree of sequence identity between sequences, it is useful to perform a computer program for pairwise or multiple alignment of sequences, such as EMBOSS Needle or EMBOSSstretcher (both Rice, P. et al., Trends Genet., 16, (6) pp276-277, 2000) can be used for paired sequence alignments, while Clustal Omega (Sievers F et al., Mol. Syst. Biol. 7:539, 2011) or MUSCLE (Edgar, RC, Nucleic Acids Res. 32 (5): 1792-1797, 2004) can be used for multiple sequence alignments, although any other appropriate program can be used. Whether the alignment is paired or multiple, it must be performed globally (i.e., over the entire reference sequence) rather than locally. Sequence alignments and % identity calculations can be determined using, for example, standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1. Alternatively, standard EMBOSSNeedle parameters can be used: matrix BLOSUM62, gap opening penalty 10, gap extension penalty 0.5. Any other suitable parameters may be used alternatively.

[0124] In some aspects, the present disclosure encompasses conjugated binding proteins, such as antibody drug conjugates. Thus, the present disclosure provides a conjugated binding protein comprising: (i) at least one binding protein as described above and below; and (ii) at least one agent, wherein the at least one agent is conjugated to the binding protein, wherein the binding protein and the agent are directly or indirectly conjugated.

[0125] In a further aspect, the present invention provides a pharmaceutical composition comprising a binding protein of the present invention as described above, or a conjugated binding protein as described above, or an engineered CAR cell as described above. In addition, the pharmaceutical composition further 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 delay agents that are physiologically compatible.

[0126] Preferably, the carrier or excipient is suitable for parenteral, e.g., intradermal, intravenous, intramuscular or subcutaneous administration (e.g., by injection or infusion). Depending on the route of administration, the binding protein or conjugated binding protein may be coated in a material to protect them from the action of acids and other natural conditions that may inactivate or denature them.

[0127] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in pharmaceutical compositions, test kits, and products include water, buffered water, and 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). Suitable fluidity can be maintained, for example, by using coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. In many cases, isotonic agents are preferably included, such as sugar, polyols (such as mannitol), sorbitol, sodium chloride, etc.

[0128] The pharmaceutical composition of the binding protein, the binding protein of conjugation or the binding protein of conjugation can be used one or more in multiple methods known in the art via one or more administration routes. As the technician will understand, the administration route and / or mode will change according to the desired result. Preferred administration routes include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, such as by injection or infusion, such as directly applied to the tumor site. As used herein, the phrase "parenteral administration" means the mode of administration except intestinal and topical administration, usually by injection. Alternatively, non-parenteral routes can be used, such as local, epidermal or mucosal administration routes. Preferably, topical administration includes peritumoral, paratumoral, intratumoral, intralesional, perilesional, intracavitary infusion, intracapsular administration and suction. However, the conjugate of antigen-binding proteins and engineered cells can also be systemically administered.

[0129] The appropriate dosage of the specific binding protein of the present invention, the conjugated binding protein or the pharmaceutical composition can be determined by a skilled practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition and product of the present invention can be changed to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response for a specific subject (i.e., patient) and is nontoxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific protein / conjugate employed, the route of administration, the time of administration, the excretion rate 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 treated patient, and similar factors well known in the medical field.

[0130] The suitable dosage of the associated protein of the present invention or the associated protein of conjugation can be, for example, in the range of about 0.1 μ g / kg to about 100 mg / kg of the patient's body weight to be treated. For example, a suitable dosage can be about 1 μ g / kg to about 20 mg / kg body weight per administration or about 10 μ g / kg to about 10 mg / kg body weight per administration. For the associated protein of the conjugation carrying a radioactive isotope, the dosage can be given at certain intervals, such as once every two weeks (every two weeks). For other purposes, a shorter interval (such as a daily dose) may be more suitable. The suitable intervals for different types of treatments are obvious to the technician.

[0131] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as single doses for a subject to be treated; each unit contains a predetermined amount of active compound, calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier.

[0132] The binding protein or conjugate / composition can be administered in single dose or multiple dose form. Multiple doses can be administered via the same or different routes and administered to the same or different positions. Alternatively, they can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency can vary according to the half-life of the administered substance in the patient and the required duration of treatment. The dosage and frequency of administration can also vary according to whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses can be administered at relatively infrequent intervals over a long period of time. In therapeutic applications, relatively high doses can be administered, for example, until the patient shows partial or complete improvement of disease symptoms. In an exemplary dosage regimen, in a cycle repeated 2 to 10 times, the conjugated binding protein is administered to the subject once a week, once every two weeks, or once every three weeks.

[0133] Although the splice variant CD44v6 is mainly expressed in tumor cells, homogeneous expression of CD44v6 is shown in many cancers, and it is expressed only in normal tissues expressed in a subgroup of epithelial tissues (keratinocytes), a pre-targeted therapy scheme can be used. In the pre-targeted therapy scheme, the binding protein itself does not carry a payload (agent), but it is carried by a second molecule. The binding protein comprising a molecule attachment tag in this embodiment is applied to the subject and is bound to the target (CD44v6). The unbound binding protein is then allowed to be removed from the system naturally or after the injection of a scavenger, and after the unbound binding protein leaves the circulation, a second molecule carrying a payload (i.e., a binding therapeutic agent) is administered. The second binding molecule binds to the molecule attachment tag of the binding protein that binds to the target, thereby delivering the payload to the target. Thus, a method of treating a subject in need thereof is provided, the method comprising administering a first binding protein comprising a molecular attachment tag, allowing any unbound binding protein to leave the subject's circulation, administering a therapeutically effective amount of a second molecule, wherein the second molecule is conjugated to a therapeutic agent, and wherein the second molecule binds to the first binding protein, thereby delivering the therapeutic agent to the CD44v6 epitope bound by the first binding protein.

[0134] Therefore, the content of the present disclosure enables the treatment, imaging and diagnosis of diseases associated with CD44v6 expression, such as cancer, by administering the conjugated binding proteins of the present invention. In the drawings and description, exemplary aspects of the present disclosure have been disclosed. However, many changes and modifications may be made to these aspects without substantially departing from the principles of the present disclosure. Therefore, the present disclosure should be considered illustrative rather than restrictive, and is not to be considered limited to the specific aspects discussed above. Therefore, although specific terms are employed, they are used only in a general and descriptive sense, and not for limiting purposes.

[0135] The description of the example embodiments provided herein is presented for the purpose of illustration. The description is not intended to be exhaustive or to limit the example embodiments to the precise form disclosed, and according to the above teachings, modifications and changes are possible, or can be obtained from the practice of various alternatives to the embodiments provided. The examples discussed herein are selected and described in order to explain the principles and properties of various example embodiments and their practical applications, so that those skilled in the art can utilize the example embodiments in various ways and various modifications suitable for the intended specific purposes. The features of the embodiments described herein can be combined in all possible combinations of methods, products and systems. It should be understood that the example embodiments presented herein can be practiced in any combination with each other. It should also be noted that the word "comprising" does not necessarily exclude the presence of other elements or steps other than those listed, and the word "one" or "one" before the element does not exclude the presence of multiple such elements. It should also be noted that any figure mark does not limit the scope of the claim, and the example embodiments can be implemented in the broadest sense of the claim.

[0136] Examples

[0137] In brief, CD44v6 binding antibodies have been selected and affinity matured. Candidates have been subjected to epitope mapping and extensive evaluation in vitro and in vivo, validation of radionuclide labeling, in vivo kinetics and dosimetry, and successful molecular radiotherapy experiments in several thyroid cancer mouse xenografts.

[0138] Tables 7-9 below summarize relevant in silico, in vitro, and in vivo experiments performed on selected MN114 antibodies of the present disclosure after selection and format conversion, with selected example experiments outlined in more detail below. Many of these experiments are also described in more detail in the following sections related to pharmacology, toxicology, and pharmacokinetics. If not otherwise specified below, the antibody format is IgG1 LALA.

[0139] Table 7: Experiments performed on selected non-radionuclide labeled MN114 antibodies

[0140]

[0141]

[0142] Table 8: In vitro experiments performed on selected radionuclide labeled MN114 antibodies

[0143]

[0144] Table 9: In vivo experiments performed on selected radionuclide labeled MN114 antibodies

[0145]

[0146]

[0147] Table 10: List of proteins and peptides used in the following examples

[0148]

[0149] Example 1: Phage display selection of human CD444v6 using a human scFv library

[0150] Phage display selections were performed to enable the isolation of scFv fragments specific for human CD44v6.

[0151] Phage display selection

[0152] Biopanning was performed using four rounds of enrichment using the original human synthetic scFv phage library SciLifeLib2 (SciLifeLab, Stockholm, Sweden), which was designed and constructed similarly to that reported previously ( et al., Protein Eng Des Sel (2016) 29: 427-437). In short, human germline genes IGHV3-23 and IGKV1-39 were used as library scaffolds, and Kunkel mutagenesis was used to introduce diversity into four of the six CDRs, namely VHCDR1, VHCDR2, VHCDR3, and VLCDR3. Selection was performed using chemically biotinylated human CD44v6 (referred to as bio-CD44v6) and streptavidin-coated magnetic beads (Dynabeads M-280, ThermoFisher Scientific, #11206D). The selection pressure was increased by gradually reducing the amount of antigen (200nM to 10nM) and by increasing the intensity and number of washes between different rounds. In order to remove non-specific or streptavidin binders and increase the possibility of selecting v6 specific binders, pre-selection was performed by incubating the phage stock on streptavidin beads with biotinylated human CD44 (subtype 6) (referred to as bio-CD44) before the 1st, 2nd and 3rd rounds of selection. The specifications of the various antigens used are shown in Table 10. In addition, 2% bovine serum albumin (BSA) was included as a blocking agent throughout the selection process. Antigen-bound phages were eluted using the trypsin-aprotinin method. The entire selection process was automated and performed by a Kingfisher Flex robot. The recovered phages were propagated in XL1 blue Escherichia coli and grown on agar plates at 37°C overnight (round 1) or in solution at 30°C overnight (rounds 2, 3 and 4). The phage stock was prepared by infecting with an excess of M13K07 helper phage (New England Biolabs, #N0315S) and inducing scFv expression by adding IPTG. Overnight cultures were PEG / NaCl precipitated, resuspended in selection buffer and used for the next round of selection.

[0153] Recloning and expression of scFv

[0154] To allow the production of soluble scFv fragments, phagemid DNA from rounds 3 and 4 of selection was isolated. In the pool, the gene encoding the scFv fragment was restriction enzyme digested and subcloned into a screening vector that provides a signal for secretion of the scFv fragment as well as a triple FLAG tag and a six-histidine (His) tag at the C-terminus. The construct was subsequently transformed into TOP10 E. coli. Single colonies were picked, cultured, and soluble scFv expression was induced with IPTG in a 96-well format. A total of 189 scFv clones present in the bacterial supernatant were prepared for ELISA screening.

[0155] ELISA screening

[0156] CD44v6 and two negative control proteins CD44 and streptavidin were fixed directly or indirectly through streptavidin at a concentration of 1 μg / ml to a 384-well ELISA plate. The scFv clones present in the bacterial supernatant were diluted 1:10 in blocking buffer (PBS supplemented with 0.5% BSA + 0.05% Tween20) and allowed to bind to the coated protein. Binding can be detected by HRP-conjugated α-FLAG M2 antibody (Sigma-Aldrich #A8592) followed by incubation with TMB-ELSA substrate (ThermoFisherScientific #34029). The generation of the colorimetric signal was stopped by adding 1M sulfuric acid and the plate was analyzed at a wavelength of 450nm. All samples were measured in duplicate.

[0157] DNA Sequencing

[0158] 95 scFv clones showing binding to CD44v6 and / or CD44 were sent to Eurofins / GATC Biotech (Cologne, Germany) for Sanger DNA sequencing.

[0159] result

[0160] Phage display selections were performed against the antigen human CD44v6 using the scFv phage library SciLifeLib 2. A total of 189 scFv clones were picked from the 3rd and 4th rounds of selection after recloning the selected scFv fragments into the screening vector. ELISA screening generated 95 potential scFv positive hits. DNA sequencing of these hits led to the identification of 17 sequence-unique scFv clones.

[0161] Example 2: Kinetic screening of 17 scFvs with unique sequences by SPR

[0162] A kinetic-based screening approach was performed on 17 sequence-unique scFv clones from Example 1 by surface plasmon resonance (SPR) to enable ranking of the different clones.

[0163] Materials and Methods

[0164] Kinetic screening was performed on a Biacore T200 instrument (Cytiva). According to the manufacturer's recommendations, the α-FLAG M2 antibody (Sigma-Aldrich # F1804) used as a capture ligand was immobilized on all four surfaces of a CM5-S amine sensor chip. The scFv clones present in the bacterial supernatant were injected and captured on the chip surface, followed by injection of 50nM CD44v6 or 50nM CD44 (negative control). The surface was regenerated with 10mM glycine-HCl pH 2.1. All experiments were performed at 25°C in running buffer (HBS supplemented with 0.05% Tween20, pH 7.5). The response curve sensorgram was obtained by subtracting the response curves of the reference surface (α-FLAG M2 antibody immobilized surface) and the blank run (injection of running buffer instead of antigen). The data were analyzed using Biacore T200 Evaluation 3.1 software and a 1:1 Langmuir binding model.

[0165] result

[0166] Eleven of the 17 clones analyzed were considered promising based on binding to CD44v6. More specifically, high binding responses and favorable slow off-rates were considered.

[0167] Example 3, conversion to full antibody format

[0168] Eleven scFv clones that appeared to specifically bind to the v6 region of CD44v6 were selected for conversion to a full-length human IgG4S228P (EU numbering) antibody format. The rationale for including these specific clones was based on performance in a panel of binding assays (see Examples 1 and 2). In addition, the positive control BIWA-4 and isotype control antibodies were similarly converted.

[0169] Fusion cloning, transfection into HEK293, expression and purification

[0170] The VH and VL regions of the selected scFv clones were PCR amplified using the In-Fusion HD Plus cloning kit (Clontech #638909) and inserted into the internally constructed vector pHAT-hIgG4-S241P. Transfection of plasmid DNA into expiHEK293 cells was performed in 4 ml culture using the ExpiFectamineTM 293 Transfection Kit (ThermoFisher Scientific #A14525). Cultures were harvested after 5 days of cultivation (37°C, 6% CO2, 80% rH, 400rpm) and antibodies were purified on protein A-conjugated magnetic beads (ThermoFisher Scientific #88846) using a Kingfisher Flex instrument. The buffer was exchanged for PBS, 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 antibodies, and the concentration was determined using an Implen NP80 UV-Vis spectrophotometer.

[0171] ELISA

[0172] CD44v6, human (hm) v6 peptide and negative control CD44 and streptavidin were fixed directly or indirectly to 384-well ELISA plates at a concentration of 1 μg / ml through streptavidin. Purified IgG4 S228P clones were diluted to 1, 0.2 or 0.04 μg / ml in blocking buffer (PBS supplemented with 0.5% BSA+0.05% Tween20) and bound to the coated protein. Binding can be detected by incubation with HRP-conjugated α-human κ antibody (Southern Biotech #9230) followed by TMB-ELISA substrate (ThermoFisher Scientific #34029). The generation of colorimetric signals was stopped by adding 1M sulfuric acid and the plate was analyzed at a wavelength of 450nm. All samples were measured in duplicate.

[0173] result

[0174] All 11 scFv clones and BIWA-4 were successfully re-cloned into human IgG4 S228P format, expressed in expiHEK293 cells and purified by protein A-conjugated magnetic beads on a Kingfisher Flex instrument. All antibodies had the expected molecular weight and acceptable purity levels by SDS-PAGE analysis and ELISA confirmed retention of binding to the v6 region of CD44v6, i.e. positive signals were obtained for CD44v6 and hm v6 peptides, whereas no binding was detected on CD44 and streptavidin. No binding was detected for the isotype control.

[0175] Example 4, Kinetic Measurement of Novel Anti-CD44v6 IgG4 Antibody

[0176] The kinetic constants of purified IgG4 clones (Example 3) for CD44 and v6 peptides were determined by surface plasmon resonance (SPR) using the single cycle kinetic (SCK) method.

[0177] Materials and Methods

[0178] Kinetic measurements were performed on a BIAcore T200 instrument (Cytiva) using the SCK method. According to the manufacturer's recommendations, α-human κ antibodies (GE Healthcare #28958325) used as capture ligands were fixed to all four surfaces of the CM5-S amine sensor chip. Injection and capture antibodies were performed in equal response units (RU). A four-fold dilution series of CD44v6 was prepared in running buffer, consisting of five concentrations ranging from 80nM to 0.3nM, and injected onto the chip surface in sequence. A single injection of 100nM CD44 (negative control) was also performed.

[0179] For kinetic measurements of human v6 peptide, v6 peptide was immobilized at approximately 20 RU using a streptavidin (SA) sensor chip. A five-fold dilution series of each antibody, consisting of five concentrations ranging from 50 nM to 0.08 nM, was prepared in running buffer and sequentially injected onto the chip surface.

[0180] 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. The response curve sensorgram was obtained by subtracting the response curve of the reference surface (α-human κ antibody immobilized surface or streptavidin immobilized surface) and the blank run (injection of running buffer instead of antigen or antibody). Data were analyzed using Biacore T200 Evaluation 3.1 software and 1:1 Langmuir binding model.

[0181] result

[0182] The transformed U-MN114-19 showed retained binding to CD44v6 and hm v 6 peptides. As expected, the reference antibody BIWA-4 hIgG4 also showed binding to CD44v6 and hm v 6 peptides. The apparent affinity (expressed as appKD) was in the low to sub-nanomolar range. U-MN114-19 bound to CD44v6 and hm v 6 peptides with higher affinity than the reference BIWA4 hIgG4, Table 11.

[0183] Table 11: Measured kinetic parameters ka (M-1s-1), kd (s-1), Kd (s-1) of U-MN114-19 IgG4 and BIWA-4 IgG4 against CD44v6 and hm v6 peptides D (M).

[0184]

[0185] Example 5, IgG4 Cell binding of U-MN114-19

[0186] After radiolabeling, all clones that were successfully transformed and produced as IgG4 were assessed for cell binding. For simplicity, data from U-MN114-19 and BIWA4 will be presented in this example.

[0187] Materials and Methods

[0188] 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 PBS) Pierce iodination tube (ThermoFisher) containing 50 μL PBS. The iodine was incubated in the tube for 6 min, gently swirled every 30 s, and then transferred to an Eppendorf tube containing 10 μg of MN114 antibody (hIgG4). The antibody / iodine reaction was incubated at 37°C and 350 rpm for 15 min. The labeling yield was determined using ITLC. 177 Lu-labeling: U-MN114-19 and BIWA-4 were first conjugated with DOTA using p-SCN-Bn-DOTA according to the manufacturer's instructions. Ultra 0.5mL centrifugal filters (Sigma) were used to convert PBS to Na 2 HPO 4 (0.1M, pH 7.5-7.9, metal-free H 2 0) buffer switch. A ten-fold molar excess of 2 HPO 4(0.1M, ph 7.5-7.9, metal-free H 2 0) was added to the antibody and incubated at 37°C and 350 rpm for 4 h. Ultra filter to purify the antibody from excess p-SCN-Bn-DOTA. 177 Lu (Curium Pharma) was added to 50 μg of antibody and incubated at 42°C and 350 rpm for 2 h. 177 Lu radiolabeling. Labeling yield was determined by ITLC.

[0189] All LigandTracer experiments were performed according to the manufacturer's standard protocol. 5 BHT-101 or ACT-1 cells were seeded in 10 cm culture dishes and incubated at 37 °C and 5% CO before the start of the experiment. 2 The culture dish was placed in a LigandTracer instrument (grey or yellow) and a baseline was established for approximately 30 min before the first concentration was added. Each concentration (1 nM and 3 nM or 1 nM, 3 nM and 10 nM for U-MN114-19 and 1 nM, 3 nM and 10 nM for BIWA-4) was run for approximately 90 min at room temperature. All culture media containing radioactive antibodies were removed and retention was started in 3 mL of fresh culture media. For kinetic measurements, the total experimental run time was 12 h.

[0190] result

[0191] In use 125 I and 177 After Lu labeling, U-MN114-19 exhibited low affinity binding to CD44v6-positive cell lines with different antigen expression levels. Representative examples of iodinated antibodies on BHT-101 cells are shown in Figure 2. Figure 7 Among all cell lines, U-MN114-19 showed lower or equal K compared with BIWA-4. D On high antigen expressing cell lines, the difference was minimal, but on low / medium antigen expressing cell lines (e.g. BHT-101), U-MN119-14 performed better ( Figure 7 ).

[0192] Example 6, Comparison of biodistribution between U-MN114-19 and BIWA-4

[0193] The lead candidate for U-MN114 antibody selection (U-MN114-19) was evaluated in vivo in balb / c nu / nu mice in direct comparison with BIWA-4.

[0194] Materials and Methods

[0195] Animal studies were performed in accordance with Swedish laws and regulations using ethical approvals C33 / 16, C9 / 16 and 10966 / 20. For inoculation of tumor cells (ACT-1), approximately 10 mice were placed in the 1% sera per mouse after harvesting the cells with trypsin. 7 Cells were injected into 100 μL serum-free medium in the right hind flank of mice. Radiolabeling was performed as described in Example 5. 8-10 days after inoculation of tumor cells, radiolabeled U-MN114-19 or BIWA4 (both in IgG4 form) was injected intravenously (iv) in the tail vein. A total of 15 μg U-MN114-19 or BIWA4 was injected, consisting of 1-2 μg radiolabeled antibody and 13-14 μg non-radiolabeled antibody, a total of 15 μg / 50 μL per mouse. The activity injected per mouse was between 100-300 kBq. Animals were euthanized and dissected 24h, 48h and 192h (8 days) after injection (pi). Organs were analyzed in a Wizard 1460 well counter (PerkinElmer) and the %ID of organ weight (g) was calculated.

[0196] result

[0197] Repeated evaluation 125 The biodistribution of IU-MN114-19 was measured and reproducible results were obtained. 125 Direct comparison of I-BIWA-4 ( Figure 8a and 8b )middle, 125 The peak tumor uptake of IU-MN114-19 was significantly higher than 125 I-BIWA4, and with 125 Compared with I-BIWA4, 125 The total tumor area under the curve was significantly greater for IU-MN114-19.

[0198] in conclusion

[0199] In ACT-1 tumor-bearing mice, 125 IU-MN114-19 showed superior 125 The tumor-to-blood ratio and greater peak tumor uptake of I-BIWA4. The biodistribution results did not reveal any off-target binding or accumulation of the antibody, indicating a stable and specific compound. The results suggest that U-MN114-19 has greater therapeutic utility than BIWA4.

[0200] Example 7, Binuclear Biodistribution of U-MN114-19

[0201] use 125 I and 177The dual-nuclide study of Lu was used to determine whether the antibody was more effective in terms of tumor targeting, total tumor dose, and safety of using halogens or radioactive metals. This data was then used for dosimetry calculations (data not shown).

[0202] Materials and Methods

[0203] Animal studies were performed 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 was injected per mouse, with 1 μg 125 IU-MN114-19 (100 kBq) and 1 μg 177 The cells were composed of Lu-U-MN114-19 (100 kBq) diluted with 13 μg of unlabeled U-MN114-19. Animals were euthanized and dissected at 1 h, 24 h, 48 h and 168 h post-injection (pi). Organs were analyzed in a Wizard 1460 well counter (PerkinElmer) and %ID of organ weight (g) was calculated.

[0204] result

[0205] At all points in time, 177 The peak tumor uptake of Lu-U-MN114-19 was significantly higher than 125 IU-MN114-19, resulting in a higher tumor-to-blood ratio ( Fig. 9 ). Validation of dosimetry assessment based on biodistribution data, 177 Lu will be a more suitable therapeutic nuclide in the future. Fig. 9 Shown; top left: 125 Biodistribution of IU-MN114-19 (IgG4), bottom left: in ACT-1 xenografts 177 Tumor to organ ratios of biodistribution of Lu-U-MN114-19 (IgG4). Top right: 177 Biodistribution of Lu-U-MN114-19 (IgG4), bottom right: 177 Tumor to organ ratio of Lu-U-MN114-19 (IgG4). Error bars represent SD, N=15.

[0206] in conclusion

[0207] 177 Lu-U-MN114-19 outperformed 125 IU-MN114-19. Dual nuclide studies have shown that 177 Lu may be the most effective therapeutic radionuclide for future research because it is 131Compared with 1, it has significantly higher tumor uptake and lower cross-fire dose to healthy tissues.

[0208] Example 8, Affinity Maturation of U-MN114-19

[0209] Clone U-MN114-19 was matured with the goal of generating a clone with improved affinity for the v6 region within CD44v6.

[0210] Library design and construction

[0211] Since U-MN114-19 was originally derived from a scFv library (Example 1), the scFv format was chosen as the scaffold for library generation. The affinity maturation library, denoted MN114-19-Lib1 (Lib1), was diversified in four of the six CDR loops; VHCDR1, VHCDR2, VLCDR1, and VLCDR2. CDRH3 of VH and VL are generally considered to be the most important regions for antigen binding. It is speculated that these loops may also be important for target interactions of U-MN114-19 and are therefore kept constant. Next-generation sequencing of the natural library showed that antibody evolution through somatic hypermutation occurs through a defined path based on the germline gene origin of the antibody (DOI: 10.3389 / fimmu.2018.01391 ). The spatial and chemical diversity introduced in Lib1 was inspired by these in vivo evolutionary patterns. The 13 most substituted positions in VHCDR1 and VHCDR2 of the natural IGHV3-23 library were targeted for mutation, and similarly, the 5 most mutated positions in VLCDR1 and VLCDR2 of IGKV1-39 were targeted. Furthermore, the amino acid composition of each of these positions was inspired by the natural library. In total, this procedure resulted in targeting 18 positions in MN114-19 Lib1, generating approximately 3.2x10 8 Combinatorial theoretical diversity of variants.

[0212] Kunkel mutagenesis was used to introduce library diversity into the scaffold genes, essentially as described (Fellouse FA, Sidhu, SS (2007)). To assess whether the expected diversity had been incorporated, a small aliquot of the generated Kunkel DNA was used to chemically transform TOP10 E. coli cells, and 96 clones were picked and sent for sequencing (GATC, Germany). The remaining DNA was then electroporated into SS320 cells (Lucigen, Middleton, WI, USA), generating a plasmid containing approximately 1.5 x 10 9The highly diverse library of each clone is measured by the number of bacterial colonies obtained after transformation. The transformed SS320 cells are harvested and stored at -80 ° C with 15% glycerol. A total of 600 ml 2xYT of antibiotics selective for both phagemid and F' appendages are inoculated with bacterial glycerol stock. Bacterial growth is until the exponential phase, and then infected with five times of infection with M13KO7 helper phage (New England Biolabs, Ipswich, MA, USA). The culture is propagated overnight, and the scFv of the display phage is harvested by standard polyethylene glycol (PEG) / NaCl precipitation.

[0213] Phage display selection

[0214] Adopt MN114-19 Lib1, use three or four rounds of enrichment to carry out phage display selection.For biotinylated target (CD44v6 and hm v6 peptide), use streptavidin coated magnetic beads to select (as described in Example 1).Similarly, use protein G coated magnetic beads (Thermofisher Scientific#10004D) to capture the CD44v6 (non-biotinylated) of Fc fusion.Selection track is designed to make only use CD44v6 or hm v6 peptide in whole selection round, or alternately use two kinds of antigens between different selection rounds, thereby produce the scheme covering four different selection tracks in total.By reducing antigen amount (50 to 1 or 0.05nM) and by increasing washing intensity and number of times (5-8) to increase the selection pressure between different selection rounds.Before the 1st round, use Bio-CD44 to carry out negative selection (pre-selection).Use trypsin-proton inhibition method to carry out the elution of antigen-bound phage. Except for the phage-target antigen incubation step, the entire phage display selection process was automated and performed using a Kingfisher Flex robot (ThermoFisher Scientific).

[0215] Recloning and expression of AL-MN114 scFv clone

[0216] Phagemid DNA from rounds 2 and 3 or 2, 3 and 4 of selection from each selection track was isolated, enzymatically digested and subcloned in a pool into the in-house screening vector pHAT-6 to enable soluble expression of AL-MN114 scFv clones fused at the C-terminus with a triple FLAG tag and a hexahistidine tag (Hisx6). The vector constructs were subsequently transformed into E. coli TOP10 cells. Single colony clones were picked, cultured and IPTG induced for soluble scFv expression in a 96-well format. A total of 468 scFv clones were prepared for assay in the primary ELISA screen.

[0217] ELISA screening

[0218] The antigen Bio-CD44v6 and hm v6 peptides were indirectly coated in 384-ELISA well plates with a negative control Bio-CD44 by streptavidin in a PBS solution of 1 μg / ml. The scFv clones of the 3xFLAG markers present in the bacterial supernatant were diluted 1:7 in a blocking buffer (PBS 0.5% BSA + 0.05% Tween20) and allowed to bind to the coated antigen. Binding was detected by incubation with a-FLAG M2 antibody (Sigma-Aldrich #A8592) conjugated with HRP or a-human κ antibody (Southernbiotech #2060-05) conjugated with HRP, followed by TMB ELISA substrate (ThermoFisher Scientific #34029). The generation of the colorimetric signal was stopped by adding 1M sulfuric acid, and the plate was analyzed at a wavelength of 450mm (abs 450nm) on a Spectramax plus instrument (Molecular Devices). All clones were assayed in duplicate from which the mean Abs 450 nm value was calculated and background was subtracted from the mean Abs 450 nm value of blank samples (blocking buffer added instead of scFv clones).

[0219] DNA Sequencing

[0220] Clones showing binding to CD44v6 and hm v6 peptides were sent to Eurofins genomics (Ebersberg, Germany) for Sanger DNA sequencing.

[0221] result

[0222] A total of 18 positions were targeted in the gene encoding U-MN114-19 scFv to generate MN114-Lib1. Sequencing of 96 randomly picked clones confirmed the introduction of the expected diversity.

[0223] After phage display selection and ELISA screening of 468 scFv clones, 354 positive hits were identified and showed binding only to the v6 region within CD44v6 and not to the negative control antigen CD44. DNA sequencing of the 354 positive hits led to the identification of 247 sequence-unique clones. These 247 clones were labeled AL-MN114 followed by a unique numerical number.

[0224] Example 9 Kinetic measurements of newly selected AL-MN114 scFv clones

[0225] In the initial kinetic screen, 247 sequence-unique AL-MN114 scFv clones (Example 8) were further analyzed by SPR for binding to the hm v6 peptide. The most promising 95 clones ranked by apparent off-rate were further subjected to more refined kinetic measurements using the SCK method for CD44 v6, human v6 peptide and cynomolgus monkey v6 peptide.

[0226] Materials and Methods

[0227] Kinetic screening and SCK measurements were performed on a BIAcore T200 instrument (Cytiva). The α-FLAG M2 antibody (Merck # F1804) used as a capture ligand was immobilized to all 4 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), and the 3xFLAG-tagged AL-MN114 scFv clones present in the bacterial supernatant were diluted in running buffer to obtain equal capture RU levels. For kinetic screening, 100 nM hmv6 peptides (Table 10, Example 13) were prepared in running buffer and injected onto the surface captured by the AL-MN114 scFv and allowed to bind. For SCK measurements, a 3-fold dilution series of hm v6 peptide and cm v6 peptide was prepared in running buffer (Table 10, Example 13), which consisted of five concentrations ranging from 200-2.5 nM. Each dilution series was injected sequentially, starting from the lowest to the highest antigen concentration. All chip surfaces were regenerated with 10 mM glycine-HCl, pH 2.1.

[0228] The response curve sensorgrams were obtained by subtracting the response curves of the reference surface (α-FLAG M2 antibody immobilized surface) and the blank run (injection of running buffer instead of antigen). The data were analyzed using Biacore T200 Evaluation 3.1 software and the 1:1 Langmuir binding model.

[0229] result

[0230] The SCK measurements of the clones correlated well with the apparent affinities obtained during the kinetic screening. The most promising clones showed an almost 5-fold improvement in off-rate compared to the parental clone U-MN114-19. The twenty most promising AL-MN114 scFv clones were converted to hIgG4 format and further characterized as previously described (Example 3).

[0231] Example 10 Characterization of AL-MN114hIgG4 clones on cancer cells

[0232] This experiment describes the time-resolved interaction analysis (LigandTracer) and kinetic properties of radiolabeled U-MN114-19, AL-MN114 antibody and BIWA4 assessed.

[0233] Materials and Methods

[0234] Radiolabeling, LigandTracer cell seeding and experiments were performed as described in Example 5.

[0235] result

[0236] LigandTracer evaluation of affinity-matured AL-MN114 antibody (hIgG4) using the anaplastic thyroid cancer cell line BHT-101. Cell lines were selected based on their CD44v6 antigen levels (middle) to better detect affinity differences. All 125 I-MN114 antibody is superior to 125 I-BIWA4 (Figure 10). Most 125 I-AL-MN114 clone showed better performance than the parental clone 125 IU-MN114-19 was more excellently retained (Table 12). As for the A431 cells (high antigen expressing cell line) measured by LigandTracer 177 Lu-labeled antibodies, U-MN114-19 and AL-MN114-465, both had better affinity than BIWA4 (Table 13). 125 I / 177 Lu-AL-MN114-465 had excellent retention for both.

[0237] Table 12: BHT-101 cells 125 Kinetic evaluation of LigandTracer data for I-labeled MN114 antibody and BIWA-4. *BIWA4 affinity was calculated based on three spike concentrations instead of two (1 nM, 3 nM and 10 nM).

[0238]

[0239]

[0240] Table 13: A431 cells (high antigen expression cell line) 177 Kinetic evaluation and comparison of LigandTracer data for Lu-labeled U-MN114-19, AL-MN114-465, and BIWA4.

[0241] Antibody <![CDATA[k a ]]> <![CDATA[k d ]]> <![CDATA[K D ]]> Model <![CDATA[ 177 Lu-BIWA4]]> <![CDATA[3*10 4 ]]> <![CDATA[10*10 -6 ]]> <![CDATA[3*10 -10 ]]> 1:1 <![CDATA[ 177 Lu-AL-MN114-465]]> <![CDATA[2*10 5 ]]> <![CDATA[5*10 -6 ]]> <![CDATA[3*10 -11 ]]> 1:1 <![CDATA[ 177 Lu-U-MN114-19]]> <![CDATA[1*10 5 ]]> <![CDATA[1*10 -5 ]]> <![CDATA[9*10 -11 ]]> 1:1

[0242] in conclusion

[0243] Affinity maturation resulted in improved affinity that exceeded that of the parental clone as well as the comparator antibody BIWA4.Four AL-MN114 clones were selected for hIgG1 conversion and small-scale production: AL-MN114-71, AL-MN114-132, AL-MN114-444 and AL-MN114-465.

[0244] Example 11, conversion to full antibody IgG1 format

[0245] AL-MN114-71, AL-MN114-132, AL-MN114-444, AL-MN114-465, U-MN114-19 and BIWA4 were converted to human IgG1 LALA and / or human IgG1 LALA IAHA format, see Table 14. For simplicity, conversion to IgG1 LALA format is described below.

[0246] Fusion cloning, transfection into HEK293, expression and purification

[0247] The VH and VL regions of AL-MN114-132, AL-MN114-465 and BIWA4 were PCR amplified using the In-Fusion HD Plus Cloning Kit (Clontech #638909) and inserted into the in-house constructed vector pHAT-hIgG1-LALA. Transfection of plasmid DNA into expiHEK293 cells was performed in 230 mL culture using the ExpiFectamineTM 293 Transfection Kit (ThermoFisher Scientific #A14525). Cultures were harvested after 5 days of culture (37°C, 7% CO2, 70% rH, 105 rpm) and antibodies were purified by affinity chromatography using HiTrap PrismA columns (Cytiva), followed by buffer exchange to PBS, pH 7.4 using HiTrap desalting columns. Endotoxin levels were <0.25 EU / mg as determined by LALA chromogenic endotoxin. SDS-PAGE was performed to determine the purity and integrity of the purified antibodies, and the concentration was determined using an Implen NP80 UV-Vis spectrophotometer. Additionally, size exclusion chromatography was performed on an Agilent Bio SEC-3 for each of the purified antibodies.

[0248] Table 14: Clones converted to full format IgG1 LALA and / or IgG1 LALA IAHA.

[0249] Clone Name IgG1 format U-MN114-19 IgG1 LALA,IgG LALA IAHA AL-MN114-71 IgG1 LALA IAHA AL-MN114-132 IgG1 LALA,IgG LALA IAHA AlLMN114-444 IgG1 LALA IAHA AL-MN114-465 IgG1 LALA,IgG LALA IAHA BIWA4 IgG1 LALA,IgG LALA IAHA

[0250] result

[0251] Selected clones were successfully transformed into IgG1 LALA and / or IgG1 LALA IAHA formats and were produced and purified.

[0252] Example 12, Epitope Mapping

[0253] The 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 1 amino acid shift, carrying a biotin moiety at the N-terminus.

[0254] Materials and Methods

[0255] The peptide array and full-length human v6 peptide (43aa) were coated into 384-ELISA well plates with 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 allowed to bind to the coated peptides. Binding was detected by HRP-conjugated α-human κ antibody (Southern Biotech #9230) followed by incubation with TMB-ELISA substrate (ThermoFisher Scientific #34029). The generation of the colorimetric signal was stopped by adding 1M sulfuric acid and the plate was analyzed at a wavelength of 450nm. Each sample was measured in duplicate, from which the average absorbance value was calculated and the background was subtracted from the average absorbance value of the blank wells (blocking buffer was added instead of antibody).

[0256] result

[0257] The results showed that all MN114 clones displayed the same six amino acid long epitope, namely amino acids WFGNRW located at positions 18-23 within the human v6 region (SEQ ID NO: 7). BIWA-4 displayed a ten amino acid long partially overlapping epitope, namely amino acids WFGNRWHEGY located at positions 18-27 within the human v6 region (SEQ ID NO: 5).

[0258] in conclusion

[0259] The evaluated MN114 antibody clones shared the same six amino acid long epitope, which partially overlapped with the ten amino acid long epitope of BIWA-4.

[0260] Example 13, Kinetic Measurements of IgG1 Clones

[0261] Kinetic measurements of AL-MN114-71, -132, -444, -465 and BIWA4 after conversion to IgG1 format were performed by SPR using the SCK method. The parental clone U-MN114-19 was also included as an IgG1.

[0262] Materials and Methods

[0263] SPR measurements were performed on a BIAcore T200 instrument (Cytiva) using the SCK method. 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 1500RU. All experiments were performed in running buffer (HBS, 0.05% Tween20, pH 7.5) at 25°C. A three-fold dilution series of CD44v6 and negative control CD44 was prepared in running buffer, consisting of five concentrations ranging from 10-0.12 nM. A four-fold dilution series of human, cynomolgus monkey, and rabbit v6 peptides was also prepared in running buffer, consisting of five concentrations ranging from 100–0.39 nM. Starting with the lowest concentration first, each dilution series was sequentially injected onto the immobilized antibody clone and the chip surface was regenerated with 10 mM glycine-HCl, pH 2.1. The specifications of the antigens used in the SPR measurements are shown in Table 10.

[0264] Response curve sensorgrams were obtained by subtracting the response curves of the reference surface (activated and deactivated chip surface) and the blank run (injection of running buffer instead of antigen).Data were analyzed using Biacore T200 Evaluation 3.1 software and the 1:1 Langmuir binding model.

[0265] result

[0266] All antibody clones showed binding to CD44v6 but not to CD44 (control antigen). app K D In the sub-nanomolar range, app K D =1-2 nM, Table 15. Binding of the antibody clones to human (hm) v6 peptide, cynomolgus monkey (cm) v6 peptide and rabbit (rb) v6 peptide was also detected, in contrast to BIWA4 which showed no binding to the rb-v6 peptide. This data correlates well with the epitope mapping performed (Example 12), which demonstrates that all novel antibodies share a six amino acid long epitope present in both cynomolgus monkey and rabbit v6 regions. The epitope found for BIWA4 is not present in the rabbit v6 region, only in the cynomolgus monkey v6 region. The introduced LALA or LALA IAHA Fc changes are not expected to have an impact on the antigen binding portion of the antibody.

[0267] Table 15: Kinetic parameters ka (1 / Ms), kd (1 / s), K of IgG1 antibodies measured against CD44v6, CD44, hm v6 peptide, cm v6 peptide and rb v6 peptide D (M) or app K D (M).

[0268]

[0269]

[0270]

[0271] -- No binding detected

[0272] in conclusion

[0273] 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.

[0274] Example 14, Specificity after radiolabeling

[0275] Two ATC cell lines were used to assess the specificity of the radiolabeled MN114 antibody as evaluated by a specificity assay in which the radiolabeled antibody competes for antigen binding with a molar excess of unlabeled antibody.

[0276] Materials and Methods

[0277] As described in Example 5, 125 Radioiodination and use of I 177 Radiolabeling of Lu. At least 24 h before the start of the experiment, add ACT-1 and BHT-101 (3-5*10 4 cells) were seeded into 48-well plates and incubated at 37°C and 5% CO 2 30 nM radiolabeled antibody or a solution of 30 nM radiolabeled antibody and 3 μM excess unlabeled antibody was added to each well (100 μL) and incubated at 37°C and 5% CcO 2 Incubate for 24 h at 4 °C. After incubation, cells were washed three times with PBS and harvested using 100 μL of trypsin per well. Cells were counted and the CPM as measured in Wizard 1460 well counter (PerkinElmer) was adjusted for cell count to obtain data expressed as CPM / 100,000 cells. Ligand Tracer culture plates were inoculated as described in Example 7. For competition assays, 10 nM125 IU-MN114-19 and 125 I-AL-MN114-444 was incubated for 2 h, and then 30 nM of unlabeled antibody was added.

[0278] result

[0279] All radiolabeled antibodies were used 125 I and 177 Lu labeling retained specificity on both ACT-1 and BHT-101 cells ( Fig.11 ), where the specificity of AL-MN114-465 was performed in the presence of a 100-fold molar excess of the parental antibody, U-MN114-19. The total CPM / 100,000 cells for AL-MN114-465 was greater than both BIWA4 and U-MN114-19, indicating that AL-MN114-465 had greater uptake in both cell lines regardless of the radiolabeling method used. Similarly, affinity-matured AL-MN114-444 had a slower off-rate than U-MN114-19, again indicating that the affinity-matured clone had superior retention and affinity ( Fig.12 ).

[0280] in conclusion

[0281] Both U-MN114-19 and the affinity matured variant (Al-MN114-465) bind with higher specificity and superior affinity than BIWA4, and in an antigen-dependent manner. In addition, AL-MN114-444 has improved retention in the presence of a three-fold molar excess of unlabeled antibody compared to U-MN114-19.

[0282] Example 15. Biodistribution of IgG1LALA and LALA / IAHA Forms in Mice

[0283] use 125 I and 177 Four selected AL-MN114 clones were evaluated in vivo in tumor xenografts in both LALA and LALA / IAHA formats.

[0284] Materials and methods

[0285] Animal studies were performed as described in Example 6 using either the ACT-1 or A431 xenograft models. Radiolabeling was performed as in Example 5. 125 I comparison, each mouse was injected with a total of 15 μg, consisting of 1 μg 125IU-MN114-19 (100 kBq), 14 μg unlabeled U-MN114-19 (IgG1 LALA or IgG1 LALA / IAHA). Animals were euthanized and dissected at 1h, 24h, 48h and 168h after injection (IgG1 LALA) and at 1h, 4h, 24h, 48h, 72h, 96h and 168h after injection (IgG1 LALA / IAHA). Organs were analyzed in a Wizard 1460 well counter (PerkinElmer) and the %ID of organ weight (g) was calculated. For dual nuclide studies of U-MN114-19 and AL-MN114-71, 132, 444 and 465, a total of 15 μg was injected per mouse, consisting of 1 μg in the same injection. 125 IU-MN114-19 (100 kBq) and 1 μg 177 Lu-U-MN114-19 (100 kBq) was diluted with 13 μg of unlabeled U-MN114-19. 177 Lu-labeled IgG1 LALA antibody, 1 μg labeled antibody and 14 μg unlabeled antibody were injected per animal at a concentration of 50 μL. Animals were euthanized and dissected at 24h, 48h, 96h and 168h post-injection (pi). Organs were analyzed in a Wizard 1460 well counter (PerkinElmer) and the %ID of organ weight (g) was calculated.

[0286] result

[0287] The IgG1 LALA / IAHA format showed a higher tumor-to-blood ratio compared with the IgG1 LALA format, while the absolute tumor dose was higher with the LALA format ( Fig.13 ). Both the affinity-matured IgG1 clone and U-MN114-19 evaluated in vivo showed good tumor uptake and retention. 125 Compared with I-labeled antibodies, 177 Lu-labeled antibodies have higher peak tumor uptake and improved retention ( Fig.14 ).

[0288] use 177 Lu evaluated two AL-MN114 clones in vivo as IgG1 LALA and compared them to U-MN114-19IgG1 LALA, all of which showed favorable biodistribution characteristics with higher tumor uptake compared to blood. The affinity-matured variants showed better tumor-to-blood ratios ( Fig.15 ).

[0289] in conclusion

[0290] All affinity-matured IgG1 clones evaluated showed good tumor uptake and retention in vivo. 125 Compared with I-labeled antibodies, 177 Lu-labeled antibodies showed improved tumor uptake and retention.

[0291] Example 16, Therapeutic Studies in Mice

[0292] Two independent studies evaluating the use of two different xenograft models, ACT-1 and BHT-101, 177 Therapeutic capacity of Lu-labeled U-MN114-19.

[0293] Materials and methods

[0294] Animal studies were performed as described in Example 6 using ACT-1 and BHT-101 xenograft models. Xenograft models were selected based on their antigen expression levels. It was estimated that ACT-1 expressed CD44v6 at a level 10-fold higher than BHT-101. For ACT-1 xenografts, radiolabeling was performed as in Example 5, except that 177 Lu injection activity and the amount of antibody injected per antibody. For therapeutic studies, approximately 15 MBq of 177 Lu-U-MN114-19 (50 μg, IgG4). In control animals, 50 μg of unlabeled U-MN114-19 (IgG4) was injected. For BHT-101 xenografts, 10 7 For BHT-101 tumor therapy, each animal was injected with approximately 7 MBq of 177 Lu-U-MN114-19 (50 μg, IgG4). For the control, each animal was injected with approximately 7 MBq of 177 Lu-isotope control ab (IgG4). Tumors were measured and tumor volume was calculated by (HxLxW)*0.52. Animal weight was monitored to understand the overall health status.

[0295] result

[0296] In the ACT-1 study, a single dose (15 MBq) resulted in complete remission in all treated animals, with no signs of tumor regrowth at the end of the study. In the BHT-101 study, a dose of 7 MBq resulted in delayed tumor growth and nearly doubled median tumor survival compared to isotope controls ( Fig.16 ).

[0297] in conclusion

[0298] Therapeutic studies demonstrated therapeutic potential in high (ACT-1) and medium (BHT-101) tumor models.

[0299] Example 17, comparison with BIWA4

[0300] A series of experiments were performed to determine the efficiency of the current antibodies compared to existing technologies. For example, affinity and therapeutic efficiency were tested.

[0301] A. Affinity

[0302] Regarding affinity, the approximate dissociation constant K was estimated D ( app K D ), where K D The value of K can be considered to be related to the concentration of the antibody (the amount of antibody required for a particular experiment), and thus K D The lower the value (lower the concentration), the higher the affinity of the antibody.

[0303] The parental clone MN19 (also referred to as U-MN114-19) was determined to have an approximate K of approximately 0.16 nM on BHT-101 cells (i.e., cells of the BHT-101 cell line, DSMZ number ACC 279, derived from a thyroid carcinoma). D , app K D All affinity matured variants of parental MN19 (as defined in Tables 3-6) showed affinity below 0.2 nM as measured on BHT-101. app K D On the other hand, BIWA4 had a 9 nM activity on the BHT-101 cell line. app K D , which is significantly higher. In this regard it can be noted that, in order to enable comparison, the BIWA4 tested in these experiments was carried out using the BIWA4 antibody of the same format / construct as the antibody of the present disclosure. On SPR, the affinity is higher than on living cells.

[0304] B. Biodistribution

[0305] Regarding the therapeutic efficiency of the antibody, the biodistribution of the injected dose (ID) was determined as % ID / g of conjugated antibody. 125 IU-MN114-19 and iodinated BIWA4 125 The biodistribution of I-BIWA4 showed that 125 Compared with I-BIWA4, 125 Tumor uptake of IU-MN114-19 was significantly better, as Fig.17 shown. Fig.17 ACT-1 xenografts 125 IU-MN114-19 and 125Comparison of tumor uptake of I-BIWA4. Assuming that the injected activity in the tumor is 0% at t = 0, then 125 The calculated area under the curve (AUC) of IU-MN114-19 was significantly greater than 125 I-BIWA4

[0306] C. Response time and effect on tumor growth

[0307] In an animal study using xenograft-bearing mice, the BHT-101 xenografts were tested side by side. 177 Lu-BIWA4 and 177 Lu-AL-MN114-465. A single dose (10 MBq / 50 μg) was injected into the tail vein of mice and caliper measurements were taken based on xenograft growth, while animal weight and behavior were used to monitor animal health. Results are shown in Fig.18 and 19 middle.

[0308] Fig.18 demonstrated the use of 10 MBq in BHT-101 xenografts. 177 Lu-AL-MN114-465 or 177 Tumor growth after Lu-BIWA4 treatment. 177 Lu-AL-MN114-465 and 177 The survival rate of Lu-BIWA4 treated animals was 100%, while all controls were euthanized on day 12 after injection. Fig.19 The time to complete reaction is shown, where Fig.19 (Top) shows the expression of BHT-101 xenografts 177 Lu-AL-MN114-465 or 177 The time it takes for Lu-BIWA4 to fully react, and Fig.19 (Bottom) shows the expression of BHT-101 xenografts in 177 Lu-AL-MN114-465 or 177 Lu-BIWA4 to partial reaction time. It can be seen that in BHT-101 xenografts, 177 Compared with Lu-BIWA4, 177 Complete and partial responses were faster in Lu-AL-MN114-465 treated animals.

[0309] In a 3D multicellular tumor spheroid assay using BHT-101 cells, compared with untreated controls and cells treated with 60 kBq 177 Compared with the spheroids treated with Lu-isotope control antibody, the spheroids treated with 60 kBq 177Lu-AL-MN114-465 treatment resulted in significantly smaller spheres at day 10 post-treatment. Similarly, treatment with 60 kBq 177 Lu-BIWA4 treatment resulted in significantly smaller spheres, but was comparable to that of 60 kBq 177 Thus, although the spheroids treated with 60 kBq Lu isotope control antibody showed no significant difference compared to the untreated control. 177 Lu-AL-MN114-465 and 177 Lu-BIWA4 treatment had a significant effect on spheroid growth, but BIWA4 failed to have a significantly greater effect than the isotope control. This suggests that AL-MN114-465 is superior to BIWA4 in this 3D setting ( Fig. 20 ).

[0310] Fig. 20 The size / growth ratio of the tumor is shown, where Fig. 20 (Top) shows the use of 60kBq 177 Growth ratio of 3D multicellular tumor spheroids of BHT-101 cells treated with Lu-AL-MN114-465, BIWA4 or isotope control (ISO-c) antibodies. Spheroids were measured over time and the growth ratio was defined as the relative size compared to the size at day 0 (start of treatment). Fig. 20 (Bottom) Shows a one-way ANOVA of size ratios at day 10 post-treatment, demonstrating that while spheroids treated with AL-MN114-465 were not significantly different from those treated with BIWA4, they did differ from the isotope control (**), whereas spheroids treated with BIWA4 failed to do so.

[0311] in conclusion

[0312] All binding proteins, parental clones and mature clones of the present disclosure as defined in Tables 3-6 showed higher affinity compared to BIWA4. In general, the dissociation constant K as measured for cells from the BHT-101 cell line was D Below 9 nM, such as below 8, 7, 6, 5, 4, 3, 2 or 1 nM, preferably below 1 nM, such as below 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, such that the KD value of the disclosed binding protein is at most 0.2 nM. D <0.2nM.

[0313] Furthermore, it can be seen that the binding protein disclosed herein produces a faster response and a greater therapeutic effect in treatment. Therefore, compared with the BIWA4 antibody of the prior art, the binding protein of the present invention has a significant technical effect. Specific embodiments

[0315] 1. A binding protein that specifically binds to CD44v6 and comprises a binding domain of an antibody, the binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) or a derivative thereof, each VL and VH comprising three complementarity determining regions (CDRs), wherein the amino acid sequence of the CDRs is selected from the group consisting of:

[0316] VHCDR1 as defined by SEQ ID NO: 1;

[0317] VHCDR2 as defined by SEQ ID NO: 2;

[0318] VHCDR3 as defined by SEQ ID NO:3;

[0319] VLCDR1 as defined in SEQ ID NO:4;

[0320] Such as X 1 VLCDR2 defined by AS, where X 1 Can be T, A or S;

[0321] VLCDR3 as defined in SEQ ID NO:6;

[0322] and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto, wherein the binding protein recognizes an epitope of CD44v6 as defined in SEQ ID NO:7.

[0323] 2. The binding protein according to item 1, wherein the VHCDR1 is composed of the amino acid sequence GFX 3 FX 5 X 6 X 7 A is defined, and where X 3 For S, X 5 G, X 6 S and / or X 7 is Y.

[0324] 3. The binding protein according to item 1 or 2, wherein the VHCDR2 is composed of the amino acid sequence ISX 3 X 4 GX 6 ST definition, and where X 3 A, X 4G and / or X 6 For S.

[0325] 4. The binding protein according to items 1 to 3, wherein the VLCDR1 is composed of the amino acid sequence QX 2 IX 4 X 5 Y is defined, and where X 2 For S, X 4 S and / or X 5 For S.

[0326] 5. The binding protein according to items 1 to 4, wherein the VLCDR2 is composed of the amino acid sequence X 1 AS definition, and where X 1 It is T or S.

[0327] 6. The binding protein according to item 1, wherein VHCDR1, VHCDR2 and VLCDR2 are present next to specific framework amino acids, wherein the CDR and framework amino acid (faa) sequences are selected from the group comprising:

[0328] VHCDR1 and faa defined by SEQ ID NO:8;

[0329] VHCDR2 and faa as defined in SEQ ID NO:9;

[0330] VLCDR2 and faa as defined in SEQ ID NO: 10;

[0331] and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto.

[0332] 7. The binding protein according to items 1 to 5, wherein the CDRs are individually selected from the group comprising:

[0333] VHCDR1 is selected from SEQ ID NO: 11-18;

[0334] VHCDR2 is selected from SEQ ID NO: 19-25, 32;

[0335] VHCDR3 as defined by SEQ ID NO:3;

[0336] VLCDR1 is selected from SEQ ID NO: 26-31;

[0337] VLCDR2 is selected from TAS, SAS, and AAS;

[0338] VLCDR3 as defined in SEQ ID NO:6;

[0339] and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto.

[0340] 8. The binding protein according to items 1 to 5 and 7, wherein the CDR is selected from the group comprising:

[0341] VHCDR1 as defined by SEQ ID NO: 11;

[0342] VHCDR2 is selected from SEQ ID NO: 19-25;

[0343] VHCDR3 as defined by SEQ ID NO:3;

[0344] VLCDR1 as defined in SEQ ID NO:26;

[0345] VLCDR2 is selected from TAS, SAS, and AAS;

[0346] VLCDR3 as defined in SEQ ID NO:6;

[0347] and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto.

[0348] 9. The binding protein according to items 1 to 5 and 7 to 8, wherein the amino acid sequence of the CDR is selected from the group comprising:

[0349] i) a binding protein having

[0350] VHCDR1 as defined by SEQ ID NO: 11;

[0351] VHCDR2 as defined by SEQ ID NO: 19;

[0352] VHCDR3 as defined by SEQ ID NO:3;

[0353] VLCDR1 as defined in SEQ ID NO:26;

[0354] VLCDR2 as defined in TAS; and

[0355] VLCDR3 as defined in SEQ ID NO:6;

[0356] ii) a binding protein having

[0357] VHCDR1 as defined by SEQ ID NO: 11;

[0358] VHCDR2 as defined by SEQ ID NO:20;

[0359] VHCDR3 as defined by SEQ ID NO:3;

[0360] VLCDR1 as defined in SEQ ID NO:26;

[0361] VLCDR2 as defined by SAS; and

[0362] VLCDR3 as defined in SEQ ID NO:6;

[0363] iii) a binding protein having

[0364] VHCDR1 as defined by SEQ ID NO: 12;

[0365] VHCDR2 as defined by SEQ ID NO:20;

[0366] VHCDR3 as defined by SEQ ID NO:3;

[0367] VLCDR1 as defined in SEQ ID NO:26;

[0368] VLCDR2 as defined by the AAS; and

[0369] VLCDR3 as defined in SEQ ID NO:6;

[0370] iv) a binding protein having

[0371] VHCDR1 as defined by SEQ ID NO: 16;

[0372] VHCDR2 as defined by SEQ ID NO:20;

[0373] VHCDR3 as defined by SEQ ID NO:3;

[0374] VLCDR1 as defined in SEQ ID NO:26;

[0375] VLCDR2 as defined by the AAS; and

[0376] VLCDR3 as defined in SEQ ID NO:6;

[0377] v) a binding protein having

[0378] VHCDR1 as defined by SEQ ID NO: 17;

[0379] VHCDR2 as defined by SEQ ID NO:20;

[0380] VHCDR3 as defined by SEQ ID NO:3;

[0381] VLCDR1 as defined in SEQ ID NO:27;

[0382] VLCDR2 as defined by SAS; and

[0383] VLCDR3 as defined in SEQ ID NO:6;

[0384] vi) a binding protein having

[0385] VHCDR1 as defined by SEQ ID NO: 12;

[0386] VHCDR2 as defined by SEQ ID NO:21;

[0387] VHCDR3 as defined by SEQ ID NO:3;

[0388] VLCDR1 as defined in SEQ ID NO:26;

[0389] VLCDR2 as defined by the AAS; and

[0390] VLCDR3 as defined in SEQ ID NO:6;

[0391] vii) a binding protein having

[0392] VHCDR1 as defined by SEQ ID NO: 12;

[0393] VHCDR2 as defined by SEQ ID NO:22;

[0394] VHCDR3 as defined by SEQ ID NO:3;

[0395] VLCDR1 as defined in SEQ ID NO:27;

[0396] VLCDR2 as defined in TAS; and

[0397] VLCDR3 as defined in SEQ ID NO:6;

[0398] viii) a binding protein having

[0399] VHCDR1 as defined in SEQ ID NO: 13;

[0400] VHCDR2 as defined by SEQ ID NO: 19;

[0401] VHCDR3 as defined by SEQ ID NO:3;

[0402] VLCDR1 as defined in SEQ ID NO:27;

[0403] VLCDR2 as defined by SAS; and

[0404] VLCDR3 as defined in SEQ ID NO:6;

[0405] ix) a binding protein having

[0406] VHCDR1 as defined by SEQ ID NO: 11;

[0407] VHCDR2 as defined by SEQ ID NO: 19;

[0408] VHCDR3 as defined by SEQ ID NO:3;

[0409] VLCDR1 as defined in SEQ ID NO:27;

[0410] VLCDR2 as defined by SAS; and

[0411] VLCDR3 as defined in SEQ ID NO:6;

[0412] x) a binding protein having

[0413] VHCDR1 as defined by SEQ ID NO:14;

[0414] VHCDR2 as defined by SEQ ID NO: 19;

[0415] VHCDR3 as defined by SEQ ID NO:3;

[0416] VLCDR1 as defined in SEQ ID NO:27;

[0417] VLCDR2 as defined by SAS; and

[0418] VLCDR3 as defined in SEQ ID NO:6;

[0419] xi) a binding protein having

[0420] VHCDR1 as defined by SEQ ID NO:14;

[0421] VHCDR2 as defined by SEQ ID NO: 19;

[0422] VHCDR3 as defined by SEQ ID NO:3;

[0423] VLCDR1 as defined in SEQ ID NO:26;

[0424] VLCDR2 as defined by the AAS; and

[0425] VLCDR3 as defined in SEQ ID NO:6;

[0426] xii) a binding protein having

[0427] VHCDR1 as defined by SEQ ID NO: 12;

[0428] VHCDR2 as defined by SEQ ID NO: 19;

[0429] VHCDR3 as defined by SEQ ID NO:3;

[0430] VLCDR1 as defined in SEQ ID NO:26;

[0431] VLCDR2 as defined by the AAS; and

[0432] VLCDR3 as defined in SEQ ID NO:6;

[0433] xiii) a binding protein having

[0434] VHCDR1 as defined in SEQ ID NO: 15;

[0435] VHCDR2 as defined by SEQ ID NO: 19;

[0436] VHCDR3 as defined by SEQ ID NO:3;

[0437] VLCDR1 as defined in SEQ ID NO:26;

[0438] VLCDR2 as defined by the AAS; and

[0439] VLCDR3 as defined in SEQ ID NO:6;

[0440] xiv) a binding protein having

[0441] VHCDR1 as defined by SEQ ID NO: 17;

[0442] VHCDR2 as defined by SEQ ID NO: 19;

[0443] VHCDR3 as defined by SEQ ID NO:3;

[0444] VLCDR1 as defined in SEQ ID NO:28;

[0445] VLCDR2 as defined by the AAS; and

[0446] VLCDR3 as defined in SEQ ID NO:6;

[0447] xv) a binding protein having

[0448] VHCDR1 as defined in SEQ ID NO: 15;

[0449] VHCDR2 as defined by SEQ ID NO: 19;

[0450] VHCDR3 as defined by SEQ ID NO:3;

[0451] VLCDR1 as defined in SEQ ID NO:26;

[0452] VLCDR2 as defined by the AAS; and

[0453] VLCDR3 as defined in SEQ ID NO:6;

[0454] xvi) a binding protein having

[0455] VHCDR1 as defined by SEQ ID NO: 12;

[0456] VHCDR2 as defined by SEQ ID NO: 19;

[0457] VHCDR3 as defined by SEQ ID NO:3;

[0458] VLCDR1 as defined in SEQ ID NO:26;

[0459] VLCDR2 as defined by the AAS; and

[0460] VLCDR3 as defined in SEQ ID NO:6;

[0461] xvii) a binding protein having

[0462] VHCDR1 as defined by SEQ ID NO: 12;

[0463] VHCDR2 as defined by SEQ ID NO:23;

[0464] VHCDR3 as defined by SEQ ID NO:3;

[0465] VLCDR1 as defined in SEQ ID NO:29;

[0466] VLCDR2 as defined by SAS; and

[0467] VLCDR3 as defined in SEQ ID NO:6;

[0468] xviii) a binding protein having

[0469] VHCDR1 as defined in SEQ ID NO: 15;

[0470] VHCDR2 as defined by SEQ ID NO:20;

[0471] VHCDR3 as defined by SEQ ID NO:3;

[0472] VLCDR1 as defined in SEQ ID NO:30;

[0473] VLCDR2 as defined in TAS; and

[0474] VLCDR3 as defined in SEQ ID NO:6;

[0475] xix) a binding protein having

[0476] VHCDR1 as defined by SEQ ID NO:18;

[0477] VHCDR2 as defined by SEQ ID NO:24;

[0478] VHCDR3 as defined by SEQ ID NO:3;

[0479] VLCDR1 as defined in SEQ ID NO:31;

[0480] VLCDR2 as defined by SAS; and

[0481] VLCDR3 as defined in SEQ ID NO:6;

[0482] xx) a binding protein having

[0483] VHCDR1 as defined by SEQ ID NO: 12;

[0484] VHCDR2 as defined by SEQ ID NO:25;

[0485] VHCDR3 as defined by SEQ ID NO:3;

[0486] VLCDR1 as defined in SEQ ID NO:26;

[0487] VLCDR2 as defined by the AAS; and

[0488] VLCDR3 as defined in SEQ ID NO:6;

[0489] xxi) a binding protein having

[0490] VHCDR1 as defined by SEQ ID NO: 12;

[0491] VHCDR2 as defined by SEQ ID NO:32;

[0492] VHCDR3 as defined by SEQ ID NO:3;

[0493] VLCDR1 as defined in SEQ ID NO:26;

[0494] VLCDR2 as defined by the AAS; and

[0495] VLCDR3 as defined in SEQ ID NO:6;

[0496] and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto.

[0497] 10. The binding protein according to item 6, wherein the sequence of the CDR including the framework amino acids (faa) is selected from the group consisting of:

[0498] i) a binding protein having

[0499] VHCDR1 and faa defined by SEQ ID NO: 121;

[0500] VHCDR2 and faa as defined in SEQ ID NO: 129; and

[0501] VLCDR2 and faa as defined in SEQ ID NO: 139;

[0502] ii) a binding protein having

[0503] VHCDR1 and faa defined by SEQ ID NO: 121;

[0504] VHCDR2 and faa as defined in SEQ ID NO: 130; and

[0505] VLCDR2 and faa as defined in SEQ ID NO: 140;

[0506] iii) a binding protein having

[0507] VHCDR1 and faa defined by SEQ ID NO: 122;

[0508] VHCDR2 and faa as defined in SEQ ID NO: 131; and

[0509] VLCDR2 and faa as defined in SEQ ID NO: 141;

[0510] iv) a binding protein having

[0511] VHCDR1 and faa defined by SEQ ID NO: 126;

[0512] VHCDR2 and faa as defined in SEQ ID NO: 130; and

[0513] VLCDR2 and faa as defined in SEQ ID NO: 144;

[0514] v) a binding protein having

[0515] VHCDR1 and faa defined by SEQ ID NO: 127;

[0516] VHCDR2 and faa as defined in SEQ ID NO: 130; and

[0517] VLCDR2 and faa as defined in SEQ ID NO: 140;

[0518] vi) a binding protein having

[0519] VHCDR1 and faa defined by SEQ ID NO: 122;

[0520] VHCDR2 and faa as defined in SEQ ID NO: 133; and

[0521] VLCDR2 and faa as defined in SEQ ID NO: 141;

[0522] vii) a binding protein having

[0523] VHCDR1 and faa defined by SEQ ID NO: 122;

[0524] VHCDR2 and faa as defined in SEQ ID NO: 134; and

[0525] VLCDR2 and faa as defined in SEQ ID NO: 142;

[0526] viii) a binding protein having

[0527] VHCDR1 and faa defined by SEQ ID NO: 123;

[0528] VHCDR2 and faa as defined in SEQ ID NO: 129; and

[0529] VLCDR2 and faa as defined in SEQ ID NO: 140;

[0530] ix) a binding protein having

[0531] VHCDR1 and faa defined by SEQ ID NO: 121;

[0532] VHCDR2 and faa as defined in SEQ ID NO: 132; and

[0533] VLCDR2 and faa as defined in SEQ ID NO: 140;

[0534] x) a binding protein having

[0535] VHCDR1 and faa defined by SEQ ID NO: 124;

[0536] VHCDR2 and faa as defined in SEQ ID NO: 129; and

[0537] VLCDR2 and faa as defined in SEQ ID NO: 143;

[0538] xi) a binding protein having

[0539] VHCDR1 and faa defined by SEQ ID NO: 124;

[0540] VHCDR2 and faa as defined in SEQ ID NO: 132; and

[0541] VLCDR2 and faa as defined in SEQ ID NO: 141;

[0542] xii) a binding protein having

[0543] VHCDR1 and faa defined by SEQ ID NO: 122;

[0544] VHCDR2 and faa as defined in SEQ ID NO: 129; and

[0545] VLCDR2 and faa as defined in SEQ ID NO: 141;

[0546] xiii) a binding protein having

[0547] VHCDR1 and faa defined by SEQ ID NO: 125;

[0548] VHCDR2 and faa as defined in SEQ ID NO: 129; and

[0549] VLCDR2 and faa as defined in SEQ ID NO: 141;

[0550] xiv) a binding protein having

[0551] VHCDR1 and faa defined by SEQ ID NO: 127;

[0552] VHCDR2 and faa as defined in SEQ ID NO: 135; and

[0553] VLCDR2 and faa as defined in SEQ ID NO: 144;

[0554] xv) a binding protein having

[0555] VHCDR1 and faa defined by SEQ ID NO: 125;

[0556] VHCDR2 and faa as defined in SEQ ID NO: 132; and

[0557] VLCDR2 and faa as defined in SEQ ID NO: 141;

[0558] xvi) a binding protein having

[0559] VHCDR1 and faa defined by SEQ ID NO: 122;

[0560] VHCDR2 and faa as defined in SEQ ID NO: 132; and

[0561] VLCDR2 and faa as defined in SEQ ID NO: 141;

[0562] xvii) a binding protein having

[0563] VHCDR1 and faa defined by SEQ ID NO: 122;

[0564] VHCDR2 and faa as defined in SEQ ID NO: 136; and

[0565] VLCDR2 and faa as defined in SEQ ID NO: 145;

[0566] xviii) a binding protein having

[0567] VHCDR1 and faa defined by SEQ ID NO: 125;

[0568] VHCDR2 and faa as defined in SEQ ID NO: 131; and

[0569] VLCDR2 and faa as defined in SEQ ID NO: 146;

[0570] xix) a binding protein having

[0571] VHCDR1 and faa defined by SEQ ID NO: 128;

[0572] VHCDR2 and faa as defined in SEQ ID NO: 137; and

[0573] VLCDR2 and faa as defined in SEQ ID NO: 145;

[0574] xx) a binding protein having

[0575] VHCDR1 and faa defined by SEQ ID NO: 122;

[0576] VHCDR2 and faa as defined in SEQ ID NO: 138; and

[0577] VLCDR2 and faa as defined in SEQ ID NO: 141;

[0578] xxi) a binding protein having

[0579] VHCDR1 and faa defined by SEQ ID NO: 122;

[0580] VHCDR2 and faa as defined in SEQ ID NO:33; and

[0581] VLCDR2 and faa as defined in SEQ ID NO: 141;

[0582] and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto.

[0583] 11. A binding protein according to items 1 to 5 and 7 to 10, wherein the VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35-54, and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto, and wherein the VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55-74, and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto.

[0584] 12. The binding protein according to item 11, wherein the CDR sequence contains no variation in the amino acid sequence, or wherein the sequence variation of the CDR amino acid sequence is at most 5%, such as 94%, 3%, 2%, 1% or less.

[0585] 13. The binding protein according to items 11 to 12, wherein the amino acid sequences of VH and VL are selected from the group comprising:

[0586] i) a binding protein having

[0587] VH as defined in SEQ ID NO:35; and

[0588] VL as defined in SEQ ID NO:55;

[0589] ii) a binding protein having

[0590] VH as defined in SEQ ID NO:36; and

[0591] VL as defined in SEQ ID NO:56;

[0592] iii) a binding protein having

[0593] VH as defined by SEQ ID NO:37; and

[0594] VL as defined in SEQ ID NO:57;

[0595] iv) a binding protein having

[0596] VH as defined by SEQ ID NO:46; and

[0597] VL as defined in SEQ ID NO:66;

[0598] v) a binding protein having

[0599] VH as defined by SEQ ID NO:54; and

[0600] VL as defined in SEQ ID NO:74;

[0601] vi) a binding protein having

[0602] VH as defined in SEQ ID NO:38; and

[0603] VL as defined in SEQ ID NO:58;

[0604] vii) a binding protein having

[0605] VH as defined by SEQ ID NO:39; and

[0606] VL as defined in SEQ ID NO:59;

[0607] viii) a binding protein having

[0608] VH as defined in SEQ ID NO:40; and

[0609] VL as defined in SEQ ID NO:60;

[0610] ix) a binding protein having

[0611] VH as defined by SEQ ID NO:41; and

[0612] VL as defined in SEQ ID NO:61;

[0613] x) a binding protein having

[0614] VH as defined in SEQ ID NO:42; and

[0615] VL as defined in SEQ ID NO:62;

[0616] xi) a binding protein having

[0617] VH as defined by SEQ ID NO:43; and

[0618] VL as defined in SEQ ID NO:63;

[0619] xii) a binding protein having

[0620] VH as defined by SEQ ID NO:44; and

[0621] VL as defined in SEQ ID NO:64;

[0622] xiii) a binding protein having

[0623] VH as defined in SEQ ID NO:45; and

[0624] VL as defined in SEQ ID NO:65;

[0625] xiv) a binding protein having

[0626] VH as defined by SEQ ID NO:47; and

[0627] VL as defined in SEQ ID NO:67;

[0628] xv) a binding protein having

[0629] VH as defined by SEQ ID NO:48; and

[0630] VL as defined in SEQ ID NO:68;

[0631] xvi) a binding protein having

[0632] VH as defined by SEQ ID NO:49; and

[0633] VL as defined in SEQ ID NO:69;

[0634] xvii) a binding protein having

[0635] VH as defined in SEQ ID NO:50; and

[0636] VL as defined in SEQ ID NO:70;

[0637] xviii) a binding protein having

[0638] VH as defined in SEQ ID NO:51; and

[0639] VL as defined in SEQ ID NO:71;

[0640] xix) a binding protein having

[0641] VH as defined in SEQ ID NO:52; and

[0642] VL as defined in SEQ ID NO:72;

[0643] xx) a binding protein having

[0644] VH as defined by SEQ ID NO:53; and

[0645] VL as defined in SEQ ID NO:73;

[0646] xxi) a binding protein having

[0647] VH as defined by SEQ ID NO: 147; and

[0648] VL as defined in SEQ ID NO: 148;

[0649] and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto.

[0650] 14. The binding protein according to items 1 to 13, wherein the binding protein is a monoclonal antibody, or an antigen binding fragment selected from the group consisting of an Fv fragment, a Fab-like fragment and a domain antibody.

[0651] 15. The binding protein according to item 14, wherein the Fv fragment is a scFv fragment.

[0652] 16. The binding protein according to item 14, wherein the Fab-like fragment is Fab or F(ab') 2 Fragment.

[0653] 17. The binding protein according to items 1 to 14, wherein the binding molecule is a monoclonal antibody of the IgG1 isotype,

[0654] Such as IgG1 LALA antibody or IgG1 LALA IAHA antibody, or a monoclonal antibody of the IgG4 isotype.

[0655] 18. The binding protein according to items 1 to 17, wherein the binding protein is human or of human origin.

[0656] 19. The binding protein according to items 17 to 18, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 75-93 and 149, and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto; and the light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-113 and 150, and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto.

[0657] 20. The binding protein according to item 19, wherein the amino acid sequences of the heavy chain and the light chain are selected from the group comprising:

[0658] i) a binding protein having

[0659] A heavy chain as defined in SEQ ID NO:75; and

[0660] A light chain as defined in SEQ ID NO:94;

[0661] ii) a binding protein having

[0662] A heavy chain as defined in SEQ ID NO:76; and

[0663] A light chain as defined in SEQ ID NO:95;

[0664] iii) a binding protein having

[0665] A heavy chain as defined by SEQ ID NO:77; and

[0666] A light chain as defined in SEQ ID NO:96;

[0667] iv) a binding protein having

[0668] A heavy chain as defined in SEQ ID NO:34; and

[0669] A light chain as defined in SEQ ID NO: 105;

[0670] v) a binding protein having

[0671] A heavy chain as defined in SEQ ID NO:93; and

[0672] A light chain as defined in SEQ ID NO: 113;

[0673] vi) a binding protein having

[0674] A heavy chain as defined by SEQ ID NO:78; and

[0675] A light chain as defined in SEQ ID NO:97;

[0676] vii) a binding protein having

[0677] A heavy chain as defined by SEQ ID NO:79; and

[0678] A light chain as defined in SEQ ID NO:98;

[0679] viii) a binding protein having

[0680] A heavy chain as defined in SEQ ID NO: 80; and

[0681] A light chain as defined in SEQ ID NO:99;

[0682] ix) a binding protein having

[0683] A heavy chain as defined in SEQ ID NO: 81; and

[0684] A light chain as defined in SEQ ID NO: 100;

[0685] x) a binding protein having

[0686] A heavy chain as defined in SEQ ID NO: 82; and

[0687] A light chain as defined in SEQ ID NO: 101;

[0688] xi) a binding protein having

[0689] A heavy chain as defined in SEQ ID NO: 83; and

[0690] A light chain as defined in SEQ ID NO: 102;

[0691] xii) a binding protein having

[0692] A heavy chain as defined by SEQ ID NO: 84; and

[0693] A light chain as defined in SEQ ID NO: 103;

[0694] xiii) a binding protein having

[0695] A heavy chain as defined by SEQ ID NO:85; and a light chain as defined by SEQ ID NO:104;

[0696] xiv) a binding protein having

[0697] A heavy chain as defined in SEQ ID NO: 86; and

[0698] A light chain as defined in SEQ ID NO: 106;

[0699] xv) a binding protein having

[0700] A heavy chain as defined by SEQ ID NO: 87; and

[0701] A light chain as defined in SEQ ID NO: 107;

[0702] xvi) a binding protein having

[0703] A heavy chain as defined in SEQ ID NO: 88; and

[0704] A light chain as defined in SEQ ID NO: 108;

[0705] xviii) a binding protein having

[0706] A heavy chain as defined in SEQ ID NO: 89; and

[0707] A light chain as defined in SEQ ID NO: 109;

[0708] xviii) a binding protein having

[0709] A heavy chain as defined in SEQ ID NO:90; and

[0710] A light chain as defined in SEQ ID NO: 110;

[0711] xix) a binding protein having

[0712] A heavy chain as defined in SEQ ID NO:91; and

[0713] A light chain as defined in SEQ ID NO: 111;

[0714] xx) a binding protein having

[0715] A heavy chain as defined in SEQ ID NO:92; and

[0716] A light chain as defined in SEQ ID NO: 112;

[0717] xxi) a binding protein having

[0718] A heavy chain as defined in SEQ ID NO: 149; and

[0719] A light chain as defined in SEQ ID NO: 150;

[0720] and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto.

[0721] 21. The binding protein according to items 20 to 21, wherein the CDR sequence contains no variation in the amino acid sequence, or wherein the sequence variation of the CDR amino acid sequence is at most 5%, such as 4%, 3%, 2%, 1% or less.

[0722] 22. A conjugated binding protein comprising:

[0723] (i) at least one binding protein as defined in any one of items 1 to 21; and

[0724] (ii) at least one reagent.

[0725] 23. A conjugated binding protein according to item 22, wherein at least one of the reagents is conjugated to the binding protein, wherein the binding protein and the reagent are directly or indirectly conjugated.

[0726] 24. A conjugated binding protein according to item 22 or 23, wherein the agent is a radioactive isotope, a photoactivated compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytotoxic agent, a prodrug, a binding molecule with different specificity, a cytokine or another immunomodulatory polypeptide.

[0727] 25. The conjugated binding protein according to items 22 to 24, wherein the agent is a therapeutic agent.

[0728] 26. A conjugated binding protein according to item 25, wherein the therapeutic agent is a cytotoxic agent comprising or consisting of one or more radioactive isotopes.

[0729] 27. The conjugated binding protein according to item 24 or 26, wherein the one or more radioactive isotopes are each independently selected from the group consisting of β-emitters, Auger emitters, conversion electron emitters, α-emitters and low photon energy emitters.

[0730] 28. A conjugated binding protein according to item 27, wherein the one or more radioactive isotopes each independently have an emission mode that locally absorbs energy that produces a high dose of absorbance in the vicinity of the agent.

[0731] 29. The conjugated binding protein according to item 27 or 28, wherein the one or more radioisotopes are each independently selected from the group consisting of: long-range beta emitters such as 90 Y. 32 P. 186 Re / 188 Re; 166 Ho, 76 As / 77 As, 153 Sm; medium-range-beta emitters such as 131 I. 177 Lu, 67 Cu, 161 Tb, 47 Sc; low energy beta emitters, such as 45 Ca, 35 S or14 C; conversion or Auger emitters, such as 51 Cr, 67 Ga, 99 TC m , 111 In, 123 I. 125 I. 201 TI, as well as α-emitters such as 212 Bi, 212 Pb, 213 Bi, 223 Ac, 225 Ac, 227 Th, 149 Tb and 211 At.

[0732] 30. The conjugated binding protein according to item 24 or 26 to 29, wherein the radioisotope is 177 Lu.

[0733] 31. The conjugated binding protein according to item 25, wherein the therapeutic agent is a cytotoxic agent comprising or consisting of one or more cytotoxic drugs.

[0734] 32. A conjugated binding protein according to item 31, wherein the one or more cytotoxic agents are selected from cytostatic drugs, toxins and chemotherapeutic agents.

[0735] 33. The conjugated binding protein according to items 22 to 24, wherein the agent is a detectable agent.

[0736] 34. The conjugated binding protein according to item 33, wherein the detectable agent is selected from the group consisting of radioisotopes, enzymes, fluorescent molecules, dyes, digoxigenin and biotin.

[0737] 35. A conjugated binding protein according to item 33 or 34, wherein the detectable agent comprises or consists of a radioactive isotope.

[0738] 36. The conjugated binding protein according to item 34 or 35, wherein the radioisotope is selected from 111 In, 99m Tc, 67 Ga, 68 Ga, 72 As, 89 Zr, 123 I. 125 I. 124 I. 47 Sc and 201 A group composed of TI.

[0739] 37. A conjugated binding protein according to items 24, 26 to 28 or 34 to 35, wherein the conjugated binding protein comprises a pair of detectable and cytotoxic radioisotopes, such as 86 γ / 90 γ, 111 In / 177 Lu or 125 I / 211 At.

[0740] 38. A conjugated binding protein according to item 37, wherein the radioisotope is capable of acting simultaneously as a detectable agent and as a cytotoxic agent in a multimodal manner.

[0741] 39. The conjugated binding protein according to items 33 to 38, wherein the detectable agent is detectable by imaging techniques such as SPECT, PET, MRI, optical or ultrasound imaging.

[0742] 40. The conjugated binding protein according to item 22 or 23, wherein the agent is indirectly conjugated to the binding protein via a linker.

[0743] 41. A conjugated binding protein according to item 40, wherein the linker is in the form of a chelator.

[0744] 42. A conjugated binding protein according to item 41, wherein the chelator is 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- Derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NODAGA), and derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0745] 43. A conjugated binding protein according to item 40, wherein the linker is a molecular attachment tag for pre-targeted therapy using a second binding molecule, wherein the second binding molecule is conjugated to the agent, and wherein the second binding molecule binds to the attachment tag of the binding protein, thereby connecting the agent to the binding protein to form a conjugated binding protein.

[0746] 44. A cell engineered 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 co-stimulatory domains, wherein the antigen binding domain comprises a binding protein according to any one of items 1 to 15.

[0747] 45. The cell according to item 44, wherein the cell is a human cell.

[0748] 46. ​​A cell according to item 44 or 45, wherein the cell is an immune effector cell, such as a T cell, a NK cell or a macrophage.

[0749] 47. A pharmaceutical composition comprising a binding protein according to any one of items 1 to 21, a conjugated binding protein according to any one of items 22 to 32 or 37 to 43, or a cell according to any one of items 44 to 46, and a pharmaceutically acceptable carrier or excipient.

[0750] 48. A binding protein according to any one of items 1 to 21, a conjugated 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 use in therapy.

[0751] 49. The binding protein, conjugated binding protein, cell or pharmaceutical composition according to item 48, for use in cancer therapy.

[0752] 50. A method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a binding protein according to any one of items 1 to 21, a conjugated 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.

[0753] 51. A method of treating a subject in need thereof, the method comprising:

[0754] 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 attachment tag;

[0755] allowing any unbound binding protein to leave the circulation of said subject;

[0756] A therapeutically effective amount of a second molecule is administered, wherein the second molecule is conjugated to the therapeutic agent, and wherein the second molecule binds to the first binding protein, thereby delivering the therapeutic agent to the CD44v6 epitope bound by the first binding protein.

[0757] 52. The method according to item 50 or 51, wherein the subject suffers from a disease characterized by expression of the CD44 variant CD44v6

[0758] 53. The method according to item 52, wherein the disease is cancer or another angiogenesis-related disease.

[0759] 54. Use of a binding protein according to any one of items 1 to 21, a conjugated 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.

[0760] 55. A binding protein, a conjugated binding protein, a cell or a pharmaceutical composition according to item 49, a method according to item 53 or a use according to item 54, wherein the 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 brain metastasis.

[0761] 56. An in vitro method for detecting the expression of CD44 variant CD44v6, the method comprising:

[0762] (i) contacting a binding protein according to items 1 to 21 or a conjugated 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, such that if CD44v6 as defined in SEQ ID NO: 7 is present in the biological sample, the binding protein or the conjugated binding protein binds to its epitope;

[0763] (ii) washing the biological sample to remove unbound binding protein or conjugated binding protein; and

[0764] (iii) detecting any binding protein or conjugated binding protein in said biological sample that has bound to said epitope.

[0765] 57. An in vivo method for detecting the expression of CD44 variant CD44v6, the method comprising:

[0766] (i) administering to a subject a conjugated binding protein according to items 22 to 24 or 33 to 43, wherein the conjugated binding protein binds to an epitope of CD44v6 as defined in SEQ ID NO: 7; and

[0767] (ii) detecting that the conjugated binding protein has bound to cells expressing the epitope.

[0768] 58. The in vivo method according to item 57, wherein the conjugated binding protein comprises one or more 111 In radioactive isotope atoms.

[0769] 59. A conjugated binding protein comprising:

[0770] (iii) at least one binding protein as defined in any one of items 1 to 14; and

[0771] (iv) at least one 177 Lu radioisotope atom, which is bound to the binding protein;

[0772] It is used in the treatment of advanced thyroid cancer.

[0773] 60. The binding protein according to items 1 to 21, wherein the dissociation constant K as measured on cells from the BHT-101 cell line is D Below 9 nM, such as below 8, 7, 6, 5, 4, 3, 2 or 1 nM, preferably below 1 nM, such as below 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2 nM.

[0774] 61. The binding protein according to item 60, wherein the dissociation constant K as measured on cells from the BHT-101 cell line is D Below 0.2 nM, making K D <0.2nM.

Claims

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

7.

2. The binding protein according to claim 1, wherein VHCDR1, VHCDR2 and VLCDR2 are present next to specific framework amino acids, wherein the CDR and framework amino acid (faa) sequences are selected from the group comprising: VHCDR1 and faa defined by SEQ ID NO:8; VHCDR2 and faa as defined in SEQ ID NO:9; VLCDR2 and faa as defined in SEQ ID NO: 10; and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto.

3. The binding protein according to claim 1, wherein the CDRs are individually selected from the group comprising: VHCDR1 is selected from SEQ ID NO: 11-18; VHCDR2 is selected from SEQ ID NO: 19-25, 32; VHCDR3 as defined by SEQ ID NO:3; VLCDR1 is selected from SEQ ID NO: 26-31; VLCDR2 is selected from TAS, SAS, and AAS; VLCDR3 as defined in SEQ ID NO:6; and CDR sequences having 95% or more, such as 96%, 97%, 98%, 99% or more identity thereto.

4. The binding protein according to claims 1 to 3, wherein the VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-54 and 147, and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto, and wherein the VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-74 and 148, and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto.

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

6. The binding protein according to claims 1 to 5, wherein the binding molecule is a monoclonal antibody of the IgG1 isotype, such as an IgG1 LALA antibody or an IgG1 LALAIAHA antibody.

7. The binding protein according to claims 1 to 6, wherein the binding protein is human or of human origin.

8. The binding protein according to claims 5 to 7, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 75-93 and 149, and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto; and the light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-113 and 150, and sequences having 80% or more, such as 85%, 90%, 95% or more identity thereto.

9. The binding protein according to claim 4 or 8, wherein the CDR sequence contains no variation in the amino acid sequence, or wherein the sequence variation of the CDR amino acid sequence is at most 5%, such as 4%, 3%, 2%, 1% or less.

10. The binding protein according to claims 1 to 9, wherein the binding protein binds to BHT-101 cells such that the KD value for the interaction is at most 1 nM, such as preferably at most 0.2 nM.

11. A conjugated binding protein comprising: (v) at least one binding protein as defined in any one of claims 1 to 10; and (vi) at least one reagent.

12. The conjugated binding protein of claim 11, wherein the at least one agent is a therapeutic agent or a detectable agent.

13. The conjugated binding protein of claim 12, wherein the at least one therapeutic agent is one or more cytotoxic agents, such as radioisotopes, cytostatic drugs, toxins or chemotherapeutic agents, and wherein the at least one detectable agent is one or more radioisotopes, enzymes, fluorescent molecules, dyes, digoxigenin or biotin.

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

15. The conjugated binding protein according to claims 11 to 14, wherein the agent is indirectly attached to the binding protein via a linker such as a chelating agent, wherein the chelating agent is 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 Derivatives, derivatives of (tBu)4(1-(1-carboxy-3-tert-butoxypropyl)-4,7,10-(tert-butoxymethyl)-1,4,7,10-tetraazacyclododecane) (DOTAGA), derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), derivatives of 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-acetic acid (NODAGA), and derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

16. A cell engineered 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, wherein the antigen binding domain comprises a binding protein according to any one of claims 1 to 5.

17. A pharmaceutical composition comprising the binding protein according to any one of claims 1 to 10, the conjugated binding protein according to any one of claims 11 to 15 or the cell according to claim 16, and a pharmaceutically acceptable carrier or excipient.

18. The binding protein of any one of claims 1 to 10, the conjugated binding protein of any one of claims 11 to 15, the cell of claim 16 or the pharmaceutical composition of claim 17 for use in therapy.

19. The binding protein, conjugated binding protein, cell or pharmaceutical composition according to claim 18, for use in cancer therapy.

20. The binding protein, conjugated binding protein, cell or pharmaceutical composition for use according to claim 19, wherein the cancer is advanced thyroid cancer.

21. An in vitro method for detecting the expression of CD44 variant CD44v6, the method comprising: (i) contacting a binding protein according to claims 1 to 10 or a conjugated binding protein according to claims 11 to 15 with a biological sample such as a tissue sample or a liquid obtained from a subject, such that if CD44v6 as defined in SEQ ID NO: 7 is present in the biological sample, the binding protein or the conjugated binding protein binds to its epitope; (ii) washing the biological sample to remove unbound binding protein or conjugated binding protein; as well as (iii) detecting any binding protein or conjugated binding protein in said biological sample that has bound to said epitope.

22. An in vivo method for detecting the expression of CD44 variant CD44v6, the method comprising: The conjugated binding protein according to claims 11 to 15 is detected to have bound to cells expressing the epitope of CD44v6 as defined in SEQ ID NO:7.

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