WO2024047172A1 - Conjugué anticorps-médicament d'intégrine alpha10 - Google Patents

Conjugué anticorps-médicament d'intégrine alpha10 Download PDF

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WO2024047172A1
WO2024047172A1 PCT/EP2023/073919 EP2023073919W WO2024047172A1 WO 2024047172 A1 WO2024047172 A1 WO 2024047172A1 EP 2023073919 W EP2023073919 W EP 2023073919W WO 2024047172 A1 WO2024047172 A1 WO 2024047172A1
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antibody
seq
drug conjugate
antigen
binding fragment
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PCT/EP2023/073919
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Evy LUNDGREN ÅKERLUND
Katarzyna CHMIELARSKA MASOUMI
Xiaoli Huang
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Targinta Ab
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to an integrin alphalO antibody drug conjugate and medical uses thereof.
  • ADCs Antibodydrug conjugates
  • tumour-selective drug delivery that may improve efficacy and reduce off-target toxicity [1]
  • the ADC approach builds on a potent cytotoxic agent, a so called “payload”, conjugated to a tumour-targeting antibody.
  • payload conjugated to a tumour-targeting antibody.
  • the majority of ADCs follow a similar mode of action that involves internalization and subsequent payload release. This typically kills the target cell, and sometimes also nearby cells, which is referred to as the bystander effect.
  • Integrin alphalO (gene name ITGA10) is a cell surface protein that belongs to the collagen-binding integrin subfamily, which consists of integrin alphalbetal, alpha2beta1, alpha10beta1 , and alpha11beta1 [3], Sequence analysis shows that the integrin alphalO subunit has the highest identity with integrin alpha 11 (43%) and an identity of 33 and 31% with integrin alphal and alpha2, respectively.
  • Integrin alpha10beta1 is normally expressed on chondrocytes in articular cartilage, in the vertebral column, in trachea and in the cartilage supporting the bronchi and on some cells in specialised fibrous tissues such as periosteum and perichondrium that likely represent mesenchymal stem cells [3-7], However, integrin alphal Obetal is highly expressed in various aggressive cancers such as triple negative breast cancer, glioblastoma, prostate cancer, pancreatic cancer and lung cancer [8,9] (WO 2020/212416). Expression of integrin alpha10beta1 is associated with metastasis and increased tumour aggressiveness (WO 2020/212416).
  • the present disclosure relates to an antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO, wherein the antibody or antigen-binding fragment comprises: a light chain variable region comprising a) a CDR-L1 comprising or consisting of an amino acid sequence of SEQ ID NO:
  • a heavy chain variable region comprising d) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; e) a CDR-H2 comprising or consisting of an amino acid sequence of SEQ ID NO: 8; and f) a CDR-H3 comprising or consisting of an amino acid sequence of SEQ ID NO: 9.
  • Another aspect of the present disclosure provides a polynucleotide encoding an antibody or antigen-binding fragment thereof according to the present disclosure or a component polypeptide chain thereof.
  • Another aspect of the present disclosure provides a vector comprising a polynucleotide disclosed herein.
  • Another aspect of the present disclosure provides a recombinant host cell comprising a polynucleotide disclosed herein.
  • Another aspect of the present disclosure provides a method for producing an antibody or antigen-binding fragment thereof according to the present disclosure, the method comprising culturing a host cell comprising the polynucleotide or the vector according to the present disclosure, under conditions which permit expression of the encoded antibody or antigen-binding fragment thereof.
  • Another aspect of the present disclosure provides for an in vitro method for the detection of cells expressing integrin alpha10beta1 in a subject, the method comprising: a) providing a sample of cells from a subject to be tested, such as biopsy tissue or blood sample; b) optionally, extracting and/or purifying the cells present in the sample; c) contacting an antibody or antigen-binding fragment thereof disclosed herein with cells present in the sample; d) determining whether the antibody or antigen-binding fragment thereof binds to the cells wherein the binding of the antibody or antigen-binding fragment thereof to the cells is indicative of the presence of a disease or disorder associated with cells expression integrin alphalO in the tissue of a subject.
  • Another aspect of the present disclosure provides for an in vitro method for identifying a patient with a disease or disorder associated with cells expressing integrin alphalO who would benefit from treatment with an antibody or antigen-binding fragment thereof according to the present disclosure, the method comprising: a) providing a sample, such as biopsy tissue or blood sample from a patient to be tested; b) optionally, extracting and/or purifying the cells present in the sample; c) contacting an antibody or antigen-binding fragment thereof according to the present disclosure with the sample; d) determining whether the antibody or antigen-binding fragment thereof binds to an integrin alphalO subunit in the sample, wherein the binding of the antibody or antigen-binding fragment thereof to an integrin alphal 0 subunit is indicative of a patient who would benefit from treatment with an antibody or antigen-binding fragment thereof according to the present disclosure.
  • Another aspect of the present disclosure provides for an in vitro method the detection of cells expressing integrin alphalO, the method comprising: a) contacting an antibody or antigen-binding fragment thereof according to the present disclosure with cells to be analysed for their expression of integrin alphalO; b) determining whether the antibody or antigen-binding fragment thereof binds to the cells wherein the binding of the antibody or antigen-binding fragment thereof to the cells is indicative of the presence of a disease or disorder associated with cells expression integrin alphalO in the tissue of a subject.
  • Another aspect of the present disclosure provides for in vivo imaging the expression of the integrin alpha10beta1 in a mammal, the method comprising the steps of a) Providing a mammal, b) Providing an antibody or antigen-binding fragment thereof according to the present disclosure, c) administering the antibody or antigen-binding fragment thereof according to the present disclosure to the mammal so as to allow the antibody or a fragment thereof to bind to an extracellular domain of integrin alphal Obetal of cells in said mammal, d) optionally adding a second labelled antibody or a fragment thereof to the sample, wherein the second antibody or a fragment thereof binds to the antibody or a fragment thereof in c), e) detecting the antibody or antigen-binding fragment thereof according to the present disclosure of said cells in c), or optionally detecting the second labelled antibody or a fragment thereof in d) bound to the antibody or a fragment thereof, and f) creating an image of the detected antibody or a fragment thereof,
  • the present disclosure provides for an antibody-drug conjugate directed against integrin alphalO comprising: a) an antibody or antigen-binding fragment thereof according to the present disclosure, b) an active agent, and c) optionally, a linker which links a) to b).
  • the present disclosure provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof or the antibody-drug conjugate according to the present disclosure, and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.
  • the present disclosure provides a method for delivery of an active agent to a cell expressing alpha10beta1 comprising administering to said cell the antibody-drug conjugate or the pharmaceutical composition according to the present disclosure, such that the active agent is delivered to said cell.
  • the present disclosure provides for the antibody-drug conjugate as described herein, for use as a medicament.
  • the present disclosure provides for the antibody-drug conjugate as described herein, for use in treating a patient with a disease or disorder associated with cells expressing integrin alphalO.
  • the present disclosure provides for the antibody-drug conjugate as described herein, for use in the treatment of a neoplastic disease or disorder.
  • the present disclosure provides a method for treatment of a disease characterized by expression of integrin alpha10beta1 in a subject, comprising administering to the subject the antibody-drug conjugate according to or the pharmaceutical composition according to the present disclosure.
  • the present disclosure provides a use of the antibody-drug conjugate or the pharmaceutical composition according to the present disclosure for treatment of a disease.
  • the present disclosure provides a kit comprising the antibody-drug conjugate or the pharmaceutical composition according to the present disclosure optionally further comprising means for administering the antibody-drug conjugate to a subject and/or instructions for use.
  • Figure 1 bis-mPEG-Glu-(Val-cit-PAB-DMEDA-PNU159682)-6’-amino-p- cyclodextrin.
  • the structure shows the conjugation technology, the linker and payload used in the antibody drug conjugates of the present disclosure. The structure is described more in detail in the section “Antibody-drug conjugate” herein.
  • the curvy line indicates the attachment with an antibody of the present disclosure.
  • FIG. 2 Internalization of five lead humanized antibody variants. Internalization of five antibody leads (Th-Ab9, Th-Ab11, Th-Ab12, Th-Ab14 and Th-Ab15) as well as a chimeric antibody (Th-AbO) and the mouse antibody (TM-Ab) was performed in C2C12alpha10 cells. The figure shows the percentage of antibody internalization after 4 hours incubation at 37 °C.
  • Figure 3 Binding specificity and affinity of Th-Ab12-ADC to integrin alphalO.
  • Binding specificity of Th-Ab12-ADC and the isotype control ADC (Ctrl-ADC) to C2C12alpha10 and C2C12alpha11 cells were investigated by flow cytometry (A). Binding affinity to C2C12alpha10 cells (B), triple negative breast cancer cell lines BT549 (C) and Hs578T (D), and glioblastoma cell lines U3046MG (E) and U3054MG
  • FIG. 4 In vitro cytotoxicity of Th-Ab12-ADC. Cytotoxicity of Th-Ab12-ADC in comparison with Ctrl-ADC and payload only (PNll-159682) was studied in C2C12alpha10 cells (A), C2C12alpha11 cells in monolayer culture (B), triple negative breast cancer cell lines BT549 (C) and Hs578T (D) cultured as sphere, glioblastoma cell lines U3046MG (E) and U3054MG (F) cultured as sphere, rhabdoid tumour A204
  • G cultured as monolayer
  • osteosarcoma SJSA-1 H cultured as sphere.
  • the cells were treated at the indicated concentrations for 5 days (C2C12alpha10, C2C12alpha11, A204, SJSA-1 cells) or 10 days (breast cancer and glioblastoma cells).
  • the level of cytotoxicity was measured by WST-1 assay.
  • FIG. 5 Internalization of Th-Ab12-ADC. Internalization of Th-Ab12-ADC and unconjugated antibody Th-Ab12 was performed in C2C12alpha10, triple negative breast cancer cells (BT549 and Hs578T), and glioblastoma cells (U3054MG and U3046MG). The figure shows the percentage of internalization after 90 min and after 4 hours of incubation at 37°C, compared to internalization at 4°C.
  • FIG. 6 Effect of Th-Ab12-ADC on cell cycle distribution.
  • C2C12alpha10 cells were treated with Th-Ab12-ADC or Ctrl-ADC at 0.02 nM for 5 days and the distribution (%) of the four indicated cell cycle stages was analysed using propidium iodide staining and flow cytometry.
  • FIG. 8 In vivo efficacy of Th-Ab12-ADC.
  • Figures A and C show that Th-Ab12-ADC reduced tumour growth compared to Ctrl-ADC and PBS, measured as reduction of tumour volume, measured by calliper.
  • Figures B and D show that neither of the treatments had a negative effect on body weight.
  • C2C12a10 cells were incubated with primary antibody against integrin a10 (Th-Ab12 or Tm-Ab) at a concentration of 10 pg/ml. After 30 min incubation with primary antibody, cells were washed twice with FACS buffer. Secondary antibody was then added according to Table 3 and incubated for 30 min. After incubation cells were washed twice with FACS buffer. Finally, a third antibody was added to samples 4 and 5 for the last incubation according to Table 3.
  • FIGS A and B show that in C2C12a10 cells, when Th-Ab12 antibody was incubated first (sample 4; 4 on X-axis), Th-Ab12 showed the same binding degree as the single staining (sample 2; 2 on X-axis) while Tm-Ab did not show any binding (sample 4; 4 on X-axis).
  • Tm-Ab was incubated first (sample 5; 5 on X-axis)
  • addition of Th-Ab12 caused the binding signal for Tm-Ab to decrease of 20% and Th-Ab12 bound to 40% (Fig. 9B).
  • an antibody includes a plurality of such antibodies.
  • an anti-integrin alphalO antibody can also refer to “Anti-integrin alphalO antibodies”, as for example the antibody variants described in Examples 1 to 8.
  • “Integrin alphalO” or “Integrin alphalO subunit” or “Integrin alphalO polypeptide” as used herein refers to the alphalO subunit of the heterodimeric protein integrin alpha10beta1. This denotation does not exclude the presence of the betal subunit bound to the alphalO subunit thus forming the integrin alpha10beta1 heterodimer. “Alpha” and “a”, as well as “alphalO” and “alphalO” are equivalent terms. “Integrin alphalO” as used herein may also refer to the polynucleotide transcript encoding the
  • SUBSTITUTE SHEET (RULE 26) alphalO subunit of the heterodimeric protein integrin alpha10beta1, and fragments thereof.
  • Anti-integrin alphalO antibody or “Integrin alphalO antibody” or “Anti-integrin alphalO subunit antibody” as used herein refers to an antibody capable of recognizing and binding to at least the alphalO integrin of the heterodimeric protein integrin alpha10beta1. These antibodies may be antibodies that recognize an epitope of the heterodimeric protein integrin alpha10beta1 , wherein the epitope comprises amino acid residues of both the alphalO and the betal integrin polypeptides.
  • the antibody or antigen-binding fragment of the invention/disclosure can be referred to as “the polypeptide of the invention/disclosure” or “the antibody polypeptide, or antigen-binding fragment thereof”, since an antibody and fragments thereof are polypeptides.
  • an antibody or an antigen-binding fragment thereof includes substantially intact antibodies as well as fragments and derivatives of antibodies.
  • An intact antibody can be regarded as an antibody comprising variable light regions, variable heavy regions, constant light regions and constant heavy regions. It further includes chimeric antibodies, humanized antibodies, isolated human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigenbinding fragments and derivatives of the same.
  • Suitable antigen-binding fragments and derivatives include, but are not necessarily limited to, Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g.
  • Fab fragments, Fab’ fragments and F(ab)2 fragments single variable domains (e.g. VH and VL domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).
  • dAbs including single and dual formats [i.e. dAb-linker-dAb]).
  • the potential advantages of using antibody fragments, rather than whole antibodies, are several-fold.
  • the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue.
  • antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • the term the “antibody” or “antigen-binding fragment of the invention/disclosure” is also intended to encompass antibody mimics (for example, non-antibody scaffold structures that have a high degree of stability yet allow variability to be introduced at certain positions).
  • antibody mimics for example, non-antibody scaffold structures that have a high degree of stability yet allow variability to be introduced at certain positions.
  • Exemplary antibody mimics include: affibodies (also called Trinectins; Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins; Innovations Pharmac. Technol.
  • adnectins also called monobodies; Meth. Mol. Biol., 352 (2007), 95-109); anticalins (Drug Discovery Today (2005), 10, 23-33); DARPins (ankyrins; Nat. Biotechnol. (2004), 22, 575-582); avimers (Nat. Biotechnol. (2005), 23, 1556-1561); microbodies (FEBS J, (2007), 274, 86-95); peptide aptamers (Expert.
  • amino acid as used herein includes the standard twenty genetically- encoded amino acids and their corresponding stereoisomers in the ‘D’ form (as compared to the natural 1’ form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g. a,a-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids as described herein.
  • unconventional amino acids e.g. a,a-disubstituted amino acids, N-alkyl amino acids, etc.
  • amino acid when an amino acid is being specifically enumerated, such as “alanine” or “Ala” or “A”, the term refers to both L -alanine and D -alanine unless explicitly stated otherwise.
  • polypeptides the antibody or antigen-binding fragment thereof
  • Other unconventional amino acids may also be suitable components for polypeptides (the antibody or antigen-binding fragment thereof) of the present disclosure, as long as the desired functional property is retained by the antibody or antigen-binding fragment.
  • amino acid sequences shown each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.
  • expression vector refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host.
  • control sequences may, e.g., include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and
  • SUBSTITUTE SHEET (RULE 26) sequences which control termination of transcription and translation.
  • the vector may, e.g., be a plasmid, a phage or simply a potential genomic insert. Once transformed into a suitable host, the vector may, e.g., replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
  • Expression vectors are designed, for example, as described in Li et al. (Construction strategies for developing expression vectors for recombinant monoclonal antibody production in CHO cells, Mol Biol Rep. 2018 Dec;45(6):2907-2912.)
  • Subject denotes a mammal, such as a rodent, a feline, a canine, an equine and a primate.
  • a subject according to the disclosure is a human.
  • sample encompasses any subject and a variety of sample types obtained from any subject.
  • samples useful in the disclosed methods include but are not limited to a subject, a liquid tissue sample such as blood, or a solid tissue sample such as biopsy material or tissue cultures or cells derived there from and the progeny thereof.
  • biological samples include cells obtained from a tissue sample collected from a subject.
  • samples encompass clinical samples, cells in culture, cell supernatants, cell lysates, and tissue samples, e.g. tissue samples from breast tissue, lung tissue, prostate tissue, pancreatic tissue, bone tissue, cartilage tissue, fat tissue, muscle tissue and connective tissue.
  • a “cancer” as used herein refers to any malignant and/or invasive growth or tumor caused by abnormal cell growth.
  • cancer refers to tumors named for the type of cells that form them.
  • a cancer or tumor comprise of tumor cells or cancer cells.
  • a cancer or tumor comprises also the cancer or tumor microenvironment, which can also comprise MSCs, fibroblasts, endothelial cells, pericytes, adipocytes, immune cells, tumor associated macrophages TAMs.
  • Part of a cancer or tumor might be stroma cells, for example connective tissue cells such as fibroblasts. Examples of solid tumors include but are not limited to sarcomas and carcinomas.
  • cancer includes but is not limited to a primary cancer that originates at a specific site in the body, a metastatic cancer that has spread from the place in which it started to other parts of the body, a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a person with a history of previous cancer of different type from latter one. Cancer, tumor, neoplasm are used herein as synonyms.
  • SUBSTITUTE SHEET (RULE 26) “Detection”, “detect”, “detecting” as used herein includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control, and further refers to the identification of the presence, absence or quantity of a given target, specifically the target of an integrin alphalO subunit.
  • disorder associated with cells expressing integrin alpha10beta1 we include such diseases or disorders wherein the pathological cells which are responsible, directly or indirectly, for the disorder express integrin alpha10beta1 on the cell surface.
  • the cells expressing integrin alpha10beta1 may be immune cells, cells of the connective tissue such as fibroblasts or neoplastic cells (cancer cells), e.g. tumor cells, per se.
  • cancer cells e.g. tumor cells
  • progenitor cells which are responsible, directly or indirectly, for the development of a neoplastic disease or disorder in an individual. Examples of CSCs are disclosed in Visvader & Lindeman, 2008, Nat Rev Cancer 8:755-768, the disclosures of which are incorporated herein by reference.
  • the cells expressing integrin alpha10beta1 may be associated indirectly with the neoplastic disease or disorder, for example, they may mediate cellular processes required for the cells to survive.
  • the present disclosure relates to an antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO, wherein the antibody or antigen-binding fragment comprises: a light chain variable region comprising a) a CDR-L1 comprising or consisting of an amino acid sequence of SEQ ID NO: 1 ; b) a CDR-L2 comprising or consisting of an amino acid sequence of SEQ ID NO: 2; and c) a CDR-L3 comprising or consisting of an amino acid sequence of SEQ ID NO:
  • a heavy chain variable region comprising d) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7;
  • SUBSTITUTE SHEET (RULE 26) e) a CDR-H2 comprising or consisting of an amino acid sequence of SEQ ID NO: 8; and f) a CDR-H3 comprising or consisting of an amino acid sequence of SEQ ID NO: 9.
  • the present disclosure relates to an antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO, wherein the antibody or antigen-binding fragment comprises: a light chain variable region comprising a) a CDR-L1 comprising or consisting of an amino acid sequence of SEQ ID NO: 1 ; b) a CDR-L2 comprising or consisting of an amino acid sequence of SEQ ID NO: 2; and c) a CDR-L3 comprising or consisting of an amino acid sequence of SEQ ID NO: 3; and a heavy chain variable region comprising d) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; e) a CDR-H2 comprising or consisting of an amino acid sequence of SEQ ID NO: 8; and f) a CDR-H3 comprising or consisting of an amino acid sequence of SEQ ID NO
  • the antibody or antigen-binding fragment of the disclosure has specificity for the alphal 0 subunit in the integrin alpha10beta1.
  • specificity we mean that the antibody or antigen-binding fragment is capable of binding to integrin alpha10beta1 in vivo, i.e., under the physiological conditions in which integrin alphal Obetal exists within the human body.
  • the antibody or antigen-binding fragment does not bind or has only minor binding to any other protein in vivo.
  • it is meant that the antibody or antigen-binding fragment is capable of binding to integrin alphal Obetal ex vivo or in vitro.
  • binding specificity may be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blots and flow cytometry using transfected cells expressing integrin alphal Obetal .
  • the antibody or antigen-binding fragment is capable of binding to integrin alpha10beta1 in vivo,
  • SUBSTITUTE SHEET (RULE 26) antibody or antigen-binding fragment is capable of binding selectively to integrin alpha10beta1 , i.e., it binds at least 10-fold more strongly to integrin alpha10beta1 than to any other proteins.
  • the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:11 and SEQ ID NO: 12 or a sequence having at least 85% sequence identity to any of SEQ ID NO: 10, SEQ ID NO:11 and SEQ ID NO:12, such as at least 95%, such as 98% or 99% sequence identity to any of SEQ ID NO: 10, SEQ ID NO:11 and SEQ ID NO:12; and/or b) an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19 or a sequence having at least 85% sequence identity to any of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:
  • the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12 and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or b) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12 and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 15; or c) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11 and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or d) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11 and an immunoglobulin
  • SUBSTITUTE SHEET (RULE 26) heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 17; or e) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12 and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin light chain variable region comprising CDR-L1 of SEQ ID NOU, CDR-L2 of SEQ ID NO:2 and CDR-L3 of SEQ ID NO:3; and b) an immunoglobulin heavy chain variable region comprising i. CDR-H1 of SEQ ID NO:4, CDR-H2 of SEQ ID NO:8 and CDR-H3 of SEQ ID NO:9, or ii. CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:8 and CDR-H3 of SEQ ID NO:9.
  • the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12; and/or b) an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising a) a CDR-L1 comprising or consisting of an amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of an amino acid sequence of SEQ ID NO: 2; and c) a CDR-L3 comprising or consisting of an amino acid sequence of SEQ ID NO: 3; and/or a heavy chain variable region comprising d) a CDR-H1 comprising or consisting of an amino acid sequence of SEQ ID NO: 4;
  • SUBSTITUTE SHEET (RULE 26) e) a CDR-H2 comprising or consisting of an amino acid sequence of SEQ ID NO: 8; and f) a CDR-H3 comprising or consisting of an amino acid sequence of SEQ ID NO: 9.
  • the antibody or antigen-binding fragment thereof comprises an IgG light chain constant region and an IgG heavy chain constant region.
  • the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin light chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO: 20; and/or b) an immunoglobulin heavy chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO: 21 .
  • the antibody or antigen-binding fragment thereof with binding specificity for the integrin alphalO polypeptide which is a part of an integrin alpha10beta1 heterodimer.
  • the antibody or antigen-binding fragment thereof with binding specificity for the integrin alpha10beta1 which is human integrin alpha10beta1.
  • the antibody or antigen-binding fragment thereof with binding specificity for the integrin alpha10beta1 which is expressed on the surface of a cell is expressed on the surface of a cell.
  • the antibody or antigen-binding fragment thereof binds to the extracellular l-domain of the integrin alphalO subunit.
  • the antibody or antigen-binding fragment thereof with binding specificity for the integrin alpha10beta1 is selected from a mouse antibody, a chimeric antibody, a human antibody, a humanised antibody, a humanised antigen-binding fragment, a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, an Fv, a single chain antibody (SCA) such as an scFv, a disulphide-bonded Fv, the variable portion of the heavy and/or light chains thereof, and a Fab miniantibody.
  • SCA single chain antibody
  • the antibody or antigen-binding fragment thereof with binding specificity for the integrin alpha10beta1 is a monoclonal antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof with binding specificity for the integrin alpha10beta1 is a humanised or fully human monoclonal antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof with binding specificity for the integrin alpha10beta1 is a recombinant antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment of the invention comprises or consists of an antibody mimic selected from the group comprising or consisting of affibodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains and affilins.
  • CTLDs tetranectins
  • adnectins monobodies
  • anticalins DARPins (ankyrins)
  • avimers iMabs, microbodies, peptide aptamers, Kunitz domains and affilins.
  • the invention also encompasses modified versions of antibodies and antigen-binding fragments thereof, whether existing now or in the future, e.g., modified by the covalent attachment of polyethylene glycol or another suitable polymer.
  • the antibody or antigen-binding fragment thereof with binding specificity for the integrin alpha10beta1 is conjugated to an additional moiety.
  • the additional moiety comprises a detectable moiety, such as a detectable moiety selected from the group consisting of a fluorophore, an enzyme and a radioactive tracer or radioisotope.
  • a detectable moiety selected from the group consisting of a fluorophore, an enzyme and a radioactive tracer or radioisotope.
  • the radioisotope is selected from the group consisting of 99mTc, 111 In, 67Ga, 68Ga, 72As, 89Zr, 1231 and 201TI.
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO comprises a pair of detectable and cytotoxic radioisotopes, such as 86Y/90Y or 1241/211 At.
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO comprises a radioisotope which is capable of simultaneously acting in a multi-modal manner as a detectable moiety and as a cytotoxic moiety.
  • the detectable moiety comprises or consists of a paramagnetic isotope.
  • the paramagnetic isotope is selected from the group consisting of 157Gd, 55Mn, 162Dy, 52Cr and 56Fe.
  • the detectable moiety is detectable by an imaging technique such as SPECT, PET, MRI, optical or ultrasound imaging.
  • the detectable moiety is joined to the antibody or antigen-binding fragment thereof indirectly, via a linking moiety.
  • the linking moiety is a chelator.
  • the chelator is selected from the group consisting of derivatives of 1 ,4, 7, 10-tetraazacyclododecane-1 , 4, 7, 10, tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic avid (DTPA), derivatives of S-2-(4- lsothiocyanatobenzyl)-1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA) and derivatives of 1 ,4,8,11-tetraazacyclodocedan-1 , 4, 8, 11 -tetraacetic acid (TETA).
  • DFA deferoxamine
  • DTPA diethylenetriaminepentaacetic avid
  • NOTA N-(4- lsothiocyanatobenzyl)-1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid
  • TETA tetraacetic acid
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO does not comprise a detectable moiety.
  • Integrins are heterodimers consisting of an alpha and a beta polypeptide.
  • the integrin alpha10beta1 heterodimer may be detected by integrin alphalO-specific antibodies and integrin alphalO binding peptides and proteins.
  • the integrin alphalO polypeptide is a part of an integrin alpha10beta1 heterodimer.
  • the integrin alphalO polypeptide is expressed on the surface of the cells.
  • the integrin alpha10beta1 was originally identified as a collagen type II binding receptor on chondrocytes in 1998 (Camper et al., 1998). In vitro studies have demonstrated its binding to other collagen subtypes and to laminin (Lundgren-Akerlund Book chapter and Thoren et al). Immunohistochemical analysis during development and in adult tissues has demonstrated a restricted localization to cartilage-containing tissues and to some fibrous tissues (Camper et al. 1998, Camper et al., 2001). Knockout mice lacking the marker have disorganized growth plates, decreased collagen in the matrix and shorter long-bones, further supporting its cell structural importance (Bengtsson et al., 2005). The amino acid sequence, variants, isoforms and sequence annotations can be found in Uniprot accession no 075578 (ITA10_HUMAN).
  • Integrin alpha10beta1 receptors transmit, upon binding to the extracellular ligand, intracellular signals that promote cell adhesion, migration, survival, proliferation, tumor growth and metastasis. Inhibition of the receptor thus impedes adhesion, migration, survival, proliferation, tumor growth and metastasis. This may be important for the treatment of many proliferative diseases such as cancer and inflammatory diseases.
  • integrin alphalO is a naturally occurring variant of integrin alphalO polypeptide, an isoform of integrin alphalO polypeptide or a splice variant of an integrin alphalO polypeptide.
  • Integrin alphalO can also be detected on nucleotide level by analyzing a sample for the presence of e.g. mRNA transcripts which upon translation generates an integrin alphalO antigen as defined herein above.
  • the antibody of the present invention is defined by its characteristic complementaritydetermining region (CDR) sequences.
  • CDR characteristic complementaritydetermining region
  • the CDRs of the antibody of the present invention have been defined using definition according to Kabat.
  • CDR-L1 , CDR-L2, CDR-L3, CDR-H1 , CDR-H2, CDR-H3) may be defined according to Kabat.
  • CDRs of the antibody of the invention by other approaches, for example by definition of CDRs according to Chothia (Al-Lazikani et al., (1997) JMB 273,927-948), Martin (Enhanced Chotia), Gelfand or Honneger. Further approaches exist, such as the AbM definition (combination of Kabat definition and Chothia definition used by Oxford Molecular's AbM antibody modelling software) or the contact definition (based on analysis of crystal structures). See, e.g., Kabat et al. (Sequences of Proteins of Immunological Interest, 1987 and 1991, NIH, Bethesda, Md.), Lefranc et al.
  • CDRs it can be beneficial to define the CDRs according to one numbering system, such as Kabat. Often, these CDR sequences are short (shorter than, for example, an approach combining numbering systems), thus providing the core sequences critical for binding. In other cases, it can be beneficial to use a combination of, for example, IMGT and Kabat CDR sequences.
  • the disclosure relates to an antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO, wherein the antibody or antigen-binding fragment comprises: a light chain variable region comprising a) a CDR-L1 comprising or consisting of an amino acid sequence of SEQ ID NO: 1 ; b) a CDR-L2 comprising or consisting of an amino acid sequence of SEQ ID NO: 2; and c) a CDR-L3 comprising or consisting of an amino acid sequence of SEQ ID NO: 3; and/or a heavy chain variable region comprising d) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; e) a CDR-H2 comprising or consisting of an amino acid sequence of SEQ ID NO: 8; and f) a CDR-H3 comprising or consisting of an amino acid sequence of SEQ
  • the antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising a) a CDR-L1 comprising or consisting of an amino acid sequence of SEQ ID NO: 1 ; b) a CDR-L2 comprising or consisting of an amino acid sequence of SEQ ID NO: 2; and c) a CDR-L3 comprising or consisting of an amino acid sequence of SEQ ID NO: 3; and/or a heavy chain variable region comprising d) a CDR-H1 comprising or consisting of an amino acid sequence of SEQ ID NO: 4;
  • SUBSTITUTE SHEET (RULE 26) e) a CDR-H2 comprising or consisting of an amino acid sequence of SEQ ID NO: 8; and f) a CDR-H3 comprising or consisting of an amino acid sequence of SEQ ID NO: 9.
  • the antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising a) a CDR-L1 comprising or consisting of an amino acid sequence of SEQ ID NO: 1 ; b) a CDR-L2 comprising or consisting of an amino acid sequence of SEQ ID NO: 2; and c) a CDR-L3 comprising or consisting of an amino acid sequence of SEQ ID NO: 3; and/or a heavy chain variable region comprising d) a CDR-H1 comprising or consisting of an amino acid sequence of SEQ ID NO: 5; e) a CDR-H2 comprising or consisting of an amino acid sequence of SEQ ID NO: 8; and f) a CDR-H3 comprising or consisting of an amino acid sequence of SEQ ID NO: 9.
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO comprises CDRs as described above (comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 9), wherein any one of the amino acids of the CDRs has been altered for another amino acid, for example, with the proviso that no more than 2 amino acids have been so altered, such as 1 amino acid.
  • the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:11 and SEQ ID NO:12 or a sequence having at least 85% sequence identity to any of SEQ ID NO: 10, SEQ ID NO:11 and SEQ ID NO:12,
  • SUBSTITUTE SHEET such as at least 95%, such as 98% or 99% sequence identity to any of SEQ ID NO: 10, SEQ ID NO:11 and SEQ ID NO:12; and/or b) an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19 or a sequence having at least 85% sequence identity to any of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, such as at least 95%, such as 98% or 99% sequence identity to any of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, such
  • the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12; and/or b) an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO comprises a light chain variable region comprising or consisting of a) the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12, for example at least 90%, 95%, 98% or 99% sequence identity; or b) an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:11 , or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO:11 , for example at least 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NO: 10 and SEQ ID NO:11.
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO comprises a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12, for example at least 90%, 95%, 98% or 99% sequence identity.
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO comprises a heavy chain variable region comprising or consisting of a) the amino acid sequence of SEQ ID NO: 16; or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 16 for example at least 90%, 95%, 98% or 99% sequence identity; or b) an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19; or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19, for example at least 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:17
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO comprises a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 16 for example at least 90%, 95%, 98% or 99% sequence identity.
  • Percent identity can be determined by, for example, the LALIGN program at the Expasy facility site (http://www.ch.embnet.org/software/LALIGN_form.html) using as parameters the global alignment option, scoring matrix BLOSUM62, opening gap penalty -14, extending gap penalty -4.
  • the percent sequence identity between two polypeptides, such as parts of an antibody may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program.
  • the parameters used may be as follows:
  • the BESTFIT program may be used to determine local sequence alignments.
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO comprises a light chain variable region and/or a heavy variable chain region as described above, wherein any one of the amino acids of the framework region of the light chain variable region and/or a heavy variable chain region has been altered for another amino acid, with the proviso that no more than 5 amino acids have been so altered, such as 4 amino acids, no more than 3 amino acids, such as 2 amino acids or no more than 1 amino acid.
  • variable light and variable heavy chains Combination of variable light and variable heavy chains
  • the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12; and/or b) an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16.
  • Another aspect of the disclosure relates to a polynucleotide encoding the antibody or antigen-binding fragment of the present disclosure, or a component polypeptide chain
  • polynucleotide we include DNA (e.g. genomic DNA or complementary DNA) and mRNA molecules, which may be single- or double-stranded.
  • the polynucleotide is an isolated polynucleotide.
  • the polynucleotide is a cDNA molecule.
  • polynucleotide may be codon- optimised for expression of the antibody or antigen-binding fragment in a particular host cell, e.g. for expression in human cells (for example, see Angov, 2011, Biotechnol. J. 6(6):650-659, the disclosures of which are incorporated herein by reference).
  • the polynucleotide encoding the antibody or antigen-binding fragment of the disclosure is encoding an antibody light chain or variable region thereof.
  • the polynucleotide encoding the antibody or antigen-binding fragment of the disclosure is encoding an antibody heavy chain or variable region thereof.
  • the polynucleotide encoding the antibody or antigen-binding fragment of the disclosure is encoding an antibody comprising: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12; and/or b) an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16.
  • Another aspect of the disclosure relates to a vector comprising the polynucleotide according to another aspect of the disclosure.
  • the vector is an expression vector.
  • expression vector is defined herein as a DNA molecule, for example linear or circular, which comprises a polynucleotide encoding a polypeptide of the present
  • SUBSTITUTE SHEET (RULE 26) invention (the antibody or antigen-binding fragment thereof) and is operably linked to additional nucleotides that provide for its expression.
  • plasmid the expression vector
  • vector vector
  • Another aspect of the disclosure relates to a recombinant host cell comprising the polynucleotide according to another aspect of the disclosure or a vector according to another aspect of the disclosure.
  • the recombinant host cell is a bacterial cell.
  • the recombinant host cell is a yeast cell.
  • the recombinant host cell is a mammalian cell.
  • the recombinant host cell is a human cell.
  • Another aspect of the disclosure relates to a method for producing the antibody or antigen-binding fragment of the another aspect of the invention, the method comprising culturing the host cell of another aspect of the disclosure comprising the polynucleotide of another aspect of the disclosure or the vector of the third aspect of the disclosure, under conditions which permit expression of the encoded antibody or antigen-binding fragment thereof.
  • Another aspect of the disclosure provides for an in vitro method for the detection of cells expressing integrin alpha10beta1 in a subject, the method comprising: a) providing a sample of cells from a subject to be tested, such as biopsy tissue or blood sample; b) optionally, extracting and/or purifying the cells present in the sample; c) contacting the antibody or antigen-binding fragment of the present disclosure with cells present in the sample; d) determining whether the antibody or antigen-binding fragment thereof binds to the cells,
  • SUBSTITUTE SHEET (RULE 26) wherein the binding of the antibody or antigen-binding fragment thereof to the cells is indicative of the presence of a disease or disorder associated with cells expression integrin alphalO in the tissue of a subject.
  • Yet another aspect of the present disclosure provides for an in vitro method for identifying a patient with a disease or disorder associated with cells expressing integrin alphalO who would benefit from treatment with an antibody or antigen-binding fragment thereof according to the present disclosure, the method comprising: a) providing a sample, such as biopsy tissue or blood sample from a patient to be tested; b) optionally, extracting and/or purifying the cells present in the sample; c) contacting an antibody or antigen-binding fragment thereof according to the present disclosure with the sample; d) determining whether the antibody or antigen-binding fragment thereof binds to an integrin alphalO subunit in the sample, wherein the binding of the antibody or antigen-binding fragment thereof to an integrin alphalO subunit is indicative of a patient who would benefit from treatment with an antibody or antigen-binding fragment thereof according to the present disclosure.
  • the antibody or antigen-binding fragment thereof according to the present disclosure can be used to detect an integrin alphalO subunit as found on the surface of cells, in particular cancer cells, expressing integrin alphalO as well as freely circulating in the blood of a patient.
  • integrin alphalO can be released from the cell membrane, for example due to the action of proteases, and end up in blood (shedding of integrin alphalO subunit).
  • the antibody or antigenbinding fragment thereof according to the present disclosure can be used to detect an integrin alphalO subunit directly in blood of a patient.
  • the present disclosure provides for a method for the detection of cells expressing integrin alphalO, the method comprising: a) contacting an antibody or antigen-binding fragment thereof according to the present disclosure with cells to be analysed for their expression of integrin alphalO; b) determining whether the antibody or antigen-binding fragment thereof binds to the cells
  • SUBSTITUTE SHEET (RULE 26) wherein the binding of the antibody or antigen-binding fragment thereof to the cells is indicative of the presence of a disease or disorder associated with cells expression integrin alphalO in the tissue of a subject.
  • said method can be is an in vivo method or an in vitro method.
  • the disclosure provides for a method for in vivo imaging the expression of the integrin alpha10beta1 in a mammal, the method comprising the steps of a) Providing a mammal, b) Providing an antibody or antigen-binding fragment thereof according to the present disclosure, c) administering the antibody or antigen-binding fragment thereof according to the present disclosure to the mammal so as to allow the antibody or a fragment thereof to bind to an extracellular domain of integrin alpha10beta1 of cells in said mammal, d) optionally adding a second labelled antibody or a fragment thereof to the sample, wherein the second antibody or a fragment thereof binds to the antibody or a fragment thereof in c), e) detecting the antibody or antigen-binding fragment thereof according to the present disclosure of said cells in c), or optionally detecting the second labelled antibody or a fragment thereof in d) bound to the antibody or a fragment thereof, and f) creating an image of the detected antibody or
  • the antibody is covalently bound to a detectable moiety, such as a detectable moiety selected from the group consisting of a fluorophore, an enzyme, a radioactive tracer or a radioisotope.
  • a detectable moiety selected from the group consisting of a fluorophore, an enzyme, a radioactive tracer or a radioisotope.
  • the integrin alphalO antigen may also be detected by detecting a peptide, protein or polypeptide other than integrin alphalO polypeptide, wherein said other peptide, protein or polypeptide is capable of specifically binding to an integrin alphalO antigen.
  • said peptide, protein or polypeptide is linked to an enzyme, a fluorophore or a radioactive tracer.
  • the radioactive tracer may e.g. be selected from a positron emitter, or a gamma emitter. Conjugation of the antibody to a detectable moiety facilitates and improves detection
  • SUBSTITUTE SHEET (RULE 26) said antibody, which in turn may facilitate detection of integrin alphalO-expressing cells in a sample and so the diagnosis of a cancer.
  • the antibody of the present disclosure can be used to detect integrin alphalO on cells, in tissues, in blood of a sample obtained from a mammal, such as in vitro, or even in vivo and/or in situ by using in vivo antibody-based detection techniques described herein and/or known to the person of skill in art.
  • the person of skill in the art is capable of selecting the standard laboratory equipment for detection of the integrin alphalO antibodies, depending on the situation and physical state of the sample.
  • the person of skill in the art would conduct the detection step using flow cytometry such as Fluorescence-Activated Cell Sorting (FACS).
  • flow cytometry such as Fluorescence-Activated Cell Sorting (FACS).
  • Typical immunological methods well known in the art include but are not limited to western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), immunofluorescent assay (IF), fluorescence in situ hybridization (FISH).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • IHC immunohistochemistry
  • IF immunofluorescent assay
  • FISH fluorescence in situ hybridization
  • Detecting integrin alphalO can be achieved using methods well known in the art of detection and imaging, such as clinical imaging, such as conventional fluorescence microscopes, confocal microscope, 2-photon microscopes, stimulated emission depletion (STED) etc.
  • clinical imaging such as conventional fluorescence microscopes, confocal microscope, 2-photon microscopes, stimulated emission depletion (STED) etc.
  • the detectable moiety is selected from the group consisting of a fluorophore, an enzyme or a radioactive tracer.
  • Typical methods for detection of cell surface antigens in vivo include but are not limited to fluorescence imaging, positron emission tomography, x- ray computed tomography (CT), magnetic resonance imaging (MRI) and functional magnetic resonance imaging (fMRI), ultrasound and single-photon emission computed tomography (SPECT).
  • CT positron emission tomography
  • MRI magnetic resonance imaging
  • fMRI functional magnetic resonance imaging
  • SPECT single-photon emission computed tomography
  • cell surface antigens can be imaged in vivo using immunolabelling with a radioactive tracer bound to an antibody or other specifically binding protein.
  • the antibodies used for in vivo imaging are antibody fragments such as Fab fragments, and single chain antibodies due to their smaller size and absence of effector function.
  • An antibody-drug conjugate is formed by covalent biochemical conjugation of a monoclonal antibody with high toxic payload via a small molecular linker.
  • ADC manufacturing is a multiple step process that can be divided into three distinct stages: cGMP production of the antibody, cGMP synthesis of the drug-linker complex, and conjugation to form an ADC.
  • the conjugated antibodies subsequently undergo an extensive purification process.
  • the present disclosure provides for an antibody-drug conjugate directed against integrin alphalO comprising: a) an antibody or antigen-binding fragment thereof as defined in the present disclosure, b) an active agent, and c) optionally, a linker which links a) to b).
  • the antibody is attached to the linker through a conjugating unit.
  • the antibody-drug conjugate of the present disclosure also comprises a functional unit, for example a unit that improved solubility of the antibodydrug conjugate.
  • the antibody drug conjugate (ADC) of the present disclosure comprises an active agent, i.e. a drug, which can be delivered intracellularly to cells expressing integrin alpha10beta1.
  • the active agent may e.g. be a therapeutic agent, a cytotoxic agent, a radioisotope or a detectable label. In a preferred embodiment the active agent is a therapeutic agent.
  • said active agent is a therapeutic agent, a cytotoxic agent, a microtubule toxin or a transcription toxin.
  • the active agent is a chemotherapeutic agent.
  • Classes of chemotherapeutic agents include alkylating agents, anthracyclines, antimetabolites, anti-microtubule/anti-mitotic agents, histone deacetylase inhibitors, kinase inhibitors, dihydrofolate reductase inhibitors, peptide antibiotics, platinum-based antineoplastics, topoisomerase inhibitors and cytotoxic antibiotics.
  • the active agent may be or comprise a radioisotope.
  • the radioisotope may serve as a radiation emitter either for treatment of affected tissues or for diagnostic purposes.
  • the radioisotope may consist of or comprise 60Co, 89Sr, 90Y, 99mTc, 1311, 137Cs, 153Sm, or 223Rd.
  • the radioisotope may be in combination with a chelator such as DOTA or EDTA or others which are well known in the art.
  • the active agent is a therapeutic agent.
  • Classes of therapeutic agents include DNA crosslinking agents, DNA alkylating agents, DNA strand scission agents, anthracyclines, antimetabolites, anti-microtubule/anti-mitotic agents, histone deacetylase inhibitors, kinase inhibitors, metabolism inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antineoplastics, topoisomerase inhibitors, DNA or RNA polymerase inhibitors, immunomodulators, nucleotide-based agents, and cytotoxic antibiotics.
  • the active agent is a cytotoxic agent.
  • the active agent is a therapeutic agent, such as a therapeutic agent selected from the group consisting of microtubule toxins, DNA toxins and transcription toxins.
  • the active agent is a microtubule toxin, such as a microtubule toxin selected from the group consisting of Auristatin-based toxins, Maytansinoid-based toxins, Tubulysins-based toxins and Eribulin.
  • the active agent is a transcription toxin, such as an RNA polymerase II and/or III inhibiting agent.
  • the active agent is a therapeutic agent, such as a therapeutic agent selected from the group consisting of alkylating agents, anthracyclines, antimetabolites, anti-microtubule/anti-mitotic agents, histone deacetylase inhibitors, kinase inhibitors, peptide antibiotics, platinum-based antineoplastics, topoisomerase inhibitors and cytotoxic antibiotics.
  • a therapeutic agent selected from the group consisting of alkylating agents, anthracyclines, antimetabolites, anti-microtubule/anti-mitotic agents, histone deacetylase inhibitors, kinase inhibitors, peptide antibiotics, platinum-based antineoplastics, topoisomerase inhibitors and cytotoxic antibiotics.
  • the active agent is a transcription toxin selected from the group consisting of Doxorubicin, Doxorubicin derivatives and Amanitin.
  • the active agent is an anthracycline, such as an anthracycline selected from Daunorubicin, doxorubicin, epirubicin, idarubicin, and 3'-deamino-3"-4'- anhydro-[2"(S)-methoxy-3"(R)-hydroxy-4''-morpholinyl]doxorubicin (PNll 159682).
  • an anthracycline selected from Daunorubicin, doxorubicin, epirubicin, idarubicin, and 3'-deamino-3"-4'- anhydro-[2"(S)-methoxy-3"(R)-hydroxy-4''-morpholinyl]doxorubicin (PNll 159682).
  • the active agent is 3'-deamino-3"-4'-anhydro-[2"(S)- methoxy-3"(R)-hydroxy-4"-morpholinyl]doxorubicin (PNU159682), which is represented in Formula X: attachment site to a linker.
  • the active agent is a DNA- or RNA-polymerase inhibitor, such as a polymerase inhibitor selected from amanitin or alpha-amanitin or derivatives thereof, actinomycin D, and aphidicolin.
  • the active agent is a nucleotide-based agent, such as an RNA- or DNA-oligonucleotide, such as a siRNA or a miRNA
  • DAR drug-to-antibody ratio
  • the DAR is between 1 and 10, such as between 2 and 8, for example between 3 and 6, such as 1, 2, 3 or 4.
  • the DAR is 1 or 2.
  • Linkers may e.g. be based on chemical motifs including disulfides, hydrazones or peptides (cleavable), or thioethers (non-cleavable), and control the distribution and delivery of the cytotoxic agent to the target cell. Cleavable and non- cleavable types of linkers have been proven to be safe in preclinical and clinical trials.
  • the ADC as disclosed herein comprises a linker that links the antibody to the active agent.
  • the antibody-drug conjugate comprises a linker selected from a cleavable and a non-cleavable linker.
  • linker cleavable or non-cleavable
  • cleavable linkers can e.g. be cleaved by enzymes in the target cell, leading to efficient intracellular release of the active agent, for example a cytotoxic agent.
  • an ADC containing a non-cleavable linker has no mechanism for drug release, and must rely on mechanisms such as degradation of the targeting antibody, for drug release.
  • the linker composition may influence critical factors such as solubility and pharmacokinetic properties of the ADC as a whole.
  • drug release is crucial for obtaining a cellular effect.
  • Drugs which are able to freely diffuse across cell membranes may escape from the targeted cell and, in a process called “bystander killing,” also attack neighbouring cells, such as cancer cells in the vicinity of the integrin alpha10beta1 expressing target cell.
  • Cleavable groups include a disulfide bond, an amide bond, a substituted amide bond in the form of a peptide bond, a thioamide, bond, an ester bond, a thioester bond, a vicinal diol bond, or a hemiacetal.
  • cleavable bonds may include enzymatically-cleavable bonds, such as peptide bonds (cleaved by peptidases), phosphate bonds (cleaved by phosphatases), nucleic acid bonds (cleaved by endonucleases), and sugar bonds (cleaved by glycosidases).
  • the linker is a cleavable linker allowing for intracellular release of the active agent inside the target cells.
  • linker from those routinely used for ADCs.
  • linkers include but not limited to Val-Cit-PAB, Fmoc-Val-Cit-PAB, Fmoc-Val-Cit-PAB-PNP, MC-Val-Cit-PAB-PNP, Phe- Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB-PNP, Ala-Ala-Asn- PAB TFA salt, Fmoc-Ala-Ala-Asn-PAB-PNP, Fmoc-Gly3-Val-Cit-PAB, Fmoc-Gly3-Val- Cit-PAB-PNP, SMCC, Py-ds-Prp-OSu, Py-ds-dmBut-OSu, Py-ds-dmBut-OSu, Py-ds-dmBut-
  • the linker is an enzyme-cleavable linker, such as a cathepsin cleavable linker.
  • the linker comprises a peptide linker.
  • the choice of peptide sequence is critical to the success of the conjugate.
  • the linker is stable to serum proteases, yet is cleaved by lysosomal enzymes in the target cell.
  • the linker is an enzyme-cleavable peptide-containing linker, such as a cathepsin cleavable peptide-containing linker.
  • Cathepsin can be one of several cathepsin types, being one of a group of lysosomal proteases.
  • the linker comprises a dipeptide, such as Valine-Citrulline (Val- Cit) or Valine-Alanine (Val-Ala).
  • the linker comprises the dipeptide Valine-Citrulline (Val-Cit, Formula V).
  • Enzymatically cleavable linkers may include a “self-immolative molecule” also referred to as “self-immolative spacer” or “self-immolative linker” to spatially separate the drug from the site of enzymatic cleavage.
  • a self-immolative spacer also referred to as “self-immolative spacer” or “self-immolative linker” to spatially separate the drug from the site of enzymatic cleavage.
  • the direct attachment of a drug to a peptide linker can result in proteolytic release of an amino acid adduct of the drug, thereby impairing its activity.
  • the use of a self-immolative spacer allows for the elimination of the fully active, chemically unmodified drug upon amide bond hydrolysis.
  • the linker comprises one or more self-immolative molecules.
  • self-immolative molecules include p-aminobenzylcarbamoyl (PAB) and N,N'-Dimethylethylenediamine (DMEDA).
  • the linker comprises a dipeptide and p-aminobenzylcarbamoyl (PAB, Formula P).
  • the linker comprises a N,N'-Dimethylethylenediamine (DMEDA, Formula D).
  • DMEDA N,N'-Dimethylethylenediamine
  • the linker comprises a dipeptide, such as Valine-Citrulline (Val-Cit) or Valine-Alanine (Val-Ala), and the self-immolative molecules p- aminobenzylcarbamoyl (PAB) and N,N'-Dimethylethylenediamine (DM EDA).
  • a dipeptide such as Valine-Citrulline (Val-Cit) or Valine-Alanine (Val-Ala)
  • PAB p- aminobenzylcarbamoyl
  • DM EDA N,N'-Dimethylethylenediamine
  • the linker comprises or consists of Val-Cit-PAB-DMEDA, as represented in Formula Y.
  • the antibody-drug conjugate of one aspect of present disclosure further comprises a conjugating unit, such as a conjugating unit derived from reactive groups such as a functionalized benzoic acid, an activated carboxylic acid derivative, an amino group, a maleimide group or derivatives thereof, N-hydroxysuccinimide, bissulfones, azides and alkynes via click chemistry, reactive attachment groups directed to modified or unmodified protein-bound carbohydrate, peptide sequences that are required for enzymatic reactions, by reaction with the antibody or a chemically or enzymatically generated derivative thereof.
  • a conjugating unit such as a conjugating unit derived from reactive groups such as a functionalized benzoic acid, an activated carboxylic acid derivative, an amino group, a maleimide group or derivatives thereof, N-hydroxysuccinimide, bissulfones, azides and alkynes via click chemistry, reactive attachment groups directed to modified or unmodified protein-bound carbohydrate, peptide sequences that are required for
  • the conjugating unit is derived from a functionalized bis-sulfone group according to Formula C below:
  • R and R’ are each independently selected form the group consisting of selected from the group consisting of Ci-Ce alkyl, a C3-C7 cycloalkyl, a C3-C7 heterocycloalkyl, phenyl, C5-C10 aryl, each of which may optionally be substituted by one or more selected from halogen, cyano, amino, Ci-Ce alkyl, Ci-Ce alkoxy, phenyl, and C5-C10 aryl.
  • the antibody or antigen-binding fragment thereof with binding specificity for integrin alphalO disclosed herein may be conjugated to the conjugating unit via a number of inter-chain thiol bridging groups across both the light chain and heavy chain constant regions of the antibody, as well as inter-chain thiol bridging groups between the heavy chain constant regions of the antibody.
  • the conjugating unit when the conjugating unit is derived from a functionalized bis- sulfone group, such as Fornula C or C’ or C”, the conjugation may occur by means of a disulfide reduction, such as by means of a reaction between one or more -SH groups of cysteines within the antibody or antigen-binding fragment thereof and the sulfone groups of the conjugating unit. Consequently, the SCh-PEGn-unit may by a leaving group which detaches from the conjugated ADC.
  • a disulfide reduction such as by means of a reaction between one or more -SH groups of cysteines within the antibody or antigen-binding fragment thereof and the sulfone groups of the conjugating unit. Consequently, the SCh-PEGn-unit may by a leaving group which detaches from the conjugated ADC.
  • the conjugating unit is derived from a functionalized bis-sulfone group according to formula C’ Formula C’ wherein * indicates an attachment site to a linker, and wherein n is an integer between 1 and 10.
  • the conjugating unit is derived from a functionalized bis-sulfone group according to formula C”, also herein referred to as bis-mPEG.
  • the conjugating unit conjugates the antibody against integrin alphalO of the present disclosure to the linker, which is attached to the active agent.
  • the conjugating unit is derived from a functionalized bis-sulfone group such as any of Formula C, C’ or C”, and is functionalized with a glutamic acid.
  • the conjugating unit is derived from a functionalized bis-sulfone group according to formula C”, also herein referred to as bison PEG , and functionalized with a glutamic acid to form a bis-mPEG-Glu, wherein the Glu is attached to the * in Formula C”.
  • the antibody-drug conjugate of one aspect of present disclosure comprises a functional unit, such as a unit that improves solubility of the antibody-drug conjugate, such as a cyclodextrin or a PEG molecule.
  • the functional unit comprises or consists of 6’-amino-p- cyclodextrin.
  • the functional unit comprises or consists of a PEG molecule having a molecular weight of 10 kDa or below, such as 8 kDa, such as 5 kDa or below, such as 4.6 kDA, such as 4 kDa, such as 1 kDa, such as 0.6 kDA, such as 0.4 KDa, such as 0.2 kDA.
  • the functional unit such as the unit that improves solubility of the antibody-drug conjugate comprises or consists of a PEG molecule consisting of 72 PEG units or less, such as 50 PEG units, such as 20 PEG units.
  • the functional unit is attached to the conjugating unit.
  • the functional unit is attached to the glutamic acid comprised in the conjugating unit.
  • the functional unit is attached to the linker.
  • the antibody-drug conjugate of the present disclosure comprises bis-mPEG-Glu-(Val-cit-PAB-DMEDA-PNU159682)-6’-amino-p-cyclodextrin, as represented in Formula A.
  • the antibody-drug conjugate of the present disclosure comprises: a) the antibody or antigen-binding fragment thereof according to another aspect of the present disclosure, wherein the antibody or antigen-binding fragment thereof comprises
  • a heavy chain variable region comprising a CDR-H1 consisting of any one of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, a CDR-H2 consisting of SEQ ID NO:5 and a CDR-H3 consisting of SEQ ID NO:6;
  • a conjugating unit such as a conjugating unit derived from a bis-sulfone group according to formula C” and functionalized with a glutamic acid (bis-mPEG- Glu);
  • a linker comprising or consisting of Val-cit-PAB-DMEDA;
  • a functional unit such as 6’-amino-p-cyclodextrin; and e) the active agent PNU159682.
  • the antibody-drug conjugate comprises an antibody or antigenbinding fragment that comprises: a light chain variable region comprising a) a CDR-L1 comprising or consisting of an amino acid sequence of SEQ ID NO: 1 ; b) a CDR-L2 comprising or consisting of an amino acid sequence of SEQ ID NO: 2; and c) a CDR-L3 comprising or consisting of an amino acid sequence of SEQ ID NO: 3; and a heavy chain variable region comprising d) a CDR-H1 comprising or consisting of an amino acid sequence of SEQ ID NO: 4; e) a CDR-H2 comprising or consisting of an amino acid sequence of SEQ ID NO: 8; and f) a CDR-H3 comprising or consisting of an amino acid sequence of SEQ ID NO: 9.
  • the antibody-drug conjugate comprises an antibody or antigenbinding fragment that comprises: a light chain variable region comprising a) a CDR-L1 comprising or consisting of an amino acid sequence of SEQ ID NO: 1 ; b) a CDR-L2 comprising or consisting of an amino acid sequence of SEQ ID NO: 2; and c) a CDR-L3 comprising or consisting of an amino acid sequence of SEQ ID NO: 3; and a heavy chain variable region comprising d) a CDR-H1 comprising or consisting of an amino acid sequence of SEQ ID NO: 5; e) a CDR-H2 comprising or consisting of an amino acid sequence of SEQ ID NO: 8; and f) a CDR-H3 comprising or consisting of an amino acid sequence of SEQ ID NO: 9.
  • the antibody-drug conjugate comprises an antibody or antigen- binding fragment that comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12 and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or b) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12 and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 15; or c) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11 and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or d) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11 and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 17; or
  • the antibody-drug conjugate comprises an antibody or antigen-binding fragment that comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12; and b) an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16.
  • the antibody-drug conjugate of the present disclosure comprises the antibody or antigen-binding fragment thereof of the present disclosure which is linked to a conjugating unit, such as bis-mPEG of Formula C” functionalized with a glutamic acid (bis-mPEG-Glu), which is linked to a linker comprising or consisting of Val-Cit-PAB-DMEDA, which is linked to the active agent PNU159682, wherein the conjugating unit is also linked to a functional unit, such as 6’-amino-p- cyclodextrin, via the glutamic acid comprised in the conjugating unit.
  • a conjugating unit such as bis-mPEG of Formula C” functionalized with a glutamic acid (bis-mPEG-Glu)
  • bis-mPEG-Glu glutamic acid
  • the conjugating unit is also linked to a functional unit, such as 6’-amino-p- cyclodextrin, via the glutamic acid comprised in the conjugating unit.
  • the antibody-drug conjugate of the present disclosure comprises the antibody or antigen-binding fragment thereof of the present disclosure which is linked to a drug conjugate as illustrated in Figure 1.
  • the ADCs directed against integrin alphalO as described herein are useful for the delivery of active agents, such as therapeutic or cytotoxic agents to cells expressing alphalO betal and thus for the treatment of a range of diseases and disorder associated with cells expressing integrin alpha10beta1.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the ADC, as described herein, together with a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.
  • compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and/or animals.
  • the pharmaceutical compositions may be lyophilised, e.g. through freeze drying, spray drying, spray cooling, or through use of particle formation from supercritical particle formation.
  • pharmaceutically acceptable we mean a non-toxic material that does not decrease the effectiveness of the anti-integrin alphalO ADC.
  • pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference).
  • buffer is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH.
  • Pharmaceutically acceptable buffers are well known in the art.
  • diluting is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the agent in the pharmaceutical preparation.
  • adjuvant is intended to mean any compound added to the formulation to increase the biological effect of the agent of the invention.
  • the adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition.
  • the adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.
  • cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.
  • the excipient may be one or more of carbohydrates, polymers, lipids and minerals.
  • carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating lyophilisation.
  • polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation.
  • lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers.
  • minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.
  • the ADCs of the present disclosure may be formulated into any type of pharmaceutical composition known in the art to be suitable for the delivery thereof.
  • the ADCs of the present disclosure or pharmaceutical compositions comprising the ADCs may be administered via any suitable route known to those skilled in the art.
  • possible routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar, buccal, oral, vaginal and rectal.
  • administration from implants is possible.
  • the pharmaceutical compositions are administered parenterally, for example, intravenously, intracerebroventricularly, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are conveniently used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multidose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the ADCs of the present disclosure are administered in situ in the body of a subject during a surgery, to the damaged site.
  • the ADCs of the present disclosure are administered intravenously.
  • the ADCs of the present disclosure are administered subcutaneously. In one embodiment the ADCs of the present disclosure are administered intracranially or intracerebrally.
  • compositions will be administered to a patient in a pharmaceutically effective amount.
  • the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent.
  • a therapeutically effective amount can be determined by the ordinarily skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
  • the administration of the pharmaceutically effective dose can be carried out both by single administration in the form of an individual dose unit, or else several smaller dose units, and also by multiple administrations of subdivided doses at specific intervals. Alternatively, the dose may be provided as a continuous infusion over a prolonged period.
  • the ADCs targeting integrin alpha10beta1 described herein may be administered alone or in combination with other therapeutic agents.
  • the ADCs targeting integrin alphalO described herein may be administered in combination with a range of anti-cancer agents, such as antimetabolites, alkylating agents, anthracyclines and other cytotoxic antibiotics, vinca alkyloids, anti-microtubule/anti-mitotic agents, histone deacetylase inhibitors, kinase inhibitors, peptide antibiotics, platinum-based antineoplastics, etoposide, taxanes, topoisomerase inhibitors, antiproliferative immunosuppressants, corticosteroids, sex hormones and hormone antagonists, cytotoxic antibiotics and other therapeutic agents.
  • anti-cancer agents such as antimetabolites, alkylating agents, anthracyclines and other cytotoxic antibiotics, vinca alkyloids, anti-microtubule/anti-mitotic agents, histone deacet
  • the ADC of the present disclosure is administered in conjunction with additional reagents and/or therapeutics that may increase the functional efficiency of the ADC, such as established or novel drugs that increase lysosomal membrane permeability, thereby facilitating molecular entry from the lysosome interior to the cytoplasm, or drugs that increase the permeability of the blood-brain barrier.
  • additional reagents and/or therapeutics that may increase the functional efficiency of the ADC, such as established or novel drugs that increase lysosomal membrane permeability, thereby facilitating molecular entry from the lysosome interior to the cytoplasm, or drugs that increase the permeability of the blood-brain barrier.
  • the present disclosure provides for a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to another aspect of the disclosure, or the antibody-drug conjugate according to the present disclosure, and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.
  • the present disclosure provides for a method for delivery an active agent to a cell expressing alpha10beta1 comprising administering to said cell the antibody-drug conjugate as described herein, or the pharmaceutical composition according to another aspect of the disclosure, such that the active agent is delivered to said cell.
  • the present disclosure provides for the antibody-drug conjugate or the pharmaceutical composition as described herein, for use as a medicament.
  • the present disclosure provides for the antibody-drug conjugate or the pharmaceutical composition as described herein, for use in treating a patient with a disease or disorder associated with cells expressing integrin alphalO.
  • the cells expressing integrin alphalO are malignant cells or tumor-associated cells, such as cancer associated fibroblast (CAFs), stromal cells, stem cells and/or stem-like cells and/or cells of the tumor microenvironment such as tumor-associated macrophages (TAMs), immune cells, endothelial cells.
  • CAFs cancer associated fibroblast
  • TAMs tumor-associated macrophages
  • immune cells endothelial cells.
  • the present disclosure provides for the antibody-drug conjugate or the pharmaceutical composition as described herein, for use in the treatment of a neoplastic disease or disorder.
  • the neoplastic disease or disorder is solid tumor or a lymphoma.
  • the neoplastic disease or disorder is a cancer.
  • the cancer is selected from the group consisting of breast cancer, brain cancer, cancer of the Central Nervous System (CNS), lung cancer, prostate cancer, pancreatic cancer, skin cancer, lymphoma, sarcoma, rhabdoid tumor, cholangiocarcinoma, or a metastasis of any one of said cancer forms.
  • cholangiocarcinoma can be an intrahepatic bile duct cancer, a perihilar bile duct cancer or a distal (extrahepatic) bile duct cancer.
  • the breast cancer is selected from the group consisting of triple negative breast cancer and inflammatory breast cancer.
  • Triple negative breast cancer is selected from the group consisting of basal-like 1 breast cancer, basal-like 2 breast cancer, claudin-low breast cancer, metaplastic breast cancer (MBC), interferon-rich breast cancer, immunomodulatory breast cancer, mesenchymal breast cancer, mesenchymal stem-like breast cancer, luminal androgen receptor breast cancer and unstable breast cancer.
  • the lung cancer is selected from the group consisting of squamous cell lung carcinoma, lung adenocarcinoma, large cell lung carcinoma and small-cell lung carcinoma.
  • the prostate cancer is small cell neuroendocrine carcinoma (SCNC) or castrate-resistant prostate cancer (CRPC).
  • SCNC small cell neuroendocrine carcinoma
  • CRPC castrate-resistant prostate cancer
  • the pancreatic cancer is an exocrine tumor or an endocrine tumor, such as wherein the pancreas cancer is an exocrine tumor selected from the group consisting of ductal adenocarcinoma, acinar cell carcinoma, adeno-squamous carcinoma, intraductal papillary mucinous neoplasm (IPMN) and Pancreatic intraepithelial neoplasia.
  • IPMN intraductal papillary mucinous neoplasm
  • Pancreatic intraepithelial neoplasia Pancreatic intraepithelial neoplasia.
  • the pancreatic cancer is an endocrine tumor selected from the group consisting of neuroendocrine tumor, gastrinoma, glucaganoma, insulinoma, somatostatinoma, VIPoma, and non-functional Islet cell tumor, such as wherein the neuroendocrine tumor is a grade I, grade II or grade III pancreatic cancer.
  • the brain cancer and/or the cancer of the CNS is selected from the group consisting of tumours of neuroepithelial tissue, tumours of cranial and paraspinal nerves, tumours of the meninges, tumours of the haematopoietic system, and tumours of the sellar region.
  • the brain cancer and/or the cancer of the CNS is an astrocytic tumour, such as an astrocytic tumor selected from Glioblastoma, Giant cell glioblastoma, Pilocytic astrocytoma, Pilomyxoid astrocytoma, Subependymal giant cell astrocytoma, Pleomorphic xanthoastrocytoma, Diffuse astrocytoma, Anaplastic astrocytoma, Gliosarcoma, Gliomatosis cerebri.
  • an astrocytic tumour such as an astrocytic tumor selected from Glioblastoma, Giant cell glioblastoma, Pilocytic astrocytoma, Pilomyxoid astrocytoma, Subependymal giant cell astrocytoma, Pleomorphic xanthoastrocytoma, Diffuse astrocytoma, Anaplastic astro
  • the brain cancer and/or the cancer of the CNS is an embryonal tumour such as a neuroblastoma, a medulloblastoma and/or a rhabdoid tumour of the brain.
  • the brain cancer and/or the cancer of the CNS is an ependymal tumour, such as an ependymal tumour selected from Subependymoma, Myxopapillary ependymoma, Ependymoma, and Anaplastic ependymoma.
  • an ependymal tumour selected from Subependymoma, Myxopapillary ependymoma, Ependymoma, and Anaplastic ependymoma.
  • the tumours of neuroepithelial tissue are selected from: a) Astrocytic tumours selected from Pilocytic astrocytoma, Pilomyxoid astrocytoma, Subependymal giant cell astrocytoma, Pleomorphic xanthoastrocytoma, Diffuse astrocytoma, Anaplastic astrocytoma, Glioblastoma, Giant cell glioblastoma, Gliosarcoma, Gliomatosis cerebri, and b) Oligodendroglial tumours selected from Oligodendroglioma and Anaplastic oligodendroglioma, and c) Oligoastrocytic tumours selected from Oligoastrocytoma and Anaplastic oligoastrocytoma, and d) Ependymal tumours selected from Subependymoma, Myxopapillary ependymoma, Ependymo
  • the tumours of cranial and paraspinal nerves are selected from: a) Schwannoma, b) Neurofibroma, c) Perineurioma, and d) Malignant peripheral nerve sheath tumour (MPNST).
  • the tumours of the meninges are selected from: a) Tumours of meningothelial cells, selected from Meningioma, Atypical meningioma, Anaplastic meningioma, b) Mesenchymal tumours selected from Lipoma, Angiolipoma, Hibernoma, Liposarcoma, Solitary fibrous tumour, Fibrosarcoma, Malignant fibrous histiocytoma, Leiomyoma, Leiomyosarcoma, Rhabdomyoma, Rhabdomyosarcoma, Chondroma, Chondrosarcoma, Osteoma, Osteosarcoma, Osteo-chondroma, Haemangioma, Epithelioid hemangioendothelioma, Haemangiopericytoma, Anaplastic haemangiopericytoma, and Angiosarcoma, Kaposi Sarcoma, Ewing Sarcoma, Kaposi Sarcom
  • the tumours of the haematopoietic system are selected from: a) Malignant Lymphomas, Plasmocytoma, and b) Granulocytic sarcoma
  • the tumours of the sellar region are selected from: a) Craniopharyngioma, b) Granular cell tumour, c) Pituicytoma, and d) Spindle cell oncocytoma of the adenohypophysis.
  • the skin cancer is a melanoma, such as malignant melanoma.
  • the sarcoma is an osteosarcoma.
  • the the antibody-drug conjugate as described herein inhibits cell division and/or inhibits cell proliferation and/or inhibits cell survival and/or induces cell death of cells expressing integrin alpha10beta1.
  • the described effect of the antibodydrug conjugate may result from blocking of microtubule polymerization, resulting in cell cycle arrest and inducing caspase-3-dependent apoptosis.
  • the cell proliferation and/or cell survival may be the result of the antibody-mediated cytotoxicity and/or binding to the epitope antigen of cancer cells and inhibits downstream signal transduction of antigen receptor which can lead to inhibition of cell survival and proliferation and induce cell apoptosis.
  • the antibody-drug conjugate as described herein may irreversibly bind to DNA and cause strong inter strand cross-linking that prevents DNA strand separation, thus destroying necessary DNA metabolic processes and finally leading to cell deaths.
  • the antibody-drug conjugate as described herein inhibits spreading of cancer cells to other sites within the same organ, as well as to other organs.
  • the antibody-drug conjugate as described herein inhibits metastasis of cancer, wherein the cancer is any one mentioned herein, such as wherein the cancer is a cancer characterized by expression of integrin alpha10beta1.
  • the the antibody-drug conjugate as described herein induces bystander effect which results in cell death of integrin alphal 0-negative cancer cells.
  • cytotoxic agent that is conjugated to the antibody by either a cleavable or non-cleavable linker has the capacity to diffuse across cell membranes after the release from the antibody and thereby cause killing of neighbouring cells.
  • the cytotoxic agent may be either the cytotoxic agent only or the cytotoxic agent with a part of the linker that has the bystander kill capacity.
  • the capacity to diffuse across cell membranes is related to the hydrophobicity of the cytotoxic agent or the combination of the cytotoxic agent and the linker.
  • bystander killing or “bystander effect” refers to the killing of target negative cells in the presence of target-positive cells, wherein killing of target-negative cells is not observed in the absence of target-positive cells.
  • Cell-to-cell contact, or at least proximity between target-positive and target- negative cells enables bystander killing. This type of killing is distinguishable from “off-target killing,” which refers to the indiscriminate killing of target-negative cells. “Off-target killing” may be observed in the absence of targetpositive cells.
  • the antibody-drug conjugate or the pharmaceutical composition according to other aspect of present disclosure is administered parenterally, for example, intravenously, intracerebroventricularly, intraarticularly, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, by infusion techniques, or in situ.
  • parenterally for example, intravenously, intracerebroventricularly, intraarticularly, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, by infusion techniques, or in situ.
  • in situ it can be understood that the antibody-drug conjugate or the pharmaceutical composition may be administered at the site of neoplasm where abnormal cells have not spread beyond where they first formed, or it can be understood that the a the antibody-drug conjugate or the pharmaceutical composition may be administered at the site, from which a neoplasm has been surgically removed.
  • the antibody-drug conjugate or the pharmaceutical composition is administered in combination with one or more further agents, such as one or more further therapeutic agents.
  • the present disclosure provides for a method for treatment of a disease characterized by expression of integrin alphal Obetal in a subject, comprising administering to the subject the antibody-drug conjugate or the pharmaceutical composition according to other aspects of the present disclosure described herein.
  • the present disclosure provides for a use of the antibody-drug conjugate or the pharmaceutical composition according other aspects of the disclosure for treatment of a disease.
  • the present disclosure provides for a kit comprising the antibody-drug conjugate or the pharmaceutical composition as described herein, optionally further comprising means for administering the antibody-drug conjugate to a subject and/or instructions for use.
  • Aim To generate human antibody sequence segments to create humanised antibody variants.
  • Structural models of the mouse antibody Tm-Ab regions were produced. Based on structure analysis, sequences of humanized variants, likely to be essential for the binding properties of the antibody, were selected and analysed in silico. Human sequence segments were identified both within and outside the CDR regions. Selected sequence segments were assembled to generate complete humanized V region sequences that were devoid of, or reduced in, significant T cell epitopes to avoid immunogenicity.
  • VH1 to VH7 The design resulted in seven heavy chain (VH1 to VH7) and three light chain (VK1 to VK3) sequences that were used for gene synthesis and expression in mammalian cells (Table 1). Seven VH were combined with 3 VK generating 21 different humanized variants.
  • Table 1 Summary table of the 21 humanized antibody variants including a chimeric antibody (Th-AbO).
  • Aim To generate small scale batches of antibodies for lead candidate selection.
  • Aim To select five lead candidates from the 21 humanized variants based on integrin a10 binding properties.
  • the mouse myoblast cell line C2C12 overexpressing either human integrin alpha10beta1 (C2C12apha10) or human integrin alpha11beta1 (C2C12alpha11) were used to study antibody binding and specificity.
  • the C2C12 cells were incubated (100 000 cells/sample) with the integrin alphalO antibodies at 1 pg/ml for 30 min followed by a secondary antibody for 30 min followed by analysis by flow cytometry.
  • results The results showed that the chimeric antibody Th-AbO as well as humanized antibody variants Th-Ab1 - Th-Ab15 had the best binding affinity to C2C12alpha10 cells (data not shown). None of the antibodies showed binding to the control C2C12alpha11 cells (data not shown). Additionally, risk analysis data from the sequence design process suggested that the antibodies Th-Ab9, Th-Ab11 , Th-Ab12, Th-Ab14 and Th-Ab15 have lower immunogenicity than the other variants (data not shown), therefore, these antibodies were selected for further internalization and thermal stability studies.
  • Aim To investigate the internalization level of the five antibody candidates in integrin alphalO expressing cell lines.
  • the mouse myoblast cell line C2C12 overexpressing the integrin alpha10beta1 (C2C12alpha10) was used in the internalization assay.
  • the cells 500 000 cells/sample) were incubated with the five lead humanized antibodies as well as the chimeric antibody Th-AbO and the original mouse antibody Tm-Ab (1 pg/sample) for 30 min at 4 °C.
  • the cells were then washed with PBS containing 2% FBS and incubated at 37 °C for 90 minutes or 4 hours and then incubated with a secondary antibody for 20 min in the dark at 4 °C.
  • the cells were washed twice with PBS prior to analysis of internalization rate (%) by flow cytometry.
  • Aim To rank the top five humanized antibody variants based on stability.
  • Thermal stability analysis of the humanized antibody lead candidates was performed using an UncleTM biostability platform and software. Samples for each variant were formulated in PBS and Sypro Orange at a final concentration of 0.5 mg/ml. Samples were subjected to a thermal ramp from 25 - 95 °C, with a ramp rate of 0.3 °C/minute and excitation at 473 nm. Monitoring of static light scattering (SLS) at 473 nm allowed the detection of protein aggregation, and calculation of T agg .
  • SLS static light scattering
  • Aim To generate and characterize Th-Ab12-ADC.
  • ADCs used for these studies were generated using a well-established conjugation approach.
  • targeting antibodies were subjected to conjugation to a “anthracycline” type of payload (Glu-(Val-cit-PAB-DMEDA- PNU159682)-6’-amino-p-cyclodextrin) by mild reduction of interchain disulfides, followed by a cysteine rebridging approach to produce ADCs with highly homogeneous drug-to-antibody ratios (DAR) of around 4.
  • DAR drug-to-antibody ratios
  • ADCs were generated with a disulfide rebridging conjugation technology (bison PEG according to Formula C” and functionalized with a glutamic acid), a cytotoxic payload PNU159682 (Formula X) and a cleavable linker Val-Cit-PAB-DMEDA (Formula Y).
  • the structure is shown in Figure 1.
  • An average DAR of the ADCs is 4.
  • the percentage of the monomeric purity were over 95% for all the ADCs and the percentage of the free payload species were not detectable. Endotoxin levels were below 0.1 EU/mg.
  • Integrin alpha10beta1 expressing cells C2C12alpha10
  • integrin alpha11beta1 overexpressing cells C2C12alpha11
  • BT549 and Hs578T triple negative breast cancer cells
  • A204 rhabdoid tumour
  • SJSA-1 osteosarcoma
  • U3046MG and U3054 MG human patient-derived glioblastoma cells
  • the cells were incubated with the IgG 1 isotype control Ctrl-ADC at 100 nM or Th-Ab12-ADC at different concentrations (0.5, 1 , 5, 10, 50, 100, 1000 nM) for 30 min at 4°C and were then incubated with a secondary antibody for 30 min at 4°C prior to the analysis by flow cytometry.
  • the binding EC50 for each cell line is within a range of 0.9 - 2.69 nM ( Figure 3B - H). There was no binding to C2C12alpha11 cells suggesting that Th- Ab12-ADC is specifically binding to integrin alphalO ( Figure 3A).
  • Th-Ab12-ADC binds to integrin alphalO in a specific manner and with high affinity, which supports its potential for successful drug development.
  • Aim To evaluate the in vitro efficacy of Th-Ab12-ADC in different cell lines.
  • Cells were seeded either as monolayer (C2C12alpha10, C2C12alpha11 and A204) or as spheres (BT549, Hs578T, U3046MG, U3054MG and SJSA-1) and treated with the IgG 1 isotype control Ctrl-ADC or Th-Ab12-ADC at different concentrations (0.13, 0.77, 4.6, 27.8, 167, 100 nM) for 5 days (C2C12alpha10, C2C12alpha11, A204 and SJSA-1) or 10 days (BT549, Hs578T, U3046MG and U3054MG).
  • the WST-1 assay is based on the cleavage of tetrazolium salt WST-1 for formazan by cellular mitochondrial dehydrogenases. The larger the number of viable cells, the higher the activity of the mitochondrial dehydrogenases, and in turn the greater the amount of formazan dye formed.
  • Th-Ab12-ADC was found to induce integrin alphalO specific toxicity in C2C12alpha10, TNBC and GB cell lines with high potency.
  • Aim To study the internalization rate of Th-Ab12-ADC in various integrin alphalO expressing cell lines.
  • C2C12alpha10, BT549, Hs578T, U3046MG and U3054MG cells were used.
  • the cells 500 000 cells/sample) were incubated with Th-Ab12-ADC or unconjugated antibody Th-Ab12 (1 pg/0.1 ml) for 30 min on ice.
  • the cells were then washed with PBS containing 2% FBS and incubated at 37 °C for 90 minutes or 4 hours and then incubated with a secondary antibody containing Alexa 488 fluorochrome for 20 min at 4 °C.
  • the cells were washed twice with PBS prior to the analysis of internalization rate (%) by flow cytometry.
  • Th-Ab12-ADC was internalized by 50 - 60% within 4 h of incubation in all the cell lines tested ( Figure 4). Overall, the internalization level was higher at 4 h than at 90 min. The internalization of Th-Ab12-ADC was slightly better than unconjugated Th- Ab12 in TNBC and GB cell lines ( Figure 4).
  • C2C12alpha10 cells (9 000 cells/well) were seeded and cultured as monolayer in a 6-well culture plate and incubated with ADCs at concentration of 0.02 nM. On day 5, the cells were harvested and stained with propidium iodide (PI) before analysis of cell cycle distribution using Propidium Iodide Flow Cytometry Kit (Abeam, ab139418) according to the manufacturer’s instructions.
  • PI propidium iodide
  • Th-Ab12-ADCs bind to the C2C12alpha10 cells, is internalized, and subsequently cleaved to release the cell toxin. The released toxin then causes DNA damage and lead to cell death/apoptosis.
  • C2C12alpha10 and C2C12alpha11 cells (with no integrin alphalO expression) were used.
  • C2C12alpha10 and C2C12alpha11 cells were seeded and cultured in a 6-well culture plate at 12 000 and 8 000 cells/well, respectively.
  • the cells were seeded at 20 000 cells/well.
  • Th-Ab12-ADC and Ctrl-ADC were added to the wells at a concentration of 0.02 nM. Viable cells were detached from the plate after 5 days of culture, and the cell number in each well was determined using a cell counter.
  • the ratio of C2C12alpha10 and C2C12alpha11 cells the cells (100 000/sample) were stained with integrin alphalO antibody conjugated with Alexa 647 and analyzed by a flow cytometry.
  • Th-Ab12-ADC has good bystander effect which means that Th-Ab12-ADC can also induce cell death in nearby cells without integrin alphalO expression. This can be beneficial for the treatment of solid tumours with heterogeneous integrin alphalO expression.
  • Aim To evaluate the in vivo efficacy of Th-Ab12-ADC in glioblastoma cell line U3046MG-derived xenograft nude mouse model.
  • Th-Ab12-ADC Treatment with Th-Ab12-ADC inhibited tumour growth compared to the Ctrl-ADC and PBS ( Figure 8A and C). The inhibitory effect was seen with both doses (0.75 mg/kg and 1 .5 mg/kg). The ADC treatments did not affect the body weights of the mice compared to the PBS group ( Figure 8B and D).
  • the results show that Th-Ab12-ADC exhibits good in vivo efficacy in glioblastoma U3046MG xenograft nude mouse model. This suggests that Th-Ab12- ADC has the potential to become an effective treatment for glioblastoma and other aggressive cancers expressing integrin alpha10beta1.
  • the mouse myoblast cell line C2C12 transduced with integrin a10 vector (C2C12a10) was cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% FBS (Gibco) and Antibiotic-Antimycotic (100 U/rnL, Gibco).
  • the C2C12a10 cells were selected using G418 (1 mg/ml, Gibco).
  • Antibodies Primary antibodies: Th-Ab12 (supernatant VH4VK3 variant) and Tm-Ab- A647 (conjugated to AlexaFluor A647, thus Tm-Ab-A647; also corresponding to: "an alternative mouse monoclonal antibody against integrin alphalO” used in Examples 2 , 3, 4, and 9 of WO 2020/212416, and to the antibody against integrin alphalO used in Munksgaard Thoren M., 2019 [8] both for FACS and conjugated to Saporin) at 10 pg/ml.
  • Secondary antibody Donkey anti human IgG Alexa 488 (T011), 1:1000 for Th- Ab12.
  • Table 3 Cells were incubated with a primary antibody against integrin a10 (Th-Ab12 or Tm-Ab- A647) at a concentration of 10 pg/ml. After 30 min incubation with primary antibody, cells were washed twice with FACS buffer; DPBS (Hyclone) containing 1% FBS (Gibco) and 0,1 % sodium azide (G Bioscience). Secondary antibody or donkey anti-human IgG AF488 was then added according to the table above and incubated for 30 min. After incubation cells were washed twice with FACS buffer. Finally, a third antibody was added to samples 4 and 5 for the last incubation according to the table above. Samples 4 and 5 were also washed twice prior to the analysis using flow cytometry.
  • a primary antibody against integrin a10 Th-Ab12 or Tm-Ab- A647
  • samples with only one integrin a10 antibody are used as single stain reference to see how the primary antibody binds to the target in absence of competition.
  • the samples with successive addition of the two antibodies against integrin a10 are the samples that will be competing for binding of target in this assay. Samples 1 and 6 are used to determine background staining.
  • Th-Ab12 has a higher affinity towards C2C12a10 cells than Tm-Ab.
  • CDR-L1 Variable light chain complementarity-determining region 1
  • SEQ ID NO: 2 KVSNRFS
  • CDR-L2 Variable light chain complementarity-determining region 2 (CDR-L2) SEQ ID NO: 3: SQSTHVPYT
  • CDR-L3 Variable light chain complementarity-determining region 3
  • CDR-H1 Variable heavy chain complementarity-determining region 1
  • CDR-H1 Variable heavy chain complementarity-determining region 1
  • CDR-H1 Variable heavy chain complementarity-determining region 1
  • CDR-H1 Variable heavy chain complementarity-determining region 1
  • SEQ ID NO: 8 WIFPGSGRTYYSEKFRG
  • CDR-H2 Variable heavy chain complementarity-determining region 2
  • CDR-H3 Variable heavy chain complementarity-determining region 3
  • VH2 Variable heavy chain - VH2
  • VH4 Variable heavy chain - VH4
  • VH5 Variable heavy chain - VH5

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Abstract

La présente invention concerne un conjugué anticorps-médicament alpha10 d'intégrine et des utilisations médicales associées.
PCT/EP2023/073919 2022-08-31 2023-08-31 Conjugué anticorps-médicament d'intégrine alpha10 WO2024047172A1 (fr)

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Citations (3)

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WO2007107774A2 (fr) * 2006-03-22 2007-09-27 Cartela R & D Ab Nouvelles méthodes de diagnostic et de traitement
WO2016133449A1 (fr) * 2015-02-16 2016-08-25 Xintela Ab Détection et traitement de tumeurs malignes dans le snc
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