WO2016118090A1 - Protéines de liaison à l'antigène spécifiques du cancer - Google Patents

Protéines de liaison à l'antigène spécifiques du cancer Download PDF

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WO2016118090A1
WO2016118090A1 PCT/SG2016/050032 SG2016050032W WO2016118090A1 WO 2016118090 A1 WO2016118090 A1 WO 2016118090A1 SG 2016050032 W SG2016050032 W SG 2016050032W WO 2016118090 A1 WO2016118090 A1 WO 2016118090A1
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antigen
binding
binding protein
binding fragment
cells
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PCT/SG2016/050032
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Boon Hwa Andre Choo
Tien Sze Louisa LUK
Robert Brown
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Agency For Science, Technology And Research
Imperial Innovations Limited
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • the present invention relates to antigen -binding proteins and fragments thereof as well as antibodies that bind to cancer cells. Specifically, the present invention relates to antigen -binding proteins and fragments thereof that bind to clathrin heavy chain 1 (CHC1) on the surface of cancer stem cells.
  • CHC1 clathrin heavy chain 1
  • Cancer stem cells are hypothesized to be a rare population of tumor cells possessing embryonic stem cell-like characteristics and are resistant to conventional chemotherapy treatments.
  • mAbs monoclonal antibodies developed against several growth factor receptors have been shown to exert therapeutic efficacy in the treatment of solid tumors, such as ovarian cancer.
  • solid tumors such as ovarian cancer.
  • bevacizumab a potent angiogenesis inhibitor
  • mAbs do not target CSC markers and therefore fail to demonstrate clinically significant effects on overall survival in cancer.
  • an antigen-binding protein or an antigen-binding fragment thereof, comprising (i) a heavy chain variable domain comprising a VHCDR1 having the amino acid sequence GFSLSRYGVY; a VHCDR2 having the amino acid sequence VIW A VGS TN YNS ALMS , and a VHCDR3 having the amino acid sequence DREYGYGLAY; and (ii) a light chain variable domain comprising a VLCDR1 having the amino acid sequence SASSSVSYIH, a VLCDR2 having the amino acid sequence DTSNLAS, and a VLCDR3 having the amino acid sequence FQGSGYPLT.
  • an antigen-binding protein or an antigen-binding fragment thereof, or an antigen-binding fragment thereof, that competes with the antigen binding protein as described herein for binding to clathrin heavy chain 1 (CHC1).
  • CHC1 clathrin heavy chain 1
  • an antigen-binding protein or an antigen-binding fragment thereof, as described herein, comprising a radioisotope or a cytotoxin conjugated thereto.
  • composition comprising a physiologically acceptable carrier and a therapeutically effective amount of the antigen-binding protein, or an antigen-binding fragment thereof as described herein.
  • an antigen-binding protein, or an antigen-binding fragment thereof as described herein in the manufacture of a medicament for treating cancer.
  • a method for detecting cancer in a subject comprising: contacting a sample obtained from the subject with an antigen-binding protein, or an antigen-binding fragment thereof as described herein in vitro; detecting the binding of the antigen-binding protein, or an antigen-binding fragment thereof in the sample; correlating the binding with a level of binding in a control sample to determine the level of binding in the sample, wherein an increase in the level of binding in the sample relative to the control sample is indicative of cancer.
  • a method for treating cancer in a subject comprising administering a therapeutically effective amount of an antigen-binding protein, or an antigen-binding fragment thereof as described herein to a subject in need thereof.
  • kits when used in the method as described herein, comprising an antigen-binding protein, or an antigen-binding fragment thereof as described herein, together with instructions for use.
  • FIG. 1 Identification of stem cell-like subpopulations in ovarian cancer cell lines.
  • A flow cytometry analysis displaying a representative diagram of the SP in IGROV1, following staining with Hoechst 33342 dye (right). Effect of the transporter blocker, verapamil, on the SP subset (left). Hoechst "blue” represents the 450 BP filter, the standard analysis wavelength for Hoechst 33342 analysis for DNA content. Hoechst "red” was detected at 675 nm.
  • B flow cytometry analysis displaying a representative diagram of Aldefluor-positive cells in IGROV1 when cells were incubated with the Aldefluor substrate, BAAA (right). The specific inhibitor of ALDH, DEAB, was used to establish the baseline fluorescence of the cells (left).
  • Fig. 2 Characterization of stem cell-like subpopulations in the IGROV1 ovarian cancer cell line.
  • A RT-qPCR analysis. Mean mRNA expression levels of stem cell markers in SP ( ⁇ ) in relation to NSP (;3 ⁇ 4 «).
  • FIG. 3 Identification of mAbs which fulfilled the selection criteria.
  • A representative flow cytometry analyses displaying reactivity of a positive antibody clone, TAG-OCS l. MAbs were screened against IGROVl Aldefluor-positive cells and positive antibody clones were then tested against five cell lines - IGROVl, PEA1, hESC, IMR90 and HEK293. (:3 ⁇ 4) represent the control samples while ( ⁇ ) represent samples incubated with the TAG-OCS l.
  • B representative immunocytochemistry data showing binding of TAG-OCS l to subpopulations of cells in the IGROVl cell line. Scale bar: 20 ⁇ .
  • FIG. 4 Reactivity of TAG-OCS l towards cell populations.
  • A flow cytometry analysis of IGROVl cells which were co-stained with TAG-OCS l and Hoechst dye.
  • B flow cytometry analyses displaying reactivity of TAG-OCS l to hESC and hESC-derived EBs.
  • C immunocytochemistry images showing binding of TAG-OCS l to hESCs. HESCs stained with Oct4 served as controls. Scale bar: 200 ⁇ .
  • D flow cytometry analyses displaying reactivity of TAG-OCS l towards cells from the normal ovarian cell lines, OSEC2 and IOSE21.
  • Fig. 5 Characterization of ovarian tumour subpopulations detected by TAG- OCS l.
  • A mean mRNA expression levels by RT-qPCR of stem cell markers in IGROVl TAG-OCS l -positive populations ( ⁇ ) in relation to TAG-OCS l -negative populations (3 ⁇ 43 ⁇ 43 ⁇ 4).
  • Fig. 6 Validating the target antigen of TAG-OCS l.
  • A validation of mass spectrometry results by immunoprecipitation and subsequent Western blotting. Immunoprecipitated eluate (IP) and membrane protein extracts (Lysate) using TAG-OCS l were subjected to Western blot and probed with rnAb to human CHC1 (mAb-CHCl, left). To confirm this result, immunoprecipitated eluate using human CHC1 was subjected to Western blot and probed with TAG-OCS l (right). Human CHC1 antibody and TAG-OCS l served as negative controls.
  • B RT-qPCR analysis.
  • D & E show staining of clinical samples by TAG-OCS l.
  • D immunofluorescence staining showing EpCam-positive patient ovarian ascites stained positively by TAG-OCS l and patient ovarian tumour sections were positively-stained by TAG-OCS l.
  • E immunohistochemistry showing patient ovarian tumour sections stained positively by TAG-OCS l.
  • F differential staining in normal and ovarian patient tumour microarray (TMA) sections. Patient TMA sections stained positively by TAG-OCS l while normal ovary sections did not exhibit any staining by TAG-OCS l.
  • FIG. 7 Effect of TAG-OCS l on cells in vitro.
  • A left, Alamar Blue assay measuring viability of IGROV1 ovarian cancer cells after addition of TAG-OCS l compared to the buffer control.
  • Right xCELLigence experiment measuring cell indexes of IGROV1 ovarian cancer cells after addition of TAG-OCS l ( ⁇ ) compared to the buffer control (;;3 ⁇ 43 ⁇ 4).
  • B left, microscopic images of IGROV1 cells after a 72 hours incubation period with buffer.
  • Right microscopic images of IGROV1 cells after a 72 hours incubation period with TAG- OCS l.
  • Scale bar 500 ⁇ .
  • the mAb also binds to 87.5% of 786-0 cells (kidney cancer cell line) and 92.1% of MCF7 cells (breast cancer cell line). Furthermore, TAG-OCS l confers cytotoxicity towards MCF7 cells.
  • F CDC assay showing percentage cytotoxicity of IGROV1 cells after incubation with complement or inactivated complement.
  • G CDC assay showing percentage cytotoxicity of 786-0 (kidney cancer) cells after incubation with complement or inactivated complement.
  • H CDC assay showing percentage cytotoxicity of MCF7 (breast cancer) cells after incubation with complement or inactivated complement.
  • I ADC assay showing luminescence levels of viable cells after IGROV1 cells were incubated with buffer, ZAP, mAb controls or mAb- ZAP.
  • J ADC assay showing luminescence levels of viable cells after MCF7 cells were incubated with buffer, ZAP, mAb controls or mAb-ZAP.
  • TAG-OCS 1 -positive cells formed increased sizes and numbers of colonies when compared to their counterparts as well as to unseparated cells.
  • TAG-OCS 1 mediates CDC activity and is successful as an ADC in PEA1 cells.
  • Fig. 11 TAG-OCS 1 heavy and light chain amino acid sequence alignment to the encoding nucleic acid sequence.
  • FIG. 12 A and B, flow cytometry experiments show that chimeric TAG-OCS 1 successfully binds to 786-0 kidney cancer cell line (A) and the MCF7 breast cancer cell line (B) at a level which is comparable to the original IgM molecule. Chimeric TAG-OCS 1 also convers cytotoxicity towards MCF7 cells.
  • C ADC assay showing luminescence levels of viable cells after IGROV1 cells were incubated with buffer, ZAP, mAb controls or mAb- ZAP.
  • D ADC assay showing luminescence levels of viable cells after PEA1 cells were incubated with buffer, ZAP, mAb controls or mAb-ZAP.
  • the present invention relates to antigen-binding proteins, or an antigen-binding fragments thereof, comprising (i) a heavy chain variable domain comprising a VHCDR1 having the amino acid sequence GFSLSRYGVY; a VHCDR2 having the amino acid sequence VIWAVGSTNYNSALMS, and a VHCDR3 having the amino acid sequence DREYGYGLAY; and (ii) a light chain variable domain comprising a VLCDR1 having the amino acid sequence SASSSVSYIH, a VLCDR2 having the amino acid sequence DTSNLAS, and a VLCDR3 having the amino acid sequence FQGSGYPLT.
  • antigen binding protein refers to antibodies, antibody fragments and other protein constructs, such as domains, which are capable of binding to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • F(ab') 2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • a Fd fragment consisting of the
  • fragment includes a reference to a nucleic acid or polypeptide molecule that encodes a constituent or is a constituent of a particular polypeptide / nucleic acid or variant thereof.
  • the fragment possesses qualitative biological activity in common with the polypeptide in question.
  • fragments of a nucleic acid sequence do not necessarily need to encode polypeptides which retain biological activity, for example, hybridisation probes or PCR primers.
  • a fragment of a nucleic acid sequence encodes a polypeptide which retains qualitative biological activity of the polypeptide.
  • the fragment may be physically derived from the full- length polypeptide / nucleic acid or alternatively may be synthesised by some other means, for example chemical synthesis.
  • antibody is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain and includes monoclonal, recombinant, polyclonal, chimeric, humanised, bispecific and heteroconjugate antibodies; a single variable domain, a domain antibody, antigen binding fragments, immunologically effective fragments, single chain Fv, diabodies, TandabsTM.
  • an antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • CDRs are defined as the complementarity determining region amino acid sequences of an antigen binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chains and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, “CDRs” as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.
  • An antigen binding fragment may be provided by means of arrangement of one or more CDRs on non-antibody protein scaffolds such as a domain.
  • the domain may be a domain antibody or may be a domain which is a derivative of a scaffold selected from the group consisting of CTLA-4, lipocalin, SpA, an Affibody, an avimer, GroEl, transferrin, GroES and fibronectin/adnectin, which has been subjected to protein engineering in order to obtain binding to an antigen, other than the natural ligand.
  • An antigen binding fragment or an immunologically effective fragment may comprise partial heavy or light chain variable sequences. Fragments are at least 5, 6, 8 or 10 amino acids in length. Alternatively the fragments are at least 15, at least 20, at least 50, at least 75, or at least 100 amino acids in length.
  • single variable domain refers to an antigen binding protein variable domain (for example, V H , V HH , V L ) that specifically binds an antigen or epitope independently of a different variable region or domain.
  • a “domain antibody” or “dAb” may be considered the same as a “single variable domain” which is capable of binding to an antigen.
  • a single variable domain may be a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent, nurse shark and Camelid V HH dAbs.
  • Camelid V HH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such V HH domains may be humanised according to standard techniques available in the art, and such domains are considered to be "domain antibodies”. As used herein V H includes camelid V HH domains.
  • domain refers to a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
  • a "single variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
  • a domain can bind an antigen or epitope independently of a different variable region or domain.
  • antigen binding protein binds to its antigen, for example, clathrin or clathrin heavy chain 1 (CHC1) with no or insignificant binding to other proteins.
  • CHC1 clathrin heavy chain 1
  • the antigen binding proteins described herein may bind to its antigen with at least 2, 5, 10, 50, 100, or 1000 fold greater affinity than they bind to closely related molecules.
  • the antigen-binding proteins or antigen-binding fragments thereof of the invention are characterized by particular functional features or properties.
  • the antibodies bind specifically to clathrin (e.g., bind to human clathrin and may cross-react with clathrin from other species, such as cynomolgus monkey).
  • an antibody of the invention binds to clathrin with high affinity, for example with a K D of 1x10 " M or less.
  • Standard assays to evaluate the binding ability of the antigen-binding proteins or antigen -binding fragments toward the antigen of interest e.g.
  • CHC1 are known in the art, including for example, ELISAs, Western blots and RIAs.
  • the binding kinetics (e.g., binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by Biacore analysis. Suitable assays for evaluating any of the above-described characteristics are described in detail in the Examples.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: l.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2
  • amino acid sequences disclosed in the present invention are encoded by nucleic acid sequences. It will also be understood to one of skill in the art that nucleic acid codons encoding each amino acid are degenerate. Accordingly, there may be more than one nucleic acid sequence for a given amino acid sequence and the alternative sequences may be determined by one of skill in the art.
  • SEQ ID NO: 1 and SEQ ID NO: 2 are provided in Table 1.
  • SEQ ID NO: 3 is one possible nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1.
  • SEQ ID NO: 4 is one possible nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2.
  • Table 1 Amino acid and nucleic acid sequences of the antigen-binding proteins of the invention.
  • an antigen-binding protein, or antigen-binding fragment thereof of the present invention may comprise heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the antigen-binding protein, or antigen-binding fragment of the invention.
  • the antigen-binding protein comprises heavy and light chain CDR regions that are about 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65% and 60% identical to the heavy and light chain CDR regions of (i) and (ii).
  • the antigen-binding protein, or an antigen-binding fragment thereof comprises heavy and light chain CDR regions that are about 80% identical to the heavy and light chain CDR regions of (i) and (ii).
  • the antigen-binding protein, or an antigen-binding fragment thereof comprises a heavy chain variable region which comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: l.
  • the antigen-binding protein, or an antigen-binding fragment thereof comprises a light chain variable region which comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the antigen-binding protein, or an antigen-binding fragment thereof is a monoclonal, recombinant, polyclonal, chimeric, humanised, bispecific and heteroconjugate antibodies; a single variable domain, a domain antibody, antigen binding fragments, immunologically effective fragments, single chain Fv, a single chain antibody, a univalent antibody lacking a hinge region, a minibody, diabodies, and TandabsTM.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • polyclonal antibody or “polyclonal antibody composition” as used herein refer to a preparation of a collection of antibody molecules, each identifying a different epitope of a specific antigen.
  • a polyclonal antibody composition displays a single binding specificity and affinity for different epitopes.
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • recombinant antibody refers to antibodies or fragments which have been produced using recombinant DNA techniques.
  • antibodies having an antigen binding site at least part of which is derived from a different antibody such as those in which hypervariable or complementarity determining regions of one antibody have been grafted into variable framework regions of a second, different, and preferably human, antibody; recombinant antibodies or fragments wherein non-Fv sequences have been substituted by non-Fv sequences from other, different antibodies; or recombinant antibodies or fragments possessing substantially the structure of a natural immunoglobulin but wherein the hinge region has a different number of cysteine residues from that found in the natural immunoglobulin, or wherein one or more cysteine residues in a surface pocket of the recombinant antibody of fragment is in the place of another amino acid residue present in the natural immunoglobulin.
  • bispecific antibody refers to antibodies or fragments thereof that can bind with at least two different antigens.
  • Bispecific antibodies may be mono-, tetra- or multivalent. Methods of generating bispecific antibodies are known in the art. For example, chemical reassociation of monovalent L and H fragments, hybrid hybridoma, or engineering knobs-into-holes complementarity into both H-chains. Tetravalent bispecific antibodies can be created by chemical crosslinking of two monoclonal antibodies (Bs(lgG)2). Using F(ab)' fragments as building blocks, multivalent bispecific antibodies can also be created by chemical crosslinking of two or more Fab' molecules (Bs(Fab')2). A genetically controlled heterodimerization of a Bs(Fab')2 molecule was previously described, where the F(ab')- molecules were fused to a fos and a jun heterodimerization domain.
  • heteroconjugate antibody refers to an antibody or fragment thereof comprising two or more covalently linked monoclonal antibodies or fragments thereof with different specificities.
  • single chain antibody or “single chain variable fragment (scFv)” refers to antibodies or fragments thereof wherein the heavy chain variable domain and light chain variable domain of an antigen binding protein or antigen binding fragment thereof are linked to form a single chain. Linkage may be by a linker peptide. Single chain antibodies or single chain Fvs may be linked to form bivalent scFvs or dimerized to form diabodies.
  • diabody refers to bivalent and bispecific antibodies or fragments thereof which comprise a heavy chain variable domain connected to a light chain variable domain by a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain to create two antigen-binding sites.
  • univalent antibody refers to antibodies or fragments thereof that have one antigen-binding site. Accordingly, the term “univalent antibody lacking a hinge region” refers to antibodies or fragments thereof that have one antigen-binding site and that lack the hinge region between the CHI and DH2 domains.
  • minibody refers to antibodies or fragments thereof comprising the variable heavy and variable light chain domains of an antigen binding protein or antigen binding fragment thereof fused to the hinge region and to the CH3 domain of the immunoglobulin molecule.
  • the antigen-binding protein, or an antigen-binding fragment thereof is a monoclonal antibody.
  • the monoclonal antibody is TAG-OCS 1.
  • the monoclonal antibody is humanised.
  • the present invention features an antigen-binding fragment thereof, or antigen-binding protein that binds to clathrin.
  • Clathrin plays a major role in the formation of coated vesicles and is involved in the intracellular trafficking of receptors and endocytosis of a variety of macromolecules.
  • Clathrin protein is composed of three heavy chains and three light chains. There are two forms of clathrin heavy chains, denoted clathrin heavy chain 1 (CHCl) and clathrin heavy chain 2 (CHC2), while there are two forms of clathrin light chains, denoted clathrin light chain A and clathrin light chain B.
  • the antigen-binding fragment thereof, or antigen- binding protein binds to clathrin heavy chain 1 (CHCl).
  • the present invention features an antigen-binding fragment thereof, or antigen-binding protein that competes with the antigen binding protein or fragment thereof as disclosed herein for binding to CHCl.
  • the term "competes with” in reference to binding of an antigen- binding fragment thereof, or antigen-binding protein refers to an antigen-binding fragment thereof, or antigen-binding protein that has a similar or higher binding affinity to an antigen than the antigen-binding fragments thereof, or antigen-binding proteins of the present invention. Binding of the antigen-binding fragment thereof, or antigen-binding protein to the target antigen inhibits binding of the antigen by the antigen-binding protein or fragments thereof of the present invention.
  • the present invention features antigen-binding protein or antigen- binding fragment thereof, for example an antigen-binding protein or fragment thereof that binds to clathrin, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells.
  • Examples include saporin, mertansine (DM1), monomethyl auristatin E (MMAE), taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • DM1 mertansine
  • MMAE monomethyl auristatin E
  • taxol examples include saporin, mertansine (DM1), monomethyl auristatin E (MMAE), taxol, cytochalasin B, gramicidin D,
  • Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vin
  • cytotoxins examples include saporin, mertansine (DM1) and monomethyl auristatin E (MMAE).
  • Cytoxins can be conjugated to antibodies of the invention using linker technology available in the art.
  • linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
  • Antibodies of the present invention also can be conjugated to a radioactive isotope, or radioisotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates.
  • radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine 131 , indium 111 , yttrium 90 and lutetium 177 .
  • Methods for preparing radioimmunconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including ZevalinTM (IDEC Pharmaceuticals) and BexxarTM (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention.
  • the radioisotope is yttrium 90 .
  • the antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-.
  • gamma gamma.
  • biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1 "), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • the present invention provides a composition, e.g., a pharmaceutical composition, comprising a physiologically acceptable carrier and a therapeutically effect amount of the antigen-binding protein, or antigen-binding fragment thereof of the present invention.
  • a composition may include one or a combination of (e.g., two or more different) antigen-binding proteins or antigen-binding fragments thereof of the invention.
  • a pharmaceutical composition of the invention can comprise a combination of antibodies that bind to different epitopes on the target antigen or that have complementary activities.
  • compositions of the invention also can be administered in combination therapy, i.e., combined with other agents or pharmaceutical ingredients.
  • the combination therapy can include an antigen-binding protein or antigen-binding fragment of the present invention (e.g., TAG-OCS 1) combined with at least one other chemo therapeutic, antiinflammatory or immunosuppressant agent.
  • therapeutic agents that can be used in combination therapy include but are not limited to infliximab, bevacizumab, trastuzumab, carboplatin, cisplatin, oxaliplatin and paclitaxel.
  • compositions of the invention are administered in combination with one or more of bevacizumab, carboplatin or paclitaxel.
  • compositions of the invention are administered together with another pharmaceutical agent
  • the other pharmaceutical agent may be administered separately, simultaneously or sequentially.
  • a “therapeutically effective amount” or “therapeutically effective dosage” include within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect, such as, a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • a “therapeutically effective dosage” preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
  • the ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors.
  • composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. Accordingly, the exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount”.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., antigen-binding fragment, antigen-binding protein or antibody may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • the pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts.
  • a "pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as ⁇ , ⁇ '-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • a pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil- soluble antioxidants such as ascorbyl palmitate, butyl
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response).
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.
  • Preferred dosage regimens for an antibody of the invention e.g.
  • TAG-OCS 1 include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.
  • the antigen-binding protein, fragment thereof or antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half -life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the antigen-binding proteins, fragments thereof, antibodies, antibody compositions and methods of the present invention have numerous in vitro and in vivo utilities involving, for example, binding to CHClor detecting CHC1 on the surface of CSCs, such as ovarian CSCs.
  • these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of situations.
  • the invention provides a use of an antigen-binding protein, or fragment thereof of the present invention in the manufacture of a medicament for treating cancer.
  • Preferred cancers which may be treated using the antibodies of the invention include cancers typically responsive to immunotherapy.
  • preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer, liver cancer (e.g. hepatocellular carcinoma, cholangiocarcinoma, fibrolamellar carcinoma, angiosarcoma and hepatoblastoma) and lung cancer (e.g. non-small cell lung cancer).
  • the invention includes refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the invention.
  • Examples of other cancers that may be treated using the methods of the invention include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic le
  • compositions or medicaments of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art.
  • routes and/or mode of administration will vary depending upon the desired results.
  • Preferred routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • an antigen-binding protein, fragment thereof or antibody of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • a non-parenteral route such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
  • compositions can be administered with medical devices known in the art.
  • the present invention also provides a method for detecting cancer in a subject, the method comprising: contacting a sample obtained from the subject with an antigen-binding protein, or an antigen-binding fragment thereof of the present invention in vitro; detecting the binding of the antigen-binding protein, or an antigen-binding fragment thereof in the sample; correlating the binding with a level of binding in a control sample to determine the level of binding in the sample, wherein an increase in the level of binding in the sample relative to the control sample is indicative of cancer.
  • the terms “detecting” or “detection” refer to the determination of the presence or absence of binding of the antigen-binding protein or antigen-binding fragments thereof of the present invention to the target antigen in a sample.
  • the terms “detecting” or “detection” may also refer to the measurement of relative levels of binding of the antigen-binding protein or antigen-binding fragments thereof of the present invention to a target antigen in a sample relative to another sample or control sample.
  • control sample is from the same subject. In another embodiment, the control sample is from a different subject.
  • the control sample may from the same type of sample, or from a different type of sample.
  • sample refers to a biological sample obtained from a subject and includes but is not limited to blood, blood plasma, serum, buccal smear, amniotic fluid, prenatal tissue, sweat, nasal swab or urine, organs, tissues, fractions, and cells isolated from mammals including humans.
  • a sample may also include sections of the biological sample including tissues (for example, sectional portions of an organ or tissue), extracts from a biological sample, for example, an antigen from a biological fluid (for example, blood or urine).
  • the term "subject” is intended to include human and non-human animals.
  • Non-human animals includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows and horses.
  • the terms "patient” or “subject” are used interchangeably.
  • Preferred subjects include human patients in need of cancer treatment.
  • the antigen -binding protein, or antigen -binding fragment thereof comprises a detectable label.
  • Detectable labels include fluorescent, chemiluminescent, bioluminescent, radioisotopes and enzyme labels. Non-limiting examples include FITC, TRITC, GFP, phycobilliproteins, luciferase ds Red, alkaline phosphatase, horseradish peroxidase, glucose oxidase, PE, Cy Dyes, biotin-strepavidin and Qdots®.
  • the detectable label is biotin, alkaline phosphatase, horseradish peroxidase, FITC, PE or Cy Dyes.
  • Detection of the detectable label may be achieved by conventional means in the art.
  • the detectable label may be detected using flow cytometry, immunohistochemistry and in tissue sections.
  • the instant disclosure provides a kit of parts comprising an antigen-binding protein or antigen-binding fragment thereof as described herein.
  • the kit may also further comprise instructions for use.
  • the antigen-binding protein or antigen-binding fragment thereof is packaged in unit dosage form.
  • the side population was defined as described in Goodell et al. (15). After which, cells were resuspended in phosphate -buffered saline (PBS) and maintained at 4°C until analysis/sorting.
  • PBS phosphate -buffered saline
  • the Aldefluor kit (STEMCELL Technologies Inc., #01700) was used to isolate CSC populations with a high aldehyde enzymatic activity according to the manufacturer's instructions.
  • IGROV1 cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature and thereafter incubated with TAG-OCS 1 culture supernatant or commercial CHC1 antibody (1: 1000 dilution; Lifespan Biosciences, #LS-C22246-100) overnight at 4°C. Binding of the antibody to the cell surface was then visualized using goat a-mouse antibody conjugated with phycoerythrin (PE; 1:500 dilution; DAKO, #R0480). Finally, mounting media (Vector Laboratories, #H1200) containing DAPI was used to counterstain cell nuclei. The same procedure was performed on hESCs with an additional permeabilisation step using Triton X- 100 for the detection of intracellular antigens.
  • Detected subpopulations were separated by sorting as described. Each subpopulation was then subjected to an invasion assay (Chemicon, #ECM554) according to the manufacturer's instructions.
  • IGROV1, HEYA8, PEA1 The ovarian cancer cell lines, IGROV1, HEYA8, PEA1, and the normal immortalized ovarian cell lines, OSEC2 and IOSE21, were all maintained in RPMI 1640 medium (Invitrogen, #11875-093) containing 10% fetal bovine serum (FBS).
  • IGROV1, PEA1 and OSEC2 were obtained from Ovarian Cancer Action, Imperial College, London, UK, while IOSE21 was obtained from Professor Frances Balkwill, Institute of Cancer, Centre for Cancer and Inflammation, Barts and The London School of Medicine and Dentistry, London.
  • HEYA8 was obtained from Professor Jean Paul Thiery, Institute of Molecular and Cell Biology, A*STAR, Singapore.
  • HES-3 human embryonic stem cell line
  • ES Cell International ESI, Singapore
  • matrigel coated organ culture dishes supplemented with conditioned medium from mouse feeders. Differentiation of hESC in vitro was induced as previously described.
  • the human embryonic kidney cell line (HEK293) and lung fibroblast cell line (IMR90) were obtained from the American Type Culture Collection (ATCC; Rockville, USA) and maintained in DMEM containing 10% FBS, 1% penicillin/streptomycin and 2 mmol/L L-glutamine.
  • mAb clones For characterization of mAb clones, cells were incubated for 30 minutes with each mAb clone (150 ⁇ ⁇ culture supernatant or 5 ⁇ g purified mAb in 200 1% BS A/PBS) or with mAb against human clathrin (CHCl; BD Transduction Laboratories, #610499). Cells were then further incubated for 15 minutes with a 1:500 dilution of goat a-mouse antibody fluorescein isothiocyanate (FITC)-conjugated (DAKO, #F0479). 1.25 mg/mL propidium iodide (PI) was added 5 minutes before analysis/sorting.
  • FITC goat a-mouse antibody fluorescein isothiocyanate
  • Table 3 Gene expression assays for the TaqMan system. The gene expression assays were selected from the Applied Biosystems website (http ://www . appliedbiosy stems .com. sg/) .
  • IGROV1 membrane protein extracts were obtained using the ProteoJET Membrane Protein Extraction Kit (Fermentas Inc., #K0321) according to the manufacturer's instructions and thereafter used for immunoprecipitation (IP). Briefly, rabbit antibody conjugated with biotin was captured onto a Streptavidin Phytip column (5 ⁇ . of resin beads). The column was functionalized with either TAG-OCS 1 or the commercially-available CHC1 antibody (BD Transduction Laboratories, #610499). Clarified membrane protein extracts were then passed through the column.
  • SDS-PAGE and Western blotting were performed by the methods of Laemmli and Towbin respectively.
  • the membrane was immunoblotted overnight at 4°C with TAG-OCS 1 culture supernatant or mouse mAb to human CHC1 followed by horseradish peroxidase- conjugated goat anti-mouse antibodies (1:10000 dilution, DAKO).
  • Binding of HRP- conjugated secondary antibodies was visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500).
  • Silver staining was performed using SilverQuest silver staining kit (Life technologies, #LC6070) according to the manufacturer's instructions and the protein band corresponding to the band on the Western Blot was manually excised for mass spectrometry (MS) analysis.
  • MS mass spectrometry
  • AlamarBlue® dye (Invitrogen, #DAL1025) was purchased commercially and the assay was performed according to the manufacturer's instructions (9,10). Briefly, 5000 cells were seeded in each well of a 96-well plate in their appropriate growth culture medium as described earlier and allowed to attach overnight. 30 of purified mAb at a final concentration of 200 ⁇ g was then added to each well. At the end of 72 hours, 10 ⁇ ⁇ of alamarBlue® was added to each well. The plate was further incubated at 37°C for 30 minutes and subsequently analyzed using a fluorescence plate reader (TECAN Infinite M200; Tecan Group Ltd.) at an excitation wavelength of 545nm and emission wavelength of 590nm.
  • TECAN Infinite M200 Tecan Group Ltd.
  • CDC experiments [00150] 5000 cells in 90 ⁇ ⁇ of non -phenol red culture media per well were plated onto a 96-well plate. Thereafter, increasing concentrations of TAG-OCS 1 were added to wells which contained either active or heat-inactivated human serum (Quidel, #A100). 100 ⁇ ⁇ of culture media served as background control for this experiment. Thereafter, lactate dehydrogenase (LDH) activity in all the wells was measured to determine cell viability using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, #G1780), according to the manufacturer's instructions. All conditions were performed in triplicates.
  • 5000 cells in 90 of culture media per well were plated onto opaque-walled 96- well plates and allowed to attach to the wells overnight. Thereafter, 0.1 of TAG-OCS 1 (1 mg/mL) and anti-M-ZAP (0.27 mg/mL) (Cat No. IT-30 Advanced Targeting Systems, San Diego, USA) were added to the test wells. The plate was then incubated for 72 hours before cytotoxicity assays were performed using the Cell Titer-Glo Luminescent Cell Viability Assay (Promega, #G757) according to the manufacturer's instructions. Controls for this experiment were buffer only, anti-M-ZAP only, and TAG-OCS 1 only. All conditions were performed in triplicates.
  • the Aldefluor activity assay yielded Aldefluor-positive populations which showed up distinctively on flow cytometry plots.
  • Incubation of cells with BAAA in the absence of the enzyme inhibitor, DEAB induced a shift in BAAA fluorescence, defining the Aldefluor-positive population (Fig. IB, right), while addition of DEAB abolished this effect (Fig. IB, left).
  • mRNA expression of stem cell markers were upregulated in Aldefluor-positive populations, with CD133 showing the highest extent of upregulation (5-fold increase) in its expression level (Fig. 2B).
  • Anchorage-independent colony forming spheroid assays showed increased numbers and sizes of spheroid in the SP (Fig. 2C) and Aldefluor-positive populations (Fig. 2D) when compared to their respective counterparts. It was further observed that the largest colonies (>64 cells) were more abundantly formed in the SP and Aldefluor- positive populations.
  • SP cells demonstrated a 7-fold increase in invasiveness when compared to NSP (Fig.
  • IGROV1 ovarian CSCs were chosen for isolation using the Aldefluor activity assay for the generation of mAbs. Isolated populations of Aldefluor-positive ovarian tumor IGROV1 cells were first injected into female BALB/c mice. Subsequently, a two-step screening process was carried out to select for mAbs which showed reactivity towards subpopulations of cells in the ovarian cancer and hESC cell lines but not towards the differentiated IMR90 and HEK293 cell lines.
  • TAG-OCS l This strong reactivity of TAG-OCS l towards hESCs was further confirmed by immunocytochemistry experiments (Fig. 4C), which suggests that the antibody may be targeting pluripotent cell types. Furthermore, TAG-OCS l did not react with cells from the normal ovarian cell lines, OSEC2 and IOSE21 (Fig. 4D). Together, the data showed that TAG-OCS l recognizes a CSC subpopulation and we sought to further characterize TAG-OCS l and the subpopulation of cells detected by TAG-OCS l in ovarian tumor cells. [00166] EXAMPLE 3
  • TAG-OCSl detects subpopulations which exhibit tumor stem cell-like characteristics
  • TAG-OCS l -positive cells exert a drug- resistance mechanism which may in part be mediated by expression of various ATP-binding cassette (ABC) transporters.
  • the CD133 marker had previously been found to be present on cells which possess cancer-initiating cell characteristics in ovarian tissues. CD133 expression in ovarian cancer has also been found to be directly regulated by epigenetic modifications. Furthermore, methylation of the CD 133 promoter may be representative of epigenetic repression of other pluripotency-associated genes.
  • TAG-OCS l -positive cells demonstrated a 3.9-fold increase in invasiveness compared to the TAG-OCS l -negative cells from the IGROVl ovarian cancer cell line (Fig. 5D).
  • This increased invasiveness of CSCs possibly relates to aggressiveness in tumors, as suggested by a previous study where putative ovarian CSCs were found to form more aggressive tumor xenografts at a lower inoculum compared to their non CSC-progeny.
  • Target antigen of TAG-OCSl is determined as clathrin heavy chain
  • IP Immunoprecipitation
  • IP eluates of the commercial CHC1 antibody were resolved by SDS-PAGE and then probed with TAG-OCS l.
  • target antigen bands of identical sizes ⁇ 180kDa
  • TAG-OCS l target antigen bands of identical sizes
  • mAb-CHCl commercially-available mAb-CHCl
  • CHC1 was upregulated by 6.9-fold and 2.9-fold in the IGROVl and HEYA8 TAG-OCS l -positive subpopulations respectively, compared to their TAG-OCS 1- negative counterparts.
  • TAG-OCS l detects a novel epitope of CHC1 which is expressed on the cell surface of ovarian CSCs.
  • CHC1 is an essential component of the clathrin molecule which mediates a major endocytosis pathway.
  • endocytic mechanisms are exhibited by both tumor- initiating and metastasizing cells where vesicular trafficking and subsequent degradation or re-assembly takes place after the disassembly of signaling and adhesion complexes.
  • factors involved in endocytosis possibly represent crucial targets of the processes driving tumor initiation and progression.
  • TAG-OCS l could potentially be useful in distinguishing early ovarian cancer from benign tumors as well as enable the early diagnosis of ovarian cancer for improved prognosis. This was confirmed by staining of ovarian clinical samples by TAG-OCS l ( Figure 6D to 6F). EpCAM-positive patient ascites and patient ovarian tumour sections showed positive staining by TAG-OCS l ( Figure 6D and 6E).
  • TMA patient tumour microarray
  • the IMR90 lung fibroblast cell line which TAG-OCS l did not bind to in FACS experiments was used as a negative control.
  • viability of IMR90 cells was not affected by the addition of TAG-OCS l.
  • Data obtained showed that incubation of cells with TAG-OCS l did not result in a decrease in cell viability of CHI ovarian cancer cells in both the Alamar Blue and xCELLigence experiments (Fig. 7C).
  • Data obtained from flow cytometry experiments revealed that TAG-OCS l binds and kills MCF7 breast cancer cells (Fig.
  • TAG-OCS l binds to 15% of the IGROVl ovarian cancer cell line, 87.5% of 786-0 cells (kidney cancer cell line) and 92.1% of MCF7 cells (breast cancer cell line). Furthermore, TAG-OCS l confers cytotoxicity towards MCF7 cells (Fig. 7E).
  • TAG-OCSl mediates CDC and functions as an ADC
  • TAG-OCS l when used as a vehicle for the specific delivery of toxins, such as the ribosome-inactivating protein, saporin, to CHC1 -expressing ovarian cancer cells was determined.
  • the mAb-ZAP complex exhibited specific cytotoxicity towards IGROV1 and PEA1 ovarian tumor cells (Fig. 7F, 71, Fig. 10), as well as towards 786-0 kidney cancer cells (Fig. 7G) and MCF7 breast cancer cells (Fig. 7H, 7J).
  • TAG-OCS 1 possesses independent cytotoxic effects in vitro, but only at concentrations higher than those used in this assay.
  • developing TAG-OCS 1 into a cytotoxic antibody-drug conjugate (ADC) in future may result in the elimination of cells which express CHC1 on the surface while sparing normal cells which express intracellular CHC1.
  • ADC cytotoxic antibody-drug conjugate
  • a baseline level of target antigen expression in normal tissues may be tolerable and resultant potential toxicities may depend on the relative ratio between tumor and normal expression levels.
  • the monoclonal antibody TAG-OCS 1 described thus far is an IgM antibody.
  • a chimeric TAG-OCS 1 (IgG) antibody was also generated using general methods known in the art and analysed. Briefly, the variable region of the antibody was cloned from the mouse hybridoma, then cloned into a construct (protein expression vector) with the constant region of a human IgGl. The chimeric TAG-OCS 1 was then expressed in a CHO cell line (in house) and purified.
  • Flow cytometry experiments show that chimeric TAG-OCS 1 successfully binds to the 786-0 kidney cancer cell line and MCF7 breast cancer cell line at a level which is comparable to the original IgM molecule (Fig. 12A and 12B). Chimeric TAG-OCS 1 also confers cytotoxicity towards MCF7 cells. [00189] Through the antibody-drug conjugate (ADC) assay, chimeric TAG-OCS 1 (at levels of lOOng/well and 200ng/well) is shown to exhibit ADC cytotoxicity towards IGROV1 (Fig. 12C) and PEA1 (Fig. 12D) ovarian cancer cells, after an incubation time of 72 hrs.
  • ADC antibody-drug conjugate
  • TAG-OCS 1 may be used to directly detect CHC1, either alone or in combination with other cancer markers, thereby allowing for the screening of early stage ovarian cancer at mass level.
  • MAb also mediates CDC in vitro and further development of TAG-OCS 1 as an ADC could prove to be highly valuable for immunotherapy of recurrent, chemoresistant disease.
  • TAG-OCS 1 represents a novel diagnostic and possible therapeutic agent with enhanced specificity against CSCs in ovarian cancer.

Abstract

La présente invention concerne des protéines de liaison à l'antigène et des fragments de liaison à l'antigène de ces dernières qui se lient à la chaîne lourde de la clathrine (CHC1) à la surface des cellules souches cancéreuses. La présente invention concerne également des compositions comprenant la protéine de liaison à l'antigène et les fragments de liaison à l'antigène de cette dernière, et l'utilisation de ces protéines de liaison à l'antigène et des fragments de liaison à l'antigène de cette dernière, ainsi que des méthodes pour détecter un cancer chez le patient comprenant l'étape consistant à mettre en contact un échantillon avec la protéine de liaison à l'antigène, et les fragments de liaison à l'antigène, de l'invention. L'invention concerne également des méthodes de traitement du cancer et des kits destinés à être utilisés dans les méthodes de l'invention.
PCT/SG2016/050032 2015-01-23 2016-01-25 Protéines de liaison à l'antigène spécifiques du cancer WO2016118090A1 (fr)

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