WO2016112423A1 - Glypican epitopes and uses thereof - Google Patents

Glypican epitopes and uses thereof Download PDF

Info

Publication number
WO2016112423A1
WO2016112423A1 PCT/AU2015/000019 AU2015000019W WO2016112423A1 WO 2016112423 A1 WO2016112423 A1 WO 2016112423A1 AU 2015000019 W AU2015000019 W AU 2015000019W WO 2016112423 A1 WO2016112423 A1 WO 2016112423A1
Authority
WO
WIPO (PCT)
Prior art keywords
epitope
seq
variant
amino acid
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2015/000019
Other languages
English (en)
French (fr)
Inventor
Douglas Campbell
Irene JUSTINIANO FUENMAYOR
Aline NOCON
Julie SOON
Quach TRUONG
Bradley Walsh
Sandra Wissmueller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Minomic International Ltd
Original Assignee
Minomic International Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CN201580076839.6A priority Critical patent/CN107531755B/zh
Priority to CA2973771A priority patent/CA2973771A1/en
Priority to DK15877381.2T priority patent/DK3245219T3/da
Priority to AU2015376851A priority patent/AU2015376851B2/en
Priority to EP15877381.2A priority patent/EP3245219B1/en
Priority to US15/543,877 priority patent/US20180002391A1/en
Priority to PCT/AU2015/000019 priority patent/WO2016112423A1/en
Priority to ES15877381T priority patent/ES2805085T3/es
Priority to JP2017537906A priority patent/JP6735005B2/ja
Priority to SG11201705808XA priority patent/SG11201705808XA/en
Application filed by Minomic International Ltd filed Critical Minomic International Ltd
Priority to KR1020177022547A priority patent/KR102435383B1/ko
Publication of WO2016112423A1 publication Critical patent/WO2016112423A1/en
Anticipated expiration legal-status Critical
Priority to US16/939,729 priority patent/US20210107958A1/en
Priority to US18/361,407 priority patent/US20240247037A1/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • 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
    • C07K16/303—Liver or Pancreas
    • 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
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4722—Proteoglycans, e.g. aggreccan

Definitions

  • the present invention relates generally to the fields of imr mology and medicine. More specifically, the present invention relates to epitopes of glvpican-l (GPC-1) and uses thereof.
  • GPC-1 glvpican-l
  • Prostate cancer is the most commonly occurring cancer in men of all races, and is second only to lung cancer in mortality among among white, black, American Indian/Alaska Native, and Hispanic men. In 20.1 1, 209,292 men in. the United States of America were diagnosed with prostate cancer and 27,970 of these died from the disease (U.S. Cancer Statistics Working Group, "United Stales Cancer Statistics: 1999-201 ⁇ incidence and Mortality Web-hased Report ", Atlanta (GA): Department of Health and Human Services, Centers for Disease Control and Prevention, and National Cancer Institute; 2014).
  • Treatment with surgery and/or radiotherapy is successful in many patients if prostate cancer is diagnosed early. However, many patients with advanced disease and a sizeable proportion of all prostate cancer patients eventually develop metastatic disease following localised therapy.
  • Glypican-i is a cell surface heparan sulfate proteoglycan with a core protein that is anchored to the cytoplasmic membrane via a glycosyl phosphatidySinositoL it is a member of a larger family of giypieans.
  • GPC-1 has been reported to be overexpressed in some forms of cancer (e.g. pancreatic cancer, breast cancer), but expression does not significantly differ in others.
  • the present inventors have recently determined that GPC- 1. is overexpressed by prostate cancer cells, and ca be used as a means of diagnosing the disease (US provisional patent application no.
  • the present inventors have determined that a series of epitopes within the GPC-i protein are preferably targeted by binding entities including, but not limited to. antibodies.
  • the present invention relates to at least the following embodiments:
  • Embodiment 1 An epitope for an anti-glypican 1 (GPC-1 ) antibody located within a portion of the GPC-1 flexible loop defined by an amino acid sequence KVNPQGPGPEEK. (SEQ ID NO: 1).
  • Embodiment s The epitope according to embodiment 1 , wherein the epitope comprises a -first segment comprising an amino acid sequence selected from:
  • VNPQGPGPEEK SEQ ID NO: 2
  • VNPQGPGPEEK (SEQ ID NO: 2), wherein the variant comprises a substituted amino acid residue only at:
  • Embodiment 3 The epitope according to embodiment 1 wherein the epitope comprises a first segment comprising an amino acid sequence selected from:
  • Embodiment 4 The variant according to embodiment 3, comprising a substituted amino acid residue only at any one or more of positions 8 and 9 of SEQ ID NO: 6.
  • Embodiment s The epitope according to embodiment 1, wherei the epitope comprises a first segment comprising an amino acid sequence selected from:
  • KVNPQGPGPE SEQ ID NO: 5
  • Embodiment 6 The variant according to embodiment 5, comprising a substituted amino acid residue only at any one or more of positions 8, 9 and 10 of SEQ ID NO: 6.
  • Embodiment 7 The variant according to embodiment 5 or embodiment 6, wherein E (giu) at position 10 is substituted with, any other amino acid.
  • Embodiment 8 The variant according to any one of embodiments 3 to 7, wherein the variant comprises a substitution only at any one or more of:
  • N (asn) is substituted with any one of H (his), P (pro) or D (asp); position 4, wherein P (pro) is substituted with any one of R (arg), (lys), W (trp), S (ser), H (his) or N (asn);
  • G (gly) is substituted with an one of D (asp), E (glu), N (asn), Q (gin), K (lys), R (arg) or A (ala);
  • P (pro) is substituted with any one of M (met), A (ala), 1 (ile), JC (lys), R (arg), Q (gin), S (ser), T (thr), or Y (tyr).
  • Embodiment 9 The epitope according to any one of embodiments 1 to 8, comprising a second segment comprising a amino acid sequence TQNARA (SEQ ID NO: 8),
  • Embodiment 10 The epitope according to any one of embodiments 1 to 8, comprising a second segment comprising an amino acid sequence TQ ARAFRD (SEQ ID NO: 7).
  • Embodiment 1 1 The epitope according to embodiment I or embodiment 2, wherein the epitope comprises a first segment comprising an amino acid sequence selected from
  • NPQGPGPEE (SEQ ID NO: 4), wherei the variant comprises a substituted amino acid residue only at any one or more of positions 1, 2, 3, 5, 7 and 9 of SEQ ID NO: 4
  • Embodiment 12 The variant according to embodiment 1 1 , comprising a substituted amino acid residue only at any one or more of positions 2, 7, and 9 of SEQ ID NO: 4.
  • Embodiment 13 The variant according to embodiment 1 1 or embodiment 12, wherein the variant comprises a substitution at any one or more of:
  • P (pro) is substituted with any one of A (ala), D (asp), C (cys), E (glu), Z (glx), G (gly), H (his), K (lys), M (met), F (phe), P (pro), S (ser), T (thr), W (trp), or Y (tyr):
  • Embodiment 14 The epitope according to any one of embodiments 1 1 to 13, wherein the epitope comprises a second segment comprising an. amino acid sequence ALSTASDDR (SEQ ID NO: 9),
  • Embodiment 15 The epitope according to embodiment 1 or embodiment 2, wherein the epitope comprises a first segment comprising an amino acid sequence selected from:
  • VNPQGPGPEE SEQ ID NO: 3
  • VNPQGPGPEE (SEQ ID NO; 3), wherein the variant comprises a substituted amino acid residue only at any one or more of positions 1 , 2, 3, 4, 5, 8 and 10 of SEQ ID NO: 3,
  • Embodiment 16 The variant according to embodiment 15, comprising a substituted amino acid residue only at any one or more of positions 1 , 2, 3, and 8 of SEQ I NO: 3.
  • Embodiment 17 The variant according to embodiment 15 or embodiment 16, wherein the variant comprises a substitution at any one or more of
  • Q (gin) is substituted with any one of Y (tyr), A (ala), E (glu), V (val), M (met), F (phe), L (leu), I (ile), T (thr), or R (arg);
  • G gly
  • A ala
  • S serotonin
  • T thr
  • H his
  • W trp
  • Y tyr
  • F phe
  • M metal
  • Embodiment 18 The epitope according to any one of embodiments 15 to .17, wherein the epitope comprises a second segment comprising:
  • Embodiment 1.9 The epitope according to any one of embodiments 15 to 1.7, wherem the epitope composes a second segment comprising an amino acid sequence PRERPP (SEQ ID NO: 10) and a third segment comprising an amino acid sequence QDASDDGSGS (SEQ ID NO: 11).
  • Embodiment 20 The epitope according to any one of embodiments 1 to 10, comprising the amino add sequence CGELYTQNARAFRDLCGNPKVNPQGPGPEEKRRRGC (SEQ ID NO: 12).
  • Embodiment 21 The epitope according to any one of embodiments 1. to 20, wherein the epitope is a linear epitope.
  • Embodiment 22 The epitope according to embodiment 19, wherein the second segment and the third segment of the epitope are discontinuous.
  • Embodiment 23 The epitope according to any one of embodiments 9, 10, 14, 18 or 22 wherein the first segment and the second segment of the epi tope are discontinuous.
  • Embodiment 24 An epitope for an anii-glypican 1 (GPC-1) antibody comprising an amino acid sequence selected from any one or a plurality of: TQNARA (SEQ ID NO: 8), ALSTASDD (SEQ ID NO: 9), PRERPP (SEQ ID NO: 10), QDASDDGSGS (SEQ ID NO: 11), LGPEC SR A VMK (SEQ. ID NO: 1.3), and TQNARAFRD (SEQ ID NO: 7).
  • GPC-1 a plurality of: TQNARA
  • ALSTASDD SEQ ID NO: 9
  • PRERPP SEQ ID NO: 10
  • QDASDDGSGS SEQ ID NO: 11
  • LGPEC SR A VMK SEQ. ID NO: 1.3
  • TQNARAFRD SEQ ID NO: 7
  • Embodiment 25 The epitope accordmg to any one of embodiments 1 to 24, wherein the epitope is an isolated polypeptide or a synthetic polypeptide.
  • Embodiment 26 An arrangement of epitopes comprising a combination of two or more distinct epitopes, wherein
  • each said distinct epitope is an epitope according to any one of embodiments 1 to 25; each said epitope is an epitope according to Table 1; or
  • Embodiment 27 An arrangement of epitopes comprising a combination of two or more distinct epitopes, wherein the arrangement comprises
  • Embodiment 28 An arrangement of epitopes comprising a combination of two or more distinct epitopes, wherein the arrangement comprises
  • Embodiment 29 A composition comprising an epitope according to any one of embodiments 1 to 25, or an arrangement of epitopes according to any one of embodiments 26 to 28, and a pharmaceutically acceptable carrier or excipient.
  • Embodiment 30 An assembly comprising an epitope according to any one of embodiments 1 to 25, or an arrangement of epitopes according to any one of embodiments 26 to 28, bound to one or more soluble or insoluble supports.
  • Embodiment 31 The assembly of embodiment 30, wherein the assembly is a component of an enzyme-linked immunosorbent, assay (ELISA).
  • Embodiment 32 A nucleic acid encoding the epitope according to any one of embodiments 1 to 25.
  • Embodiment 33 A vector comprising the nucleic acid according to embodiment 32,
  • Embodiment 34 A host cell comprising the vector according to embodiment 33.
  • Embodiment 35 An isolated binding entit capable of specifically binding to an epitope according to any one of embodiments i to 25, wherein the binding entity is not an. antibody.
  • Embodiment 36 An isolated binding entity capable of specifically binding to an epitope according to any one of embodiments 1 to 25, wherein the binding entity is art.
  • antibody and with the proviso that the antibody is not a: MIL38 antibody (CBA20140026).
  • rabbit anti-GPC-I polyclonal antibody (ab 137604, abeam), mouse anti-glypican monoclonal antibody 2600 clone 4D1 (Miilipore), or goat anti-glypican 1 antibody (AA 24-530).
  • Embodiment 7 A method for detecting prostate cancer in a subject, the method comprising obtaining a biological sample from the subject, detecting the presence of an epitope according to any one of embodiments 1 to 25 in the sample, and determining that the subject has prostate cancer or an increased likelihood of developing prostate cancer based on amount of the epitope detected in the sample.
  • the biological sample may be a body fluid sample.
  • the biological sample may be a tissue sample.
  • Embodiment 38 The method according to embodiment 37, wherein detecting the presence of the epitope in the sample comprises contacting the sample with a binding entity capable of specifically binding to an epitope according to any one of embodiments 1 to 25.
  • Embodiment 39 The method according to embodiment 38, wherein the binding entity is a populatio of antibodies.
  • Embodiment 40 The method according to embodiment 39, wherein the population of antibodies comprises any one or more of: M1L38 antibody (CBA20140026), rabbit anti-GPC- 1 polyclonal antibody (abi 37604, abeam), mouse anti-glypican monoclonal antibody 2600 clone 4D.1 (Miilipore), or goat anti-glypican 1 antibody (AA 24-530).
  • M1L38 antibody CBA20140026
  • rabbit anti-GPC- 1 polyclonal antibody abi 37604, abeam
  • mouse anti-glypican monoclonal antibody 2600 clone 4D.1 Miilipore
  • goat anti-glypican 1 antibody AA 24-530.
  • Embodiment 41 The method according to embodiment 39, wherein the populatio of antibodies does not contain any of: MIL38 antibody (CBA20140026), rabbit anti-GPC-1 polyclonal antibody (abl 37604, abeam), mouse anti-glypican monoclonal antibody 2600 clone 4D1 (Millipore), or goat anti-glypican I antibody (AA 24-530).
  • MIL38 antibody CBA20140026
  • rabbit anti-GPC-1 polyclonal antibody abl 37604, abeam
  • mouse anti-glypican monoclonal antibody 2600 clone 4D1 Millipore
  • goat anti-glypican I antibody AA 24-530.
  • Embodiment 42 The method according to any one of embodiments 37 to 41, comprising comparing the amount of epitope present in the biological sample with an amount of epitope present in a control sample, wherein the detection of an increased amount of epitope in the body fluid sample compared to an equivalent measure of the control sample is indicative of prostate cancer in the subject, or an increased likelihood of developing prostate cancer in the subject.
  • Embodiment 43 The method according to embodiment 42, wherein the amount of epitope detected in the sample is increased by more than 50% over the amount of epitope detected in the control sample.
  • Embodiment 44 The method according to any one of embodiments 37 to 43, wherein detecting the presence of the epitope comprises contacting the sample with a population of MIL38 antibodies as deposited at Cellbaiik Australia under accession number CBA20140026.
  • Embodiment 45 The method according to any one of embodiments 37 to 43, wherein detecting the presence of the epitope comprises contacting the sample with a population of antibodies that does not comprise a antibody comprising a fight chain variable region comprising: a complementarity determining region 1. (CDR1) comprising or consisting of an amino acid sequence defined by positions 48-58 of SEQ ID NO: 20; a complementarity determining region 2 (CDR2) comprising or consisting of an amino acid sequence defined by positions 74-80 of SEQ ID NO: 20: and/or a complementarity determining region 3 (CDR3) comprising or consisting of a amino acid sequence defined by positions 1 13-1.21 of SEQ ID NO: 20.
  • CDR1 complementarity determining region 1.
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • Embodiment 46 The method according to any one of embodiments 37 to 45, farther comprising determining the level of prostate-specific antigen (PSA) in the biological sample and comparing the level detected to that of the control sample.
  • PSA prostate-specific antigen
  • Embodiment 47 The method according to any one of embodiments 37 to 46, wherein the biological sample is a body fluid sample.
  • Embodiment 48 The method according to any one of embodiments 37 to 46, wherein the biological sample is a tissue sample.
  • Embodiment 49 A fusion protein comprising the epitope according to any one of embodiments 1 to 25.
  • Embodiment 50 Use of the epitope according to any one of embodiments 1 to 25, the arrangement of epitopes according to any one of embodiments 26 to 28. or the fusion protem according to embodiment 48,. as a positive control, element in a method for detecting GPC-I .
  • Embodiment 51 The use according to embodiment 50, wherein the method is the method for detecting prostate cancer according to any one of embodiments 37 to 48.
  • the positive control element when used in a detection method or detection assay may directly or indirectly provide a positive/affirmative signal, and thereby at least in part or wholly validate that the method or assay is capable of functioning correctly.
  • Figure I shows a rendering of chain B as present in Protein Data Bank identifier (PBD ID) 4AD7 (http://www.ebi.acaik/pdbsimi/4AD7);
  • Figure 2 shows box plot graphs of raw data of IL38-AM4 antibody screening.
  • the bottom and top of the boxes are the 25th and 75th percentile of the data.
  • the band near the middle of the box is the 50th percentile (the median).
  • the whiskers are at 1.5 the inter- quantile range, an indication of statistical outliers within the dataset ( cgil! et al., The American Statistician, 32: 12-16, 1978);
  • Figure 3 shows plots of the peptide number versus the recorded intensity obtained for all five of arrays (sets 1 -5, Example 1), when incubated with l ug/ml (top trace) or 10 ⁇ / ⁇ 1 of MIL38-AM4 (bottom trace);
  • Figure 4 shows rendering of chain B from Protein Data Bank identifier (PBD ID) 4AD7 ⁇ http://ww.ebi.ac.uk/pdbsutn/4AD7) with sequence TQNARAFRDLYS (SEQ ID NO; 21) in dark-coloured spheres, and residues K347, V348, G362, and K363 in light- coloured spheres.
  • the loop connecting V348 to G362 is not resolved from the X- ray diffraction;
  • FIG. 5 shows box plot graphs of raw data of polyclonal and monoclonal anti-GPC-1 and MIL3S-AM3 antibody screening. Tlie bottom and top of the boxes are the 25 th and 75th percentile of the data. The band near the middle of the box is the 50th percentile (the median). The whiskers are at 1.5 the inter-quantile range, an indication of statistical outliers withi the dataset (Mcgilt el al, The American Statistician, 32: 12-16, 1978);
  • Figure 6 shows plots of the peptide number versus the recorded intensity obtained for three commercially available anti-glypican 1 preparations tested on the array used to map MIL38-AM4, which is shown for comparison;
  • Figure 7 shows rendering of chain B from Protein Data Bank identifier (PBD ID) 4AD7 (http://www.ebi.ac.uk pdbsum 4AD7).
  • Figure 7A shows rendering of chain B with sequence PRERPP (SEQ ID NO: 10) in. light-shaded spheres, and residues K347, V348, G362, and 363 in dark-shaded, spheres. The loo connecting V348 to G362 is not resolved from the X-ray diffraction. These epitopes are recognized by rabbit anti-GPC-1. An extra epitope, QDASDDGSGS (SEQ ID NO: 11), is not resolved in the coordinate file.
  • Figure 7B shows rendering of chain B with sequence LGPECSRAVMK (SEQ ID NO: 13) in shaded spheres. This epitope is recognized by mouse anti-GPC-1 ;
  • Figure 8 shows box plot graphs of raw data of antibody screening. The bottom, and top of the boxes are the 25th and 75th percentile of the data. The band near the middle of the box is the 50th percentile (the median). The whiskers are at 1.5 the inter-qnantile range, an indication of statistical outliers within the datase (Mcgiil t i., The American Statistician, 32; 12-16, 1978);
  • Figure 9 shows a letterplot representation of rabbit polyclonal Ab 137604 probed on the substitution analysis of set 3.
  • the central/horizontal bar represents the mean intensity recorded for the base sequence. Individual mutations are drawn in the column corresponding to the position of the mutation, and symbols are plotted at the height corresponding to the recorded intensity;
  • Figure 10 shows a letterplot representation of goat polyclonal anti-GPC-1 probed on the substitution analysis of set 2 (Example 3);
  • Figure 11 shows a letterplot representation of MIL38-AM4 probed on the substitution analysis of set 3 (Example 3 );
  • peptide is residues 344 - 353 of SEQ ID NO: 1; X-2 peptide is residues 348 - 357 of SEQ ID NO: 1; X-3 peptide is residues 352 - 361 of SEQ ID NO: I; X-4 peptide is residues 356 - 365 of SEQ ID NO: 1; X-5 peptide is residues 344 - 354 of SEQ ID NO: 1; X-6 peptide is residues 348 - 358 of SEQ ID NO; 1; X-7 peptide is residues 352 - 362 of SEQ ID NO: 1; X-8 peptide is residues 356 - 366 of SEQ ID NO: 1; X-9 peptide is residues 344 - 355 of SEQ ID NO: 1; X- 10 peptide is residues 348 - 35 of SEQ ID NO: 1 ; X-l 1 peptide is residues 352 - 363 of SEQ ID NO: 1 ;
  • Y- 1 peptide is residuesl35 - 147 of SEQ ID NO: 1; Y- i l peptide is residues 131 - 144 of SEQ ID NO: 1; Y-.12 peptide is residues 135 - 148 of SEQ ID NO: 1 ; Y-13 peptide is residues 131 - 145 of SEQ ID NO: 1; Y-14 peptide is residues 135 - 149 of SEQ ID NO: 1; Y-15 peptide is residues 131 - 146 of SEQ ID NO: 1; Y-16 peptide is residues 131 - 147 of SEQ ID NO: 1 ; Y-17 peptide is residues 131 - 148 of SEQ ID NO; 1 ; Y-18 peptide is residues 131 - 149 of SEQ ID NO: 1; Y- 9 peptide is a scramble/random sequence.
  • Figure 13 is a scatter plot matrix of all results obtained on the peptides of set 1 ;
  • Figure 14 shows results of 2D electrophoresis and western blotting which demonstrate that IL38 and anti-GPC-1. antibodies show overlapping reactivity on 2D gel western blot;
  • Figure 16 shows western blots of immunoprecipitations demonstrating that IL38 antibody can detect glypican-1 in the plasma ( Figure 16 A ) of prostate cancer patients and in extracts of prostate cancers (Figure I6B).
  • Figure 1 A lanes are: 046 IP NT- IP from prostate cancer plasma; 046 IP HepI- IP from prostate cancer plasma treated with heparinase; 042 IP NT- IP from normal control plasma; 042 IP HepI- IP from normal control plasma treated with heparinase; Magic Mark-commercial protein marker as molecular weight standard.
  • an antibody also includes multiple antibodies.
  • a sample “comprising” antibody A may consist exclusively of antibody A or may include one or more additional components (e.g. antibody B).
  • multiple and “plurality” mean more than one. In certain specific aspects or embodiments, multiple or plurality may mean 2, 3, 4. 5, 6, 7, 8, 9, 10, I , 12, 13, 14, 15, 16, 17, 18, .19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or more, and any integer derivable therein, and any range derivable therein.
  • epitope refers to the specific portion(s) of an antigen which interact (e.g. bind) with one or more binding entities such as, for example, a protein, ligand, antibody, antibody fragment, or antibody derivative.
  • antibody and “antibodies” include gG (including IgGl , IgG2, IgG3, and IgG4), IgA (including IgA l and IgA2), IgD, IgE, IgM, and IgY, whole antibodies, including single-chain whole antibodies, and antigen-binding fragments thereof.
  • Antigen-binding antibody fragments include, but are not limited to, Fv, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disul fide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • the antibodies may be from any animal origin or appropriate production host.
  • Antigen-binding antibody fragments may comprise the variable region/s alone or in combination with the entire or partial of the following: hinge region, CHI , CH2, and CH3 domains. Also included are any combinations of variable region/s and hinge region, CH I, CFJ2, and CH3 domains.
  • Antibodies may be monoclonal, polyclonal, chimeric, multispecific, humanised, and human monoclonal and polyclonal antibodies which specifically bind the biological molecule.
  • the antibody may be a bi -specific antibody, avibody, diabody, tribody, tetrabody, nanobody, single domain antibody, VHH domain, human antibody, fully humanized antibody, partially humanized antibody, anticalin, adnectin, or affibody.
  • the term "monoclonal antibody” refers to an antibody that recognises a single antigenic epitope, and that is obtained from a population of substantially homogeneous antibodies which bind specifically to the same antigenic epitope, and are identical with the potent ial exception of naturally occurring mutation/s that may be present in m inor amounts.
  • humanised antibody refers to forms of antibodies that contain sequences from human antibodies as well as non-human antibodies (e.g. murine antibodies).
  • a humanised antibody can comprise substantially all of at least one and typically two variable domains, in which all/substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and ail/substantially all of the FR regions are from the human immunoglobulin sequence.
  • the humanised antibody may optionally aiso comprise at least a portion of an immunoglobulin constant region (Fc) which may typically be that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • chimeric antibody refers to an antibody which exhibits a desired biological activity, and in which a portion of the light chain and/or heavy chain is identical to or homologous with corresponding sequences in antibodies derived from a given specific species, while the remaining chain/s is/are identical to or homologous with corresponding sequences in antibodies derived from another different species.
  • a chimeric antibody may comprise variable regions that are derived from a first species and comprise constant regions that are derived from a second species.
  • Chimeric antibodies can be constructed for example by genetic engineering from immunoglobulin gene segments belonging to different species.
  • hybrida refers to a cell produced by the fusion of an immortal ceil (e.g. a multiple myeloma cell) and an antibody-producing cell (e.g. a B lymphocyte), which is capable of producing monoclonal antibodies of a single binding specificity.
  • an immortal ceil e.g. a multiple myeloma cell
  • an antibody-producing cell e.g. a B lymphocyte
  • binding specifically and “specifically binding” in reference to an antibody, antibody variant, antibody derivative, antigen binding fragment, and the like refers to its capacity to bind to a given target molecule preferentially over other non-target molecules.
  • molecule A the antibody, antibody variant, antibody derivative, or antigen binding fragment
  • molecule ⁇ the antibody, antibody variant, antibody derivative, or antigen binding fragment
  • molecule A has the capacity to discriminate between molecule B and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, molecule A will selectively bind to molecule B and other alternative potential binding partners will remain substantially unbound by molecule A.
  • molecule A will preferentially bind to molecule B at least 10-fold, preferably 50- fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners.
  • Molecule A may be capable of binding to molecules that are not molecule 8 at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from molecule B-specific binding, for example, by use of an appropriate control.
  • a “subject” includes any animal of economic, social or research importance including bovine, equine, ovine, primate, avian and rodent species.
  • a “subject” may be a mammal such as, for example, a human or a non-human mammal.
  • isolated in reference to a biological molecule (e.g. an antibody) is a biological molecule that is free from at least some of the components with which it naturally occurs.
  • protein As used herein, the terms “protein”, “peptide” and “polypeptide” each refer to a polymer made up of amino acids linked together by peptide bonds and are used interchangeably.
  • a “polypeptide” may constitute a full length protein or a portion of a full length protein, and there is no intended difference in the meaning of a “peptide” and a “polypeptide”.
  • conservative amino acid substitution refers to the replacement, of a given amino acid residue in a sequence of amino acids with another, different amino acid residue of a similar size and/or of similar chemical properties.
  • conservative amino acid substitutions include: A (Ala) substituted with S (Ser); R (arg) substituted with K (lys); N (asn) substituted with Q (gin) or H (his); D (asp) substituted with E (gin); Q (gin) substituted with N (asn); C (cys) substituted with S (ser); E (glu) substituted with D (asp): G (giy ) substituted with P (pro).
  • polynucleotide refers to a single- or double-stranded polyme of deoxyribonueleotide bases, ribonucleotide bases, known analogues or natural nucleotides, or mixtures thereof.
  • binding entity encompasses any molecule capable of binding specifically to a GPC-1. epitope or mimic thereof as described herein.
  • binding entities include polypeptides such as, for example, antibodies.
  • kits refers to any delivery system for delivering materials.
  • delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (for example labels, reference samples, supporting material, etc. in the appropriate containers) and/or supporting materials (for example, buffers, written instructions for performing an assay etc.) from one location to another.
  • reaction reagents for example labels, reference samples, supporting material, etc. in the appropriate containers
  • supporting materials for example, buffers, written instructions for performing an assay etc.
  • enclosures such as boxes, containing the relevant reaction reagents and/of supporting materials.
  • positi ve control element in the context of a detection method or detection assay refers to an element that, when used in the method or assay, directly or indirectly provides a positive/affirmative signal, and thereby at least in part or wholly validates that the method or assay is capable of functioning correctly.
  • a polypeptide of between 10 residues and 20 residues in length is inclusive of a polypeptide of 10 residues in length and a polypeptide of 20 residues in length.
  • the present inventors have identified specific epitopes within glypican-1 (GPC-1 ) advantageous for detecting and quantifying (IPC- 1 levels when using binding entities such as antibodies. Although useful for many purposes, these epitopes and combinations thereof can be targeted in diagnostic assays for prostate cancer seeking to quantify GPC-1 levels.
  • GPC-1 glypican-1
  • the present invention relates to GPC- 1 epitopes and components thereof; combinations of said epitopes and/or epitope components; binding entities capable of specifically targeting the epitopes, components and/or combinations; compositions, kits and other entities comprising the epitopes, components and/or combinations; methods for generating binding entities capable of specifically targeting the epitopes, components and/or combinations: and diagnostic methods for prostate cancer requiring detection of the epitopes, components and/or combinations.
  • the present invention arises from a series of epitopes in glypican-1 heparan sulfate proteoglycan (GPC-1 ) which are targeted by specific binding entities (e.g. antibodies).
  • GPC-1 glypican-1 heparan sulfate proteoglycan
  • the epitopes may be present in a mammalian GPC-1 protein such as, for example, a bovine, equine, ovine, primate or rodent species.
  • a mammalian GPC-1 protein such as, for example, a human or a non-human mammal (e.g. a dog GPC-1 protein).
  • the epitopes may be present in a non-mammalian GPC-1 protein such as, for example, an avian GPC-1 protein.
  • the epitopes may be present in a human GPC-1. protein (e.g. as defined by a sequence set forth in any orte of: NCBI reference sequence accession no. NP 002072.2, GenBank accession no. AAH51279,1, GenBank accession no. AAA98132.1, GenBank accession no, EAW71184.1 , UniProtKB/Swiss-Prot accession no, P35052.2, and/or SEQ ID NO: 14).
  • the epitopes may be present in a GPC-1 variant (e.g. a GPC-1 isoform, splice variant, or allotype).
  • the epitopes may be present in cell -surface bound and/or secreted forms of GPC-1.
  • the present invention provides GPC-1 epitopes and components thereof, and also includes combinations of said epitopes and/or epitope components.
  • a GPC-1 epitope according to the present invention may comprise or consist of any one or more of the epitopes set out in Table 1 below.
  • an epitope of the present invention comprises or consists of a plurality of segments. These epitopes may be linear or discontinuous. Non-limiting examples of epitopes comprising a plurality of segments are set out in Table 2 below.
  • TQNARA (or a variant or fragment thereof)
  • KVNPQOPGPEEK (or a variant or fragment thereot)
  • NPQGPGPEE (or a variant or fragment thereot)
  • VNPQGPGPEE (or a variant or fragment thereof)
  • VNPQGPGPEEK (or a variant or fragment thereof)
  • KVNPQGPGPE (or a variant or fragment thereof)
  • KVNPQOPGPEEK (or a variant or fragment thereof)
  • ⁇ 2nd segment QDASDDGSGS (or a variant or fragment thereof)
  • NPQGPGPEE (or a variant or fragment thereof)
  • VNPQGPGPEE (or a variant or fragment thereof)
  • VNPQGPGPEEK (or a variant or fragment thereof )
  • KVNPQGPGPE (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • KVNPQOPGP (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof) 1st segment: VNPQGPGPEE (or a. variant or fragment thereof) ( 2nd segment: LGPECSRAVMK (or a variant or fragment thereof) I i st segment: VNPQGPGPEEK (or a variant or fragment thereof) j 2nd segment: LGPECSRAVMK (or a variant or fragment thereof) I
  • KVNPQGPGPE (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • KVNPQGPGPEEK (or a variant or fragment thereof)
  • ⁇ i 2nd segment PRERPP or a variant or fragment thereof
  • j 1st segment KVNPQGPGP (or a variant or fragment thereof)
  • i 2nd segment PRERPP (or a variant or fragment thereof) j j 1 st segment; VNPQGPGPEE (or a variant or fragment thereof)
  • VNPQGPGPEEK (or a variant or fragment thereof)
  • i 2nd segment PRERPP (or a variant or fragment thereof)
  • KVNPQGPGPE (or a variant or fragment thereof)
  • LGPECSRAVMK LGPECSRAVMK (or a variant or fragment thereof)
  • i 1st segment PRERPP (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • j 1st segment ALSTASDDR (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • LGPECSRAVMK LGPECSRAVMK (or a variant or fragment thereof)
  • TQNARAFRD (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • TQN RAFRD (or a variant or fragment thereof)
  • I 2nd segment ALSTASDDR (or a variant or fragment thereof)
  • i 1st segment TQ A (or a variant or fragment thereof)
  • 2nd segment ALSTASDDR (or a variant or fragment thereof)
  • TQN RAFRD (or a variant or fragment thereof)
  • TQNARA (or a variant or fragment thereof)
  • 2nd segment PRERPP (or a variant, or fragment thereof)
  • QDASDDGSGS (or a variant or fragment thereof)
  • 2nd segment PRERPP (or a variant or fragment thereof)
  • TQNARAFRD (or a variant or fragment thereof)
  • TQNARA (or a variant or fragment thereof)
  • KVNPQGPGPEEK (or a variant or fragment thereof)
  • 2nd segment PRERPP (or a variant or fragment thereof)
  • st segment KVNPQGPGP (or a variant or fragment thereof)
  • NPQGPGPEE (or a variant or fragment thereof)
  • VNPQGPGPEE (or a. variant or fragment thereof)
  • 2nd segment PRERPP (or a variant or fragment thereof)
  • VNPQGPGPEE (or a variant or fragment thereof)
  • 2nd segment PRERPP (or a variant or fragment thereof)
  • V PQGPGPE (or a variant or fragment thereof)
  • P .ERPP (or a variant or fragment thereof)
  • KVNPQGPGPEEK (or a variant or fragment thereof)
  • the present in vention provides combinations of distinct epi topes that comprise or consist of a plurality of discrete epitopes. These discrete epitopes may be linear or discontinuous. Non-limiting examples of epitope combinations comprising a plurality of distinct epitopes are set out in Table 3 below.
  • KVNPQGPGPEEK (or a variant or fragment thereof)
  • TQNARAFRD (or a variant or fragment thereof)
  • KVNPQGPGP (or a variant or fragment thereof)
  • TQNARAFRD (or a variant or fragment thereof)
  • NPQGPGFEE (or a variant or fragment thereof)
  • TQNARAFRD (or a variant or fragment thereof)
  • VNPQGPGPEE (or a variant or fragment thereof)
  • TQ ARAFRD (or a variant or fragment thereof)
  • VNPQGPGPEEK (or a variant or fragment thereof)
  • TQN ARAFRD (or a variant or fragment thereof)
  • KVNPQGPGPE (or a variant or fragment thereof)
  • TQN ARAFRD (or a variant or fragment thereof)
  • KVNPQGPGPEEK (or a variant or fragment thereof)
  • TQNARA (or a variant or fragment thereof)
  • KVNPQGPGP (or a variant or fragment thereof)
  • J 2nd epitope TQNARA (or a variant or fragment thereof )
  • NPQGPGFEE (or a variant or fragment thereof)
  • TQN AR A (or a variant or fragment thereof)
  • VNPQGPGPEE (or a variant or fragment thereof)
  • TQNARA (or a variant or fragment thereof)
  • VNPQGPGPEEK (or a variant or fragment thereof) j 2nd epitope; TQNARA for a variant or fragment thereof) j
  • KVNPQGPGPEEK (or a variant or fragment thereof)
  • 2nd epitope ALSTASDDR (or a variant or fragment thereof)
  • KVNPQGPGP (or a variant or fragment thereof)
  • 2nd epitope ALSTASDDR (or a variant or fragment thereof)
  • NPQGPGPEE (or a. variant or fragment thereof)
  • 2nd epitope ALSTASDDR (or a variant or fragment thereof)
  • VNPQGPGPEE (or a variant or fragment thereof)
  • 2nd epitope ALSTASDDR (or a variant or fragment thereof)
  • VNPQGPGPEEK (or a variant or fragment thereof)
  • 2nd epitope ALSTASDDR (or a variant: or fragment thereof)
  • KVNPQGPGPE (or a variant or fragment thereof)
  • 2nd epitope ALSTASDDR (or a variant or fragment thereof)
  • KVNPQGPGPEEK (or a variant or fragment thereof)
  • QDASDDGSGS (or a variant or fragment thereof)
  • KVNPQGPGP (or a variant o fragment thereof)
  • NPQGPGPEE (or a variant or fragment thereof)
  • VNPQGPGPEE (or a variant or fragment thereof)
  • QDASDDGSGS (or a variant or fragment thereof)
  • VNPQGPGPEEK (or a variant or fragment thereof)
  • QDASDDGSGS (or a variant or fragment thereof)
  • KVNPQGPGPE (or a variant or fragment thereof;)
  • ⁇ 2nd epitope QDASDDGSGS (or a variant or fragment thereof)
  • ⁇ 1st epitope KVNPQGPGPEEK (or a variant or fragment thereof)
  • LGPECSRAVM (or a variant or fragment thereof) 1st epitope: KVNPQGPGP (or a variant or fragment thereof) j 2nd epitope: LGPECSRAVMK (or a variant or .fragment thereof) I i st epitope: NPQGPGPEE (or a variant or fragment thereof) j 2nd epitope: LGPECSRAVMK (or a variant or fragment: thereof) I
  • VNPQGPGPEE (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • NPQGPGPEE (or a variant or fragment thereof)
  • VNPQGPGPEE (or a variant or fragment thereof)
  • VNPQGPGPEEK (or a variant or fragment thereof
  • KVNPQGPGPE (o a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • PRERPP (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • ALSTASDDR (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • TQNA A (or a variant or fragment thereof)
  • LGPECSRAVMK (or a variant or fragment thereof)
  • TQNARAFRD (or a variant or fragment thereof)
  • LGPECSRAVMK LGPECSRAVMK (or a variant or fragment thereof) 7 1st epitope: TQNARAFRD (or a variant or fragment thereof) 9 2nd epitope: ALSTASDDR (or a variant or fragment thereof)
  • TQNARA (or a variant or fragment thereof)
  • PRERPP 10 1st epitope: PRERPP (or a variant or fragment thereof)
  • TQNARAFRD (or a variant or fragment thereof)
  • PRERPP (or a variant or fragment thereof)
  • TQNARA (or a variant or fragment thereof)
  • PRERPP (or a variant or fragment thereof)
  • PRERPP (or a variant or fragment thereof)
  • QDASDDGSGS (or a variant or fragment thereof)
  • KVNPQGPGPEEK (or a variant or fragment thereof)
  • PRERPP (or a variant or fragment thereof)
  • KVNPQGPGP (or a variant or fragment thereof)
  • PRERPP (or a variant or fragment thereof)
  • VNPQGPGPEE (or a variant or fragment thereof)
  • PRERPP (or a variant or fragment, thereof)
  • H i 3rd epitope QDASDDGSGS (or a variant or fragment thereof) 2 I 1st epitope: VKPQQPGPEBK. (or a variant or fragment thereof)
  • V PQGPGPE (or a variant or fragment thereof)
  • QDASDDGSGS (or a variant or fragment thereof)
  • PRERPP a variant or fragment thereof
  • QDASDDGSGS (or a variant or fragment thereof)
  • a combination of epitopes according to the present invention may comprise prostate-specific antigen (PSA), also known, as gamma-seminoprotem or kallikrein-3 (KLK3). Accordingly, a combination of epitopes according to the present invention may comprise any one of the epitopes listed in Table 1 or Table 2 i combination with PSA, or any one of the epitope combinations listed in Table 3 further combined with PSA.
  • PSA prostate- specific antigen
  • KLK3 gamma-seminoprotem or kallikrein-3
  • the present invention provides variants of the GPC-1 epitopes described herein.
  • the variants may comprise conservative or non-conservative amino acid substitution ⁇ ), as known to those of ordinary skill in the art.
  • a variant of an epitope of the present inventio may have a specified percentage of amino acid residues that are the same (percentage of "sequence identity"), over a specified region, or, when not specified, over the entire sequence. Accordingly, a 'Variant" of a GPC-1 epitope disclosed herein may share at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identit y with the sequence of a GPC-l epitope described herein.
  • the variant may retain identical, substantially identical, or altered biological activity in comparison to the GPC- 1 epitope sequence from which the variant arises.
  • the variant may be a homologue GPC-1 epitope from a different family, genus or species having identical or substantially identical biological function or activity to the GPC-1 epitope sequence from which the variant arises (e.g. those derived from other species of mammals).
  • a conservative amino acid substitution refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain, as well known to those of ordinary skill in the art.
  • the substitution of the charged amino acid glutamic acid (Gin) for die similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution.
  • the percentage of sequence identity between two sequences may be determined without difficulty using methods known to those of ordinary skill in the art.
  • the percentage of sequence identit between two sequences may be determined by comparing two optimally aligned sequences over a comparison window.
  • the portion of the sequence in the comparison window may, for example, comprise deletions or additions (i.e. gaps) in comparison to the reference sequence (for example, a GPC- 1 epitope sequence as described herein), which does not comprise deletions or additions, in order to align the two sequences optimally.
  • a percentage of sequence identity may then be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence analysis software e.g.. Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1 710 University Ave., Madison, Wis. 53705. This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
  • Computer software for measuring the degree of sequence identity between two or more sequences include, but are not limited to, CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BEST/FIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Aecelrys Inc., 9685 Scranton Road, San Diego, California, USA).
  • Table 4 provides suitable and non-limiting examples of the variants referred to. Table 4; Non-limiting examples of G PC- J epitope variants according to the present invention
  • a variant of KVNPQGFGPEE may comprise V (val) at position 2, Q (gin) at position 5, G (gly) at position 6, and P (pro) at position 7.
  • the variant may further comprise K. (lys) at position I, and/or N (asn) at position 3, and/or P (pro) at position 4.
  • the variant may further comprise G (gly) at position 8, and/or P (pro) at position 9, and/or E (glu) at position 10.
  • the variant may comprise one, two, three, four, five, six, seven or eight substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions 1, 3, 4, 8, 9, 10, 11, and/or 12 of SEQ ID NO: 1.
  • a variant of K VNPQGFGPEEK may comprise G (gly) at position 6, G (gly) at position 8, and E (glu) at position 10.
  • the variant may further comprise (asn) at position 3, and/or Q (gin) at position 5, and/or P (pro) at position 7.
  • the variant may comprise one, two, three, four, five, six. seven, eight or nine substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions .1 , 2. 3, 4, 5, 7, 9, 1 1 , and/or 12 of SEQ ID NO: 1.
  • a variant of VMPOGPGPEEK may comprise P (pro) at positio 7, G (gly) at position 8, and E (glu) at position 10, The variant may further comprise Q (gin) at position 5, and/or G (gly) at position 6, and/or E (glu) at position 1 1. Additionally or alternatively, the variant may comprise one, two, three, four, five, six, seven, eight or nine substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions 1 , 2, 3, 4, 5, 6, 9, ! 1, and/or 12 of SEQ ID NO: 1.
  • a variant of VNPQGPGPEEK may comprise V (val.) at position 1, Q (gin) at position 4, Q (gly) at position 5, and P (pro) at position 6, The variant may further comprise N (asn) at position 2, and/or P (pro) at position 3. Additionally or alternatively, the variant may further comprise G (gly) at position 7, and/or P (pro) at position 8, and/or E (glu) at position 9. Additionally or alternatively, the variant may comprise one, two, three, four, five, six, or seven substituted residues. The substituted amino acid residue(s) may be at any one or more of positions 2, 3, 7, 8, 9, 10, and/or 1 1 of SEQ ID NO: 2.
  • a variant of VNPQGPGPEEK may comprise G (gly) at position 5, G (gly) at position 7, and £ (glu) at position 9.
  • the variant may further comprise N (asn) at position 2, and/or Q (gin) at position 4, and/or P (pro) at position 6.
  • the variant may comprise one, two, three, four, five, six, seven, or eight substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions L 2, 3, 4, 6, S, 10, and/or 1 1 of SEQ I D NO: 2.
  • a variant of VNPQGPGPEEK may comprise G (gly) at position 5, G (gly) at position 7, and £ (glu) at position 9.
  • the variant may further comprise N (asn) at position 2, and/or Q (gin) at position 4, and/or P (pro) at position 6.
  • the variant may comprise one, two, three, four, five, six, seven, or eight substituted residues.
  • (SEQ ID NO: .2) may comprise P (pro) at position 6, G (gly) at positio 7, and E (glu) at position 9.
  • the variant may further comprise Q (gin) at position 4, and/or G (gly) at position 5, and/or E (glo) at position 10.
  • the variant may comprise one, two, three, four, five, six, seven, or eight substituted residues.
  • the substituted amino acid residiie(s) may be at any one or more of positions I , 2, 3, 4, 5, 8, 10, and/or 1 1 of SEQ ID NO: 2.
  • a variant of VNPQGPGPEE may comprise V (val) at position i, Q (gin) at position 4, G (gly) at position 5, and P (pro) at position 6.
  • the variant may further comprise N (asn) at position 2, and/or P (pro) at position 3.
  • the variant may further comprise G (gly) at position 7, and/or P (pro) at position 8, and/or E (glu) at position 9.
  • the variant may comprise one, two, three, four, five or six substituted residues.
  • the substituted amino acid tesidue(s) may be at any one or more of positions 2, 3, 7, 8, 9, and/or 10 of SEQ ID NO: 3,
  • a variant of VNPQGPGPEE may comprise G (gly) at position 5, G (gly) at position 7, and E (glu) at position 9.
  • the variant may further comprise N (asn) at position 2, and/or Q (glu) at position 4, and or P (pro) at position 6.
  • the variant may comprise one, two, three, four, five, six, or seven substituted residues.
  • the substituted amino acid res idite(s) ma be at any one or more of positions i , 2, 3, 4, 6, 8, and/or 10 of SEQ ID NO: 3.
  • a variant of VNPQGPGPEE may comprise P (pro) at position 6, G (gly) at position 7, and E (glu) at position 9.
  • the variant may fiirther comprise Q (gin) at position 4, and/or G (gly) at position 5, and/or E (glu) at position J O.
  • the variant may comprise one, two, three, four, five, six, or seven substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions i, 2, 3, 4, 5, 8, and/or 10 of SEQ ID NO: 3.
  • a variant of NPQGPGPEE may comprise Q (gin) at position 3, G (gl ) at position 4, and P (pro) at position 5.
  • the variant may further comprise N (asn) at position 1, and/or P (pro) at position 2.
  • the variant may further comprise G (gly) at position 6, and/or P (pro) at position 7, and/or E (glu) at position 8.
  • the variant may comprise one, two. three, four, five, or six substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions 1, 2, 6, 7, 8, and/or 9 of SEQ ID NO: 4.
  • a variant of NPQGPGPEE may comprise G (gly) at position 4, G (gly) at position 6, and E (glu) at position 8.
  • the variant may further comprise N (asn) at position 1, and/or Q (gin) at position 3, and/or P (pro) at position 5.
  • the variant may comprise one, two, three, four, five, or six substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions 1 , 2, 3, 5, 7, and/or 9 of SEQ ID NO: 4.
  • a variant of NPQGPGPEE may comprise P (pro) at position 5, G (gly) at position 6, and E (glu) at position 8.
  • the variant may further comprise Q (gin) at position 3, and/or G (gly) at position 4, and/or E (glu) at positio 9.
  • the varian may comprise one, two, three, four, five, or six, substituted residues.
  • the substituted amino acid residue(s) may be at an one or more of positions 1 , 2, 3, 4, 7, and3 ⁇ 4r 9 of SEQ ID O; 4.
  • a variant of KVNPQGPGPE may comprise V (val) at position 2, Q (gin) at position 5, G (gly) at position 6, and P (pro) at positio 7.
  • the variant may further comprise K (lys) at position L and/or N (asn) at position 3, and/or P (pro) at position 4, Additionally or alternatively, the variant may further comprise G (gly) at position 8, and/or P (pro) at position 9, and/or E (glu) at position .10. Additionally or alternatively, the variant may comprise one. two, three, four, five or six substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions 1 , 3, 4, 8, 9, and/or 10 of SEQ ID NO: 5.
  • a variant of KVNPQGPGPE may comprise G (gly) at position 6, G (gly) at position 8. and E (glu) at position 10.
  • the variant may further comprise N (asn) at position 3, and/or Q (gin) at position 5, and/or P (pro) at position 7.
  • the variant may comprise one, two, three, four, five, six, or seven substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions 1 , 2, 3, 4, 5, 7, and/or 9 of SEQ ID NO: 5.
  • a variant of K VNPQGPGPE may comprise P (pro) at position 7, G (gly) at position 8, and E (glu) at position 10.
  • the variant may further comprise Q (gin) at position 5, and/or G (gly) at position 6.
  • the variant may comprise one, two, three, four, five, six, or seven substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions 1 , 2, 3, 4, 5, 6, and/or 9 of SEQ ID NO: 5.
  • a variant of KVNPQGPGP may comprise V (val) at position 2, Q (gin) at position 5, G (gly) at. position 6, and and P (pro) at position 7.
  • the variant may further comprise K (lys) at position 1 , and/or N (asn) at position 3, and/or P (pro) at position 4.
  • the variant may further comprise G (gly) at position 8, and/or P (pro) at position 9.
  • the variant may comprise one, two, ' three, four, or five substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions 1 , 3, 4, 8, and/or 9 of S.EQ ID NO: 6.
  • a variant of KV ' PQGPGP may comprise G (gly) at position 6, and G (gly) at position 8.
  • the variant may further comprise N (asn) at position 3, and/or Q (gin) at position 5, and/or P (pro) at position 7.
  • the variant may comprise one, two, three, four, five, six, or seven substituted residues.
  • the substituted amino acid residue(s) may be at any one or more of positions 1. 2, 3, 4, 5, 7, and/or 9 of SEQ ID NO: 6,
  • a variant of K VNPQGPGP may comprise P (pro) at position 7, and G (gly) at position S.
  • the variant may further comprise Q (gin) at position 5, and/or G (gly) at positio 6.
  • th variant may comprise one, two, three, four, five, six, or seven substituted residues.
  • the substituted amino acid residne(s) may be at any one or more of positions 1, 2, 3, 4, 5, 6, and/or 9 of SEQ ID NO; 6.
  • a variant of any one of the epitopes of the present invention as set forth in SEQ ID NOs: 7-13 may comprise an amino acid substitution at any one or more position(s) of the epitope sequence.
  • the amino acid substitution may be a conservative amino-acid substitution or a non-conservative amino acid substitution, as are known to those of ordinary skill in die art.
  • the variants may comprise conservative substitution ⁇ ) only, non-conservative substitution(s) only, or a mixture of conservative substiution(s) and non-conservative substitutions).
  • the present invention provides fragments ' of the GPC-I epitopes and variants described herein.
  • a fragment of an epitope of the present invention may comprise or consist of 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Accordingly, a fragment of an epitope according to the present invention may comprise or consist of, for example, between 5 and 10, between 5 and 15, between 5 and 20, between 10 and 20, between 10 and 15, between 7 and 15, between 7 and 13, between 8 and 12, between 8 and 1.0, between 1 1 and 39, between 12 and 18, or between 13 and 17 amino acids in length. Generally, a fragment of a full GPC-l epitope disclosed herein may possess stmi lar or in some cases improved immunological properties compared to the full GPC-l epitope.
  • GPC-1 epitopes in. the form of fusion polypeptides.
  • two or more full epitopes selected from Table 1, two or more epitope segments selected from Table 2, a combination of two or more full epitopes selected from Table 3, or a combination of a full epitope from Table 1 and any one or more epitope segments selected from Table 2 may be linked together to form the fusion polypeptides.
  • the fusion polypeptides can be prepared using standard techniques known to those of ordinary skill in the art including, for example, chemical conjugation.
  • the fusion polypeptides ma also be expressed as a recombinant polypeptide in an expression system.
  • DNA sequences encoding polypeptide components of the fusion polypeptide may be assembled separately, and ligated into an appropriate expression vector.
  • the 3 * terminus of the DNA sequence encoding one polypeptide component can be ligated, with or without a peptide linker, to the 5' terminus of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in frame. This can allow translation into a single fusion polypeptide retaining the biological activity of both polypeptide components,
  • a linker sequence may be employed to separate first and second polypeptide components of the fusion protein by a distance sufficient to ensure that each polypeptide component folds into appropriate secondary and or tertiary structures.
  • suitable linkers include peptides/polypeptides, alkyJ chains or other convenient spacer molecules as known to those of ordinary skill in the art.
  • Suitable peptide linker sequences may be selected according to factors including an ability to adopt a flexible extended conformation, an inability to adopt a secondary staicture that could interact with functional epitope/s in polypeptide component's of the fusion polypeptides, and/or a lack of hydrophobic and/or charged residues that may react with functional epitope/s in polypeptide component's of the fusion polypeptide.
  • the peptide linker sequences may contain Gly, Asn and/or Ser residues. Additionally or alternatively., other near neutral amino acids such as Thr and Ala may also be used in the peptide linker sequences.
  • Further examples of amino acid sequences that may be usefully employed as linkers include those disclosed in US patent no. 4,935,233; ' US patent no. 4,751,180; Murphy ei at., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1 86; and Maratea el «/., Gene 40:39-46, 1985).
  • the linker sequence may generally be from 1 to about 50 amino acids in length.
  • the linker sequences may be 5, 10, 15, 20, 30, 40, between 10 and 50, between 10 and 40, between 10 and 30, between 20 and 30, between 1 and 5, or between 5 and 10 amino acids in length.
  • Fusion polypeptides according to the present invention may not require a linker sequence if the first and second polypeptide components of the fusion polypeptide have non-essential I -terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • the present invention also provides polynucleotides encoding GPC-i epi ' tope/s of ⁇ the present invention, segments of the GPC-1 epitopes, and fusion proteins comprising GPC- I epitopes and/or segments thereof.
  • the polynucleotides may be cloned into a vector.
  • the vector may comprise, for example, a D ' NA, RNA or complementary DNA, (cDNA) sequence encoding the GPC-1 epitopes, GPC-1 epitope segment's, and/or fusion proteins.
  • the vector may be a plasmtd vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences, their introduction into cells and the expression of the introduced sequences.
  • the vector is an expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenyiation signals and transcription termination sequences.
  • the invention also contemplates host cells transformed by such vectors.
  • the polynucleotides of the invention may be cloned into a vector which is transformed into a bacterial host cell, for example E. coii.
  • GPC- 1 epitopes of the present invention segments of the GPC- 1 epitopes, and fusion proteins comprising the GPC-1 epitopes and/or segments thereof can be manufactured according to standard methodologies well known to persons of ordinary skill in the art.
  • the epitopes, segments and fusion proteins may be prepared using any of a variety of well-known synthetic and/or recombinant techniques.
  • Polypeptides may, for example, be generated by synthetic means using methodologies well known to those of ordinary skill in the art.
  • commercially available solid-phase techniques e.g. the Merrifield solid-phase synthesis method
  • Equipment for automated synthesis of the epitopes, segments and fusion proteins disclosed herein is commercially available (e.g. Perkin Elmer/Applied BioSystems Division (Foster City, Calif.), and may be utilised according to the manufacturer's instructions.
  • the epitopes, segments and fusion proteins may be produced by any other method available to one of skill in the art.
  • recombinant means may be used in which nucleic acids encoding selected epitope/s, segment/s and/or fusion protein/s may be inserted into an expression vector using any of a variety of procedures known in the art (e.g. Sambrook et a/., 1 89, Molecular Cloning, Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (Jan.
  • the ligated nucleic acid sequences can be operably linked to suitable transcriptional or translational regulatory elements that facilitate expression of the epitope/s, segment s and/or fusion protein/s. Regulatory elements responsible for the expression of proteins may be located 5' to the coding region for the polypeptide. Stop codons that end translation and transcription termination signals may be present 3' to the nucleic acid sequence encoding the epitope/s, segment/s and/or fusion protein/s. After construction of a nucleic acid encoding the polypeptide/s of interest with the operably linked regulatory elements, the resultant expression cassette can be introduced into a host cell and the encoded pol peptide/s can be expressed.
  • GPC- 1 epitopes may be "isolated".
  • An "isolated" polypeptide is one that is removed from its original environment.
  • a naturally-occurring protein or polypeptide referred to herein is considered to be isolated if it is separated from, some or all of the co-existing materials in the natural system.
  • Isolated polypeptides referred to herein may also be purified.
  • the isolated polypeptides may be at least about .90% pure, a least about 95% pure or at least about 99% pure.
  • Polynucleotides and nucleic acids according to the present invention are generally known in the art, and are described, tor example, in Ausubel F. M. et at, (Eds) Current Protocols in Molecular Biolog (2007), John Wiley and Sons, lac; Sambrook et at. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plamview, New York; and Maniatis et at. Molecular Clo ing (1.982), 280-281.
  • Polynucleotides may be prepared, for example, by chemical synthesis techniques, for example, the phosphodiester and phosphot iester methods (see for example Narang S. A. et at, (1979) Meth.
  • Epitopes and fusion polypeptides according to the present invention may be attached to a support.
  • the support may, for example, be an insoluble material or a matrix which retains the epitope and excludes it from freely moving in the bulk of a reaction mixture. Suitable supports for immobilizing or localizing epitopes and fusion polypeptides are well known to those of ordinary skill in the art.
  • the support can be selected from, a wide variety of matrices, polymers, and the like in a variety of forms including beads convenient for use in rnieroassays, plastic and glass plates with individual wells, as well as other materials compatible with the reaction conditions.
  • the support can be a plastic material, such as plastic beads or wafers, or that of the well or tube in which a particular assay (e.g. an ELISA) is conducted.
  • the present invention provides binding entities capable of binding specifically to the GPC-1 epitopes described herein.
  • the binding entity may be any molecule capable of binding specifically to a GPC-1 epitope as described herein.
  • binding entities include polypeptides, antibodies, antibody fragments, molecular" imprints, lectins, and capture compounds.
  • the binding entity may be an agent that can bind to a GPC-1 epitope of the present invention or alternatively a different region of GPC-I, and also modify a binding interaction between the epitope and another different binding entity such as, for example, an antibody as disclosed herein.
  • Binding entities such as antibodies capable of binding specifically to GPC-i epitopes of the present invention can bind to a given target epitope, combination of epitope segments, or epitope combination preferentially over other non-target molecules.
  • the binding entity molecule A
  • molecule B molecule A
  • molecule A has ⁇ the capacity to discriminate between molecule B and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, molecule A will, selectively bind to molecule B and other alternative potential binding partners will remain substantially unbound by molecule A.
  • molecule A will preferentially bind to molecule B at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100- told more frequently than other potential binding partners.
  • Molecule A may be capable of binding to molecules that are not molecule B at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from molecule B-specific binding, for example, by use of an appropriate control.
  • Immortal, antibody- producing cell lines can be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus (see, for example, IVL Schreier el al, (1 80), “ ' Hybridoma Techniques 7 ', Cold Spring Harbor Laboratory: Hammerling ei ⁇ , ( 1981), “Monoclonal Antibodies and T-cell Hybridomas", Eisevier/ ' North- Holland Biochemical Press, Amsterdam; Keniiett ei a/., (1980), “Monoclonal Antibodies", Plenum Press).
  • a means of producing a hybridoma from which the monoclonal antibody is produced, a myeloma or other self-perpetuating ceil line can be fused with lymphocytes obtained from the spleen of a mammal hj'perimm.unised with a recognition factor -binding portion -thereof, or recognition factor, or an origin-specific D A-binding portion thereof.
  • Hybridomas producing a monoclonal antibody capable of binding speci fically to a GPC-1 epitope of the present invention are dentified by their ability to immunoreact with the epitope/s presented.
  • a monoclonal antibody useful in practicing the inventio ca be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody-containing medium is then collected.
  • the antibody molecules can then be further isolated by well known techniques.
  • polyclonal antibodies there are various procedures known in the art which may be used for the producticm of polyclonal, antibodies.
  • various host animals ca be immunised by injection with the epitopes, including, but not limited to, rabbits, chickens, mice, rats, sheep, goats, etc.
  • the target molecule can be conjugated to an immunogenic carrier (e.g., bovine serum albumin (BSA) or keyhole limpet hemocyamn ( LH)).
  • BSA bovine serum albumin
  • LH keyhole limpet hemocyamn
  • various adjuvants may be used to increase the immunological response, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as rysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • Freund's complete and incomplete
  • mineral gels such as aluminium hydroxide
  • surface active substances such as rysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • Suitable assays for iinmunospeeific binding of antibodies include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, m situ immunoassays,. Western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, hnmunoelectrophoresis assays, and the like (see, for example, Ausube!
  • Antibod binding ma be detected by virtue of a detectable label on the primary antibody.
  • the antibody may be detected by virtue of its binding with a secondary antibody or reagent which is appropriately labelled.
  • a variet of metliods for the detection of binding in an immunoassay are known in the ait and are included in the scope of the present invention.
  • tire antibodies (or fragments thereof) raised against specific GPC-1 epitope/s of interest have binding affinity for the epitope/s.
  • tire antibodies (or fragments thereof) have binding affinity or avidity greater than about 10 J M "1 , more preferably greater than about 10 6 M , still more preferably greater than about 10' M "! and most preferably greater than about 1 S M "! .
  • an antibody in terms of obtaining a suitable amount of an antibody according to the present invention, one may manufacture the antibody(s) using batch fermentation with serum free medium. After fermentation the antibody may be purified via a multistep procedure incorporating chromatography and viral inactivation/removal steps. For instance, the antibody may be first separated by Protein A affinity chromatography and then treated with solvent/detergent to inactivate any lipid enveloped viruses. Further purification, typically by anion and cation exchange chromatography may be used to remove residual proteins, solvents/detergents and nucleic acids. The purified antibody may be further purified and formulated into 0.9% saline using gel filtration columns. The formulated bulk preparation may then be sterilised and viral filtered and dispensed.
  • the antibody may be purified via a multistep procedure incorporating chromatography and viral inactivation/removal steps. For instance, the antibody may be first separated by Protein A affinity chromatography and then treated with solvent/detergent to inactivate any lipid enveloped viruses. Further pur
  • Non-limiting examples of antibodies capable to binding to one or multiple GPC-1 epitopes of the present invention include;
  • anti-Glypican l/GPCi antibody (ah 137604): a rabbit polyclonal (IgG) specific for human GPC-1 and commercially available from abeam*
  • (iv) goat anri-glypican 1 antibody (A A 24-530): a goat polyclonal specific for human GPC-1 and commercially available from Li f eSpan Biosciences, Inc. (see ht.tps://www .lsbio.com/antibodies/an.ti-gpc 1 -antibody-glypican-anttbody-aa24-530-ice- wb-westem-fiow-ls-c330760/341104).
  • compositions and kits according to the present invention may comprise:
  • GPC-1 epitope/s e.g. Table I
  • variant/s or fragment s thereof as described herein (e.g. Table I);
  • GPC-1 epitope segment's, variant/s or fragment/s thereof as described herein e.g. Table 2;
  • GPC-1 epitope combinations as described herein (e.g. Table 3);
  • fusion proteins comprising GPC-1 epitope/s, segment's of GPC-1 epitopes, and/or variant/s or fragment/s thereof, as described herein (see subsection above entitled “Fusion polypeptides”); and/or
  • binding entities one o more binding entities as described herein (see section above entitled "Binding entities ").
  • compositions may additionally comprise an acceptable carrier, adjuvant and/or diluent.
  • the carriers, diluents and adjuvants may be "acceptable” in terms of being compatible with the other ingredients of the composition, and/or "pharmaceutically acceptable 7 " in generally being not deleterious to any recipient thereof. Suitable earners, diluents and adjuvants are well known to those of ordinary skill in the art (see, for example, Oilman et ah (eds.) Goodman and Oilman's: the pharmacological basis of therapeutics, 8th Ed., Perganion Press ( 1990); Remington's Pharmaceutical Sciences, Mack Publishing Co.: Easton, Pa., 1.7th Ed, (1985)).
  • the compositions may, in some embodiments, be used for diagnostic or research purposes.
  • kits may be fragmented or combined kits.
  • a "fragmented kit” refers to a delivery system comprising two or more separate containers that each contain a sub portion of the total kit components. Any delivery system comprising two or more separate containers that each contain a sub portion of the total kit components are included withi the meaning of the term fragmented kit.
  • a “combined kit” refers to a deliver ' system containing all of the kit components in a single container (e.g. in a single box housing each, of ' the desired components). The kits may, in some embodiments, be used for diagnostic or research purposes.
  • GPC- I epitopes of the present invention can be used in diagnostic methods.
  • the present inventors have discovered that glypican-l is a new marker for prostate cancer (US. provisional patent application no. 61/928/776 entitled "Cell Surface Primate Cancer Antigen for Diagnosis " , Walsh ei l. - unpublished).
  • the present invention provides for methods for the diagnosis, prognosis, or likelihood of developing prostate cancer in subjects based on the detection of GPC-1 epitope/s . , GPC-1 epitope segments, and/dr variants and fragments of the epitopes/epitope segments as described herein. Additionally or alternatively, variants and fragments of the GPC-1 epitopes and/or GPC-1 epitope fragments may be detected in the diagnostic methods.
  • the methods comprise determining the level of GPC-1 epitope/s and/or GPC-1 epitope segments in a biological sample from a subject, to be tested. Additionally or alternatively, the level of variant s and fragment s of the GPC-1 epitopes and/or segments thereof in the biological sample may also be determined when carrying out the methods.
  • GPC-1 epitopes, GPC-1 epitope segments, and variants and fragments include those referred to in the section entitled "Epitopes" (see in particular Tables 1-3 and associated description of variants and fragments).
  • the methods may additionally comprise determining levels of prostate-specific antigen (PSA), also known as gamma-seminoprotein or kallikrein-3 (KL 3), in the biological sample, and optionally comparing the level of PSA detected with that of a control,
  • PSA prostate-specific antigen
  • KL 3 gamma-seminoprotein or kallikrein-3
  • the level of GPC-1 epitope/s, GPC-1 epitope segments, and/or variants and fragments of the epitopes/epitope segments detected in the subject's biological sample may be compared to levels determined to be present in a control sample.
  • the levels in the control sample may be determined before, during or after determining the level present in the subject's biological sample.
  • the levels present in the control sample may be determmed based on those present in an equivalent biological sample from an individual or based on mean levels present in biological samples from a population of individuals.
  • the individual or individuals may have been determined not to have cancer, and/or determined not to have prostate cancer, in general, detection of increased levels in the subject's biological sample compared to those of the control can be taken as indicati ve that the subject has prostate cancer, or an increased likelihood of developing prostate cancer. For example, an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% may be indicative that the subject has prostate cancer, or an increased likelihood of developing prostate cancer. Alternatively, detection of equivalent or decreased levels in the subject's biological sample compared to those of the control can be taken as indicative that the subject does not have prostate cancer, or does not have an increased likelihood of developing prostate cancer.
  • GPC-1 epitope/s, GPC-i epitope segments, and/or variants and fragments of the epitopes/epitope segments as described herein are used as positive controls in the diagnostic methods of the present invention. For example, they may be used in a given assa to confirm that the assay as performed is capable of detecting the GPC- i epitope/s, GPC-1 epitope segments, and/or variants and fragments.
  • the diagnostic methods of the present invention utilise fusion protein's of the present invention-
  • suitable fusion proteins are provided in the subsection entitled ""Fusion proteins".
  • the fusion protein/s may be used as a positive control in the detection methods.
  • the biological sample may be a tissue sample (e.g. a biopsy sample of prostate tissue) or a body fluid sample.
  • the body fluid sample may be a blood, serum, plasma or urine sample.
  • Non-limiting examples of prostate cancers that, may be detected with the present invention include prostatic intraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma.
  • the detection of the GPC-1 epitopes, GPC-1 epitope segment s, and variants and fragments may be by way of standard assays known to those or ordinary skill in the art including, but. not limited to Western blot analysis, Enzyme-linked immunosorbent assays (BLISAs), fluorescent activated ceil sorting (PACS), a biofilm test, or an affinity ring test (see, for example, US application 2013/016,736).
  • BLISAs Enzyme-linked immunosorbent assays
  • PES fluorescent activated ceil sorting
  • a binding entity according to the present invention may be used the assays.
  • the binding entity is an antibody. In other embodiments, the biding entity is not an antibody
  • the antibody is selected from any one or more of: (i) MIL3S antibody as deposited under the terms of the Budapest Treaty at Cellbank Australia at 214 Bawkesbiiry Road, Westmead, NSW 2145, Australia on 22 nd August 2014 under accession number CBA2GI40026
  • anti-Glypican i /GPCi antibody abl37604
  • IgG rabbit polyclonal (IgG) specific for human GPC-l and commercially available from abeam*
  • the antibody is not selected from any one or more of;
  • anti-Glypican 1/GPCl antibody (ab 137604): a rabbit polyclonal (IgG) specific for human GPC-l and commercially available from abeam*
  • the binding entity is an antibody population comprising MIL38 antibody (CBA20140026) and not comprising an anti-glypican 1 (GPC-l) antibody capable of binding to an epitope comprising an amino acid sequence selected from any one or a pluraiity of: TQNARA (SEQ I» NO: 8), ALSTASDDR (SEQ ID NO: 9), PRERPP (SEQ ID NO: 10), QDASDDGSGS (SEQ ID NO: 11), LGPECSRAVMK (SEQ ID NO: 13), and TQNA ' RAFRD (SEQ ID NO: 7).
  • TQNARA SEQ I» NO: 8
  • ALSTASDDR SEQ ID NO: 9
  • PRERPP SEQ ID NO: 10
  • QDASDDGSGS SEQ ID NO: 11
  • LGPECSRAVMK SEQ ID NO: 13
  • TQNA ' RAFRD SEQ ID NO: 7
  • Example 1 identification and characterisation of a glypican- 1 epitope bound by murine anti-hiiman glypican 1 (MIL38-AM4)
  • Murine anti human glypican 1 monoclonal antibody M1L38-AM4 was provided set out in Table 6 below.
  • the following human glypican- 1 (GPC- 1) sequence was used as a basis to generate a library of structured peptides.
  • Figure 1A shows the rendering of chain B as present under Protein Data Bank identifier (PBD ID) 4AD7 (http://wwxv.ebf ,ac.uk/pdbsiim/4AD7).
  • PBD ID Protein Data Bank identifier
  • CLIPS technology structurally fixes peptides into defined three-dimensional structures. This results in functional mimics of even the most complex binding sites, CLIPS technology is now routinely used to shape peptide libraries into single, double or triple looped structures as well as sheet- and helix-like folds.
  • the CLIPS reaction takes place between bromo groups of the CLIPS scaffold and thiol sidechains of cysteines. The reaction is fast and specific under mild conditions. Using this chemistry, native protein sequences are transformed into CLIPS constructs With a range of structures including single T2 loops, T3 double loops, conjugated T2+T3 loops, stabilized beta sheets, and stabilized alpha helixes (Timroerraan et L, h MoL Recognit 2007; 20: 283-29).
  • CLIPS library screening starts with the conversion of the target protein into a library of up to 10,000 overlapping peptide constructs, using a combinatorial matrix design.
  • a matrix of linear peptides is synthesized, which are subsequently shaped into spatially defined CLIPS constructs.
  • Constructs representing both parts of the discontinuous epitope in the correct conformation bind the antibody with high affinity, which is detected and quantified.
  • Constructs presenting the incomplete epitope bind the antibody with lower affinity, whereas constructs not containing the epitope do not bind at all.
  • Affinity information is used in iterative screens to define the sequence and conformation of epitopes in detail.
  • the target protein containing a discontinuous conformational epitope is converted into a matrix library.
  • Combinatorial peptides are synthesized on a proprietary mmicard and chemically converted into spatially defined CLIPS constructs. Binding of antibodies is quantified.
  • CLIPS Chemically Linked Peptides on Scaffolds
  • a 0,5 mM solution of the T2 CLIPS template 1,3-bis (bromomethyl) benzene was dissolved in ammonium bicarbonate (20 raM, pH 7.9)/aeetonitrile (l :!(V/v)). This solution was added onto the peptide arrays.
  • the CLIPS template bound to side-chains of two cysteines as present in the solid-phase bound peptides of the peptide-arrays (455 wells plate with 3 ⁇ wells). The peptide arrays were gently shaken in the solution for 30 to 60 minutes while completely covered in solution.
  • peptide arrays were washed extensively with excess of 3 ⁇ 4G and sonicated in disrupt-buffer containing 1 percent SDS/0.1. percent beta- mercaptoefhanoi in PBS (pH 7.2 ⁇ at 70°C for 30 minutes, followed by sonicatton in 3 ⁇ 4Q for another 45 minutes.
  • the T3 CLIPS carrying peptides were made in a similar way but with three cysteines.
  • Positions 2 - 16 contain the linear 15mers of Set 1 in which cysteines are replaced by alanine. After synthesis, peptides ate constrained by a P2 CLIPS lM linker to constrain the shape.
  • the binding of antibody to each of the synthesized peptides was tested in a PEPSCAN-based EOS A.
  • the peptide arrays were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 dilution of an antibody peroxidase conjugate (SBA, cat.nr. 2010-05) for one hour at 25°C. After washing, the peroxidase substrate 2,2 , -azino-di-3-ethylbenztl)iazoline sulfonate (ABTS) and 2 ⁇ /ml of 3 percent 3 ⁇ 4(3 ⁇ 4 were added. After one hour, the color development was measured. The color development was quantified with a charge coupled device (CCD) - camera and an image processing system.
  • CCD charge coupled device
  • the values obtained from the CCD camera ranged from 0 to 3000 mAU. similar to a standard 96-well plate ELlSA-reader. The results were quantified and stored in the Peplab database. Occasionally a well contained an air-bubble resulting in a false-positive value. The cards were manually inspected and any values caused by am air-bubble were scored as 0.
  • Table 7 summarises antibody binding conditions. For the Pepscaa Buffer and Pre- conditioning (SQ), the numbers indicate the relative amount of competing protein (a combination of horse serum and ovalbumin).
  • FIG. 2 A graphical overview of the complete dataset of raw EL. ISA results generated by the screening is shown in Figure 2. Here a box plot depicts each dataset and indicates the average ELTSA signal, the distribution and the outliers within each dataset. Dependin on experiment conditions ( mount of antibody, blocking strength etc) different distributions of ELBA data were obtained. The data enabled identification of an epitope.
  • This stud aimed to map the epitope of murine anti human glypican 1 (MIL38- AM4).
  • Empirical sets spanning the reference sequence with different conformational constraints were made, and the antibody was probed on the peptide array.
  • Significant binding indicated a discontinuous epitope, consisting of major contributions from the flexible loop . ⁇ VMPQGPGPEE ass (SEQ ID NO: 2), and minor contributions .from (residues of) the helix 135 TQNARAF Di 4 j (SEQ ID I O: 7).
  • SEQ ID NO: 2 identification and characterisation of additional glypican-1 epitopes bound by other anti-giypican-l antibodies
  • Table 8 below provides information on the antibodies used in this study. The first three antibodies listed are commercially available.
  • the human glypican-1 (GPC-1) sequence defined by SEQ ID NO: 14 was used as a basis to generate a Librar of structured peptides.
  • Figure lA shows the rendering of chain B as present under Protein Data Bank identifier (PBD ID) 4AD7 (hitp * . / w.ebt.ac.uk7pdbsutn 4AD7).
  • PBD ID Protein Data Bank identifier
  • Example 1 Peptide synthesis was performed using the methods referred in Example 1. Chemically synthesized linear and CLIPS peptides were synthesized according to the designs shown in Example 1 (sets 1 -5)..
  • Table 9 summarises antibody binding conditions. For the Pepscan Buffer and Preconditioning (SQ), the numbers indicate the relative amount of competing protein (a combination of horse serum and ovalbumin). P/Tw ( PBS-Tween) was also used to reduce stringency of binding. Table 9: screening conditions
  • FIG. 5 A graphical overview of the complete dataset of raw ELISA results generated by the screening is shown in Figure 5.
  • a box plot depicts each dataset and indicates the average ELISA signal, the distribution and the outliers within each dataset.
  • experiment conditions amount of antibody, blocking strength etc
  • different distributions of ELISA data are obtained.
  • the data enabled identification of an epitope for the monoclonal and polyclonal sera, but not for M1L38-AM3. ⁇ 38- ⁇ 3
  • Rabbit anti GPC ! yields 3 main signals, as can be seen in the corresponding panel in Figure 6. These signals correspond to the stretches 3 8VNPQGPG EEK35S (SE-Q ID NO: 2), m RERPPsTi (SEQ ID NO: 10),. and 4 7 sQDASDDGSGS4*7 (SEQ ID NO: 11, ⁇ . j 5 - Mouse Ab 2600 (Mittipore)
  • Mouse antibody 2600 recognizes one clear peak in all peptide sets, sharing the common core 242EGPECSRAV K252 (SEQ ID NO: 13). This can be seen in the corresponding panel in Figure 6.
  • Goat anti-GPC-1 yields 2 main signals, as can be seen in the corresponding panel in Figure 6. These signals correspond to the stretches 34gVNPQGPGPEEK.35s.iSEQ ID NO: 2), and 4 sALSTASDDR 4 i4 (SEQ ID NO: 9).
  • Rabbit anti GPC-1 recognizes at least three stretches in glypica 1, 348V PQGPG EE 3S8 (SEQ ID NO: 2), 366 PRERPP3?i (SEQ ID NO: 10), and 780JDASDDGSGS4S7 (SEQ ID NO: 11). Two of these stretches are resolved in
  • Mouse anti glypican mab 2600 recognizes the stretch 242 LGPECSRA VMK5 52 (SEQ ID NO: 13) in all peptide sets of the array.
  • the localization of epitope in coordinate file 4AD7.pdb is depicted in Figure 7B.
  • Goat anti glypican 1 recognizes at least two stretches in glypican 1, j 8 ' VNPQGPGP.EEK 35 8 (SEQ ID NO: 2), and 408ALSTASDDR41 (SEQ ID NO: 9). Since this is a polyclonal antibody preparation, it cannot be assessed whether the epitopes are linear or are part of a complex epitope. Neitfier stretch is resolved in tire available coordinate file.
  • the rabbit and goat polyclonal preparations both recognize a stretch 348V QGPGPEEK358 (SEQ ID NO: 2) in Glypican 1, which also forms part of the (likely discontinuous ' ) binding site of Mab MIL38-AM4, Both polyclonal preparations also recognize additional epitopes on Glypican 1, but it cannot be assessed if these epitopes form a discontinuous epitope, or are manifestations of the polyclonal nature of the sample. Neither pA.b recognizes the stretch which is thought to contribute to MIL38-AM4 binding.
  • Mouse Mab 2600 recognizes an epitope that is not shared by any other anti-GPC-1 antibody tested thus far.
  • Example 3 identification and characterisation of additional glypican-1 epitopes bound by other anti-glypican-l antibodies .1 Materials and Methods
  • Table 10 below provides information on the antibodies used in this study. Three anti-glypican I antibodies were used, each having been used in earlier experiments as described in Example 1 and/or Example 2 above.
  • the human glypican-1 (GPC-1) sequence on which this study was based is defined in SEQ 10 NO: 14. The following sequences of residues were used: GFCl_residues # 3 44 -366 GN P N P Q G PG P ⁇ ⁇ EE P.RGK LAP (SEQ ID NO: 15) GPCl_reSid es #131 - 145 GELYTQNARAFRDLYSELR (SEQ ID NO; 16)
  • PKVNPQGPGPEEKRR SEQ I D NO: 18
  • all individual amino acids were replaced by all naturally occurring amino acids, except cysteine.
  • a heat map is a graphical representation of data where the values taken by a variable in a two-dimensional map are represented as colours.
  • a two-dimensional map can be derived from the independent sequences of the first and second loops.
  • the sequences of a given series of 16 CLIPS peptides are effectively permutations of 4 unique sub-sequences in loop I and 4 unique sub-sequences in loo 2.
  • the observed ELISA data can be plotted in a 4x4 matrix, where each X coordinate corresponds to the sequence of the first loop, and each Y coordinate corresponds to the sequence of the second loop.
  • ELISA values can be replaced with a continuous gradient of shading, in this case, extremely low values are light coloured, extremely high values are darke coloured, and average values are black coloured.
  • this gradient map is applied to a data matrix, a shaded heat map is obtained.
  • Table 11 summarises antibody binding conditions. For the Pepscan Buffer and Preconditioning (SQ), the numbers indicate the relative amount of competing protein (a combination of horse serum and ovalbumin).
  • FIG. 8 A graphical overview of the complete dataset of raw ELISA results generated by the screening is shown in Figure 8.
  • a box plot depicts each dataset and indicates the average ELISA signal, the distribution and the outliers within each dataset. Depending on experiment conditions (amount of antibody, blocking strength etc) different distributions of ELISA data are obtained.
  • Example 2 the stretch M8 VNPQGPGPEEK 358 (SEQ ID NO: 2) was found to suffice for binding some IgG from rabbit polyclonal Ab 137604. In this study all constructs containing (SEQ ID NO: 3) again were bound by IgG from tins sample at 1/2500 dilution, in the matrices of Set! there is no augmentation of signal m specific constructs.
  • residues P353, G354, and E356 are essential Limited substitutions are allowed at positions Q35 1 , G352, and 1/257.
  • Example 2 the stretch M SVNPQGPGPEEK 3S S (SEQ ID NO: 2) also sufficed to bind some IgG from goat polyclonal antiGPC-l.
  • This antibody recognizes the same loop as die rabbit polyclonal, but does so with a slightly different fine specificity.
  • the most critical residues are G352, G354, and E356, and that limited substitution is allowed on positions N349, Q35 L and P353.
  • the stretch around residues 140 - 149 was not implied in the primary mapping of this antibody, distinct constructs including this stretch are better baits for binding by some IgG in this polyclone.
  • Antibody MIL38-AM4 was not implied in the primary mapping of this antibody, distinct constructs including this stretch are better baits for binding by some IgG in this polyclone.
  • the antibodies bind to or not bind to similar constructs, as can be seen in the scatter plots of Figure 13. However, many constructs are exclusively seen by one of the preparations (points along the axes, or in the lower right hand and upper left hand comers).
  • Example 2 The two leads obtained in Example 1 that point to a discontinuous epitope for MIL 38 - AM4 were used to generate a matri array in which the loops have different lengths. In addition, full substitution analyses of the individual lead sequences were made. All arrays were probed with the three antibodies listed above.
  • ail antibodies investigated in this study bind to an epitope that exclusively or mainly consists of the flexible loop between residues 347 and 358.
  • the epitope of ⁇ some IgG species present in) rabbit polyclonal Ab 137604 is the linear stretch 34sVN.PQGPGPE.E3s7 (SEQ ID NO: 3). There is no indication for the presence of antibodies that recognize a conformational epitope.
  • Table 13 represents a summary of substitutions tolerated in the epitope sequences analysed.
  • Example 4 MIL3S and antl-glypkan-I (anti-GPC-l.) antibodies show overlappin reactivity on 2D western blots.
  • Rabbit anti-GPC-l antibody ab 137604 showed reactivity with the glypican-1 core protein at a molecular weight of approximately 60 fcDa - the same molecular weight as detected by MIL38.
  • prostate cancer DU- 145 MPEK extracts were subjected to 2D electrophoresis and western blotting.
  • Membrane protein extracts of DU-145 prostate cancer cells were separated on 2D gel (pi gradient -horizontal and molecular mass vertical). Western blots using MIL38 antibody and comniercial iGPC-l rabbit polyclonal antibodies show overlapping reactivity marking a 6QKd protein (circled in Figure 14).
  • Lane D is a one dimension separation for DU-145 extracts as a control.
  • Lane M is a one dimension separation lane for molecular size markers as controls.
  • M1L38 antibody and the anti-GPC-l antibodies showed overlappmg reactivity detecting a protein with 60 kDa molecular weight and isoelectric points ranging from 5 to 7.
  • MIL38 or rabbit anti-GPC-l antibodies were used to ioimuttopiecipitate their respective antigens from DU-145 or C3 ( IL38 negative) MPEK extracts.
  • the immnnoprecipitates (IPs) were western blotted witli either MIL38 or anti-GPC- l antibody ( Figure IS).
  • MIL38 and rabbit anti-GPC-l antibodies were each used to immunoprecipitate their antigens from DU-145 prostate cancer or C3 (MIL38 negative) celt membrane protei extracts. Shown are the western blots of the immunoprecipitations detected with either MIL38 or anti-GPC- l antibody.
  • Figure ISA depicts GPCl detection of MIL38 immunoprecipitate s (left) and MIL38 detection of GPC l immun precipitates (right).
  • Figure 15B depicts MIL38 detection of MIL38 immimoprecipitates as a control.
  • Lanes are: Magic Mark-commercial protein marker as control; DU 145 MPEK- prostate cancer membrane protein extract (not immunoprecipitated); 0U145 FT- prostate cancer flow through from immune precipitation; DU1 5 IP- immunoprecipitate using antibody; C3 MPEK- (M1L38 negative) control membrane protein extract (not inimunoprecipitated); C3 FT- flow through from immune precipitation; C3 IP ehttion- (MIL38 negative) immimoprecipiiate using antibody.
  • MIL38 can detect the immunoprecipitate from rGPCl antibody and vice versa.
  • M1L38 can also bind to all controls including DU145 MPE and to IP conducted by M1L38.
  • GPC-1 can be detected by MIL38 in prostate cancer plasma sam l s and in membrane extracts from prostate cancer patients.
  • Plasma samples from one normal patient (042) and one prostate cancer patient (046) were immunoprecipitated with MIL38 antibody and the IP sample western blotted with MIL38 and anti-GPC-1 antibodies ( Figure ⁇ .6 ⁇ ).
  • Example 7 Detection of 1L38 antigen in patient urine.
  • MIL38 can detect cells in the urine of prostate cancer patients. To test the sensitivit and specificity of this detection method, 125 age-matched urine samples were obtained. Cells were spun down from the urine and analyzed by the MIL38 indirect immunofluorescence assay. A total of 47 healthy controls, 37 benign prostatic hypertrophy (BPH) and 41 biopsy-confirmed prostate cancers were analyzed.
  • BPH benign prostatic hypertrophy
  • the M1L38 immunofluorescence assay ( F A) test demonstrated a sensitivity of 73 % and a specificity of 73% in identifying prostate cancers within the cohort. The test showed 71% sensitivity and 76% specificity in identifying prostate cancers compared to BPH patients (Table 14). Table .14: Sensitivity a i specificity calculations of prostate cancer detection in patient urine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Oncology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
PCT/AU2015/000019 2015-01-16 2015-01-16 Glypican epitopes and uses thereof Ceased WO2016112423A1 (en)

Priority Applications (13)

Application Number Priority Date Filing Date Title
JP2017537906A JP6735005B2 (ja) 2015-01-16 2015-01-16 グリピカンエピトープおよびその使用
DK15877381.2T DK3245219T3 (da) 2015-01-16 2015-01-16 Glypicanepitoper og anvendelser deraf
AU2015376851A AU2015376851B2 (en) 2015-01-16 2015-01-16 Glypican epitopes and uses thereof
EP15877381.2A EP3245219B1 (en) 2015-01-16 2015-01-16 Glypican epitopes and uses thereof
US15/543,877 US20180002391A1 (en) 2015-01-16 2015-01-16 Glypican epitopes and uses thereof
PCT/AU2015/000019 WO2016112423A1 (en) 2015-01-16 2015-01-16 Glypican epitopes and uses thereof
ES15877381T ES2805085T3 (es) 2015-01-16 2015-01-16 Epítopos de glipicano y usos de éstos
CN201580076839.6A CN107531755B (zh) 2015-01-16 2015-01-16 磷脂酰肌醇蛋白聚糖表位及其用途
CA2973771A CA2973771A1 (en) 2015-01-16 2015-01-16 Glypican epitopes and uses thereof
SG11201705808XA SG11201705808XA (en) 2015-01-16 2015-01-16 Glypican epitopes and uses thereof
KR1020177022547A KR102435383B1 (ko) 2015-01-16 2015-01-16 글리피칸 에피토프 및 이의 용도
US16/939,729 US20210107958A1 (en) 2015-01-16 2020-07-27 Glypican epitopes and uses thereof
US18/361,407 US20240247037A1 (en) 2015-01-16 2023-07-28 Glypican epitopes and uses thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/AU2015/000019 WO2016112423A1 (en) 2015-01-16 2015-01-16 Glypican epitopes and uses thereof

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US15/543,877 A-371-Of-International US20180002391A1 (en) 2015-01-16 2015-01-16 Glypican epitopes and uses thereof
US16/939,729 Continuation US20210107958A1 (en) 2015-01-16 2020-07-27 Glypican epitopes and uses thereof

Publications (1)

Publication Number Publication Date
WO2016112423A1 true WO2016112423A1 (en) 2016-07-21

Family

ID=56405046

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2015/000019 Ceased WO2016112423A1 (en) 2015-01-16 2015-01-16 Glypican epitopes and uses thereof

Country Status (11)

Country Link
US (3) US20180002391A1 (enExample)
EP (1) EP3245219B1 (enExample)
JP (1) JP6735005B2 (enExample)
KR (1) KR102435383B1 (enExample)
CN (1) CN107531755B (enExample)
AU (1) AU2015376851B2 (enExample)
CA (1) CA2973771A1 (enExample)
DK (1) DK3245219T3 (enExample)
ES (1) ES2805085T3 (enExample)
SG (1) SG11201705808XA (enExample)
WO (1) WO2016112423A1 (enExample)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016168885A1 (en) * 2015-04-20 2016-10-27 Minomic International Ltd. Therapeutic antibodies and uses thereof
KR20170085051A (ko) * 2014-10-23 2017-07-21 미노믹 인터내셔널 리미티드 단클론성 항-gpc-1 항체 및 이의 용도
WO2020154150A1 (en) * 2019-01-22 2020-07-30 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services High affinity monoclonal antibodies targeting glypican-1 and methods of use
EP3617231A4 (en) * 2017-04-28 2021-04-07 National University Corporation Kochi University ANTI-GPC-1 ANTIBODY
IT202200021546A1 (it) 2022-10-19 2024-04-19 Centro Di Riferimento Oncologico Di Aviano Anticorpo monoclonale anti-GPC1, suoi usi terapeutici e diagnostici
WO2025202359A1 (en) * 2024-03-27 2025-10-02 Adcendo Aps Anti-gpc1 antibodies and uses thereof
WO2025202361A1 (en) * 2024-03-27 2025-10-02 Adcendo Aps Anti-gpc1 antibodies and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111187350A (zh) * 2019-12-30 2020-05-22 西安英创生物技术有限公司 一种与磷脂酰肌醇蛋白聚糖-1结合的抗原结合蛋白

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4356270A (en) 1977-11-08 1982-10-26 Genentech, Inc. Recombinant DNA cloning vehicle
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4751180A (en) 1985-03-28 1988-06-14 Chiron Corporation Expression using fused genes providing for protein product
US4935233A (en) 1985-12-02 1990-06-19 G. D. Searle And Company Covalently linked polypeptide cell modulators
WO1999037764A2 (en) 1998-01-27 1999-07-29 Vlaams Interuniversitair Instituut Voor Biotechnologie New members of the glypican gene family
US20040226056A1 (en) * 1998-12-22 2004-11-11 Myriad Genetics, Incorporated Compositions and methods for treating neurological disorders and diseases
US20130016736A1 (en) 2008-08-11 2013-01-17 Gavin Parnaby Method of synchronization for low power idle

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5840839A (en) * 1996-02-09 1998-11-24 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Alternative open reading frame DNA of a normal gene and a novel human cancer antigen encoded therein
CA2346264A1 (en) * 1998-10-16 2000-04-27 Regents Of The University Of California Glypicans for the detection and treatment of human carcinoma
EP2208784B1 (en) * 2001-06-22 2013-01-02 Chugai Seiyaku Kabushiki Kaisha Cell proliferation inhibitors containing anti-glypican 3 antibody
DE602005024502D1 (de) * 2004-07-09 2010-12-16 Chugai Pharmaceutical Co Ltd Anti-glypican-3-antikörper
US20070087005A1 (en) * 2005-10-14 2007-04-19 Lazar Gregory A Anti-glypican-3 antibody
CN102180969B (zh) * 2011-01-30 2013-04-10 中国人民解放军军事医学科学院微生物流行病研究所 抗肝癌活性单克隆抗体及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4356270A (en) 1977-11-08 1982-10-26 Genentech, Inc. Recombinant DNA cloning vehicle
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4751180A (en) 1985-03-28 1988-06-14 Chiron Corporation Expression using fused genes providing for protein product
US4935233A (en) 1985-12-02 1990-06-19 G. D. Searle And Company Covalently linked polypeptide cell modulators
WO1999037764A2 (en) 1998-01-27 1999-07-29 Vlaams Interuniversitair Instituut Voor Biotechnologie New members of the glypican gene family
US20040226056A1 (en) * 1998-12-22 2004-11-11 Myriad Genetics, Incorporated Compositions and methods for treating neurological disorders and diseases
US20130016736A1 (en) 2008-08-11 2013-01-17 Gavin Parnaby Method of synchronization for low power idle

Non-Patent Citations (35)

* Cited by examiner, † Cited by third party
Title
"Antibodies-A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
"Anti-GPC antibody HPA030571", 26 March 2010 (2010-03-26), XP055461315, Retrieved from the Internet <URL:http://www.proteinatlas.org/ENSG00000063660-GPC1/antibody> [retrieved on 20150211] *
"Current Protocols in Molecular Biology", 2007, JOHN WILEY AND SONS, INC.
"GenBank", Database accession no. AAA98132.1
"GenBank", Database accession no. AAH51279.1
"GenBank", Database accession no. EAW71184.1
"Goodman and Gilman's: the pharmacological basis of therapeutics", 1990, PERGAMON PRESS
"Molecular Cloning: A Laboratory Manual", COLD SPRING HARBOR LABORATORY PRESS
"NCBI", Database accession no. NP 002072.2
"Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING CO.
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1989, GREEN PUBLISHING ASSOCIATES AND WILEY INTERSCIENCE
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", vol. 1, 1994, JOHN WILEY & SONS
AUSUBEL F. M. ET AL.: "Current Protocols in Molecular Biology", 2007, JOHN WILEY AND SONS, INC
BEAUCAGE S.L ET AL., TETRAHEDRON LETTERS, vol. 22, 1981, pages 1859 - 1862
BROWN, E. L. ET AL., METH. ENZYMOL., vol. 68, 1979, pages 109
COLE ET AL.: "Monoclonal Antibodies and Cancer Therapy", 1985, ALAN R. LISS, INC., pages: 77 - 96
DATABASE UniProt 1 February 1994 (1994-02-01), XP055455319, Database accession no. P35052 *
HAMMERLING ET AL.: "Monoclonal Antibodies and T-cell Hybridomas", 1981, ELSEVIER/NORTH-HOLLAND BIOCHEMICAL PRESS
KENNETT ET AL.: "Monoclonal Antibodies", 1980, PLENUM PRESS
KOHLER ET AL.: "Continuous cultures of fused cells secreting antibody of predefined specificity", NATURE, vol. 256, 1975, pages 495 - 497, XP002024548
KOZBOR ET AL.: "The Production of Monoclonal Antibodies From Human Lymphocytes", IMMUNOLOGY TODAY, vol. 4, 1983, pages 72 - 79, XP023942945, DOI: doi:10.1016/0167-5699(83)90123-8
M. SCHREIER ET AL.: "Hybridoma Techniques", 1980, COLD SPRING HARBOR LABORATORY
MANIATIS ET AL., MOLECULAR CLONING, 1982, pages 280 - 281
MARATEA ET AL., GENE, vol. 40, 1985, pages 39 - 46
MCGILL ET AL., THE AMERICAN STATISTICIAN, vol. 32, 1978, pages 12 - 16
MERRIFIELD, J. AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2146
MURPHY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 8258 - 8262
NARANG S. A. ET AL., METH. ENZYMOL., vol. 68, 1979, pages 90
POSTHUMUS ET AL., J. VIROLOGY, vol. 64, 1990, pages 3304 - 3309
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOKRUSSELL: "Molecular Cloning: A Laboratory Manual", 15 January 2001, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP3245219A4
SUHOVSKIH, A. ET AL.: "Proteoglycan Expression in Normal Human Prostate Tissue and Prostate Cancer.", ISRN ONCOLOGY, vol. 2013, 2013, pages 680136, XP055357331 *
TIMMERMAN ET AL., J. MOL. RECOGNIT., vol. 20, 2007, pages 283 - 29

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102249981B1 (ko) 2014-10-23 2021-05-11 미노믹 인터내셔널 리미티드 단클론성 항-gpc-1 항체 및 이의 용도
EP3209686A4 (en) * 2014-10-23 2018-03-07 Minomic International Ltd. Monoclonal anti-gpc-1 antibodies and uses thereof
US10577418B2 (en) 2014-10-23 2020-03-03 Minomic International Ltd. Monoclonal anti-GPC-1 antibodies and uses thereof
KR20170085051A (ko) * 2014-10-23 2017-07-21 미노믹 인터내셔널 리미티드 단클론성 항-gpc-1 항체 및 이의 용도
WO2016168885A1 (en) * 2015-04-20 2016-10-27 Minomic International Ltd. Therapeutic antibodies and uses thereof
US11198738B2 (en) 2015-04-20 2021-12-14 Minomic International Ltd. Therapeutic antibodies and uses thereof
EP3617231A4 (en) * 2017-04-28 2021-04-07 National University Corporation Kochi University ANTI-GPC-1 ANTIBODY
WO2020154150A1 (en) * 2019-01-22 2020-07-30 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services High affinity monoclonal antibodies targeting glypican-1 and methods of use
CN113784987A (zh) * 2019-01-22 2021-12-10 美国政府(由卫生和人类服务部的部长所代表) 靶向磷脂酰肌醇蛋白聚糖-1的高亲和力单克隆抗体和使用方法
US12122843B2 (en) 2019-01-22 2024-10-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services High affinity monoclonal antibodies targeting glypican-1 and methods of use
WO2024083921A1 (en) 2022-10-19 2024-04-25 Centro Di Riferimento Oncologico Di Aviano Anti-gpc1 monoclonal antibody, therapeutic and diagnostic uses thereof
IT202200021546A1 (it) 2022-10-19 2024-04-19 Centro Di Riferimento Oncologico Di Aviano Anticorpo monoclonale anti-GPC1, suoi usi terapeutici e diagnostici
WO2025202359A1 (en) * 2024-03-27 2025-10-02 Adcendo Aps Anti-gpc1 antibodies and uses thereof
WO2025202361A1 (en) * 2024-03-27 2025-10-02 Adcendo Aps Anti-gpc1 antibodies and uses thereof

Also Published As

Publication number Publication date
US20240247037A1 (en) 2024-07-25
EP3245219B1 (en) 2020-04-08
EP3245219A1 (en) 2017-11-22
EP3245219A4 (en) 2018-06-06
CA2973771A1 (en) 2016-07-21
KR20170109584A (ko) 2017-09-29
DK3245219T3 (da) 2020-06-29
AU2015376851A1 (en) 2017-07-27
SG11201705808XA (en) 2017-08-30
US20210107958A1 (en) 2021-04-15
JP6735005B2 (ja) 2020-08-05
JP2018504903A (ja) 2018-02-22
ES2805085T3 (es) 2021-02-10
CN107531755B (zh) 2022-02-25
CN107531755A (zh) 2018-01-02
AU2015376851B2 (en) 2020-07-09
US20180002391A1 (en) 2018-01-04
KR102435383B1 (ko) 2022-08-24

Similar Documents

Publication Publication Date Title
US20240247037A1 (en) Glypican epitopes and uses thereof
US20230391886A1 (en) Compositions and methods for muc18 targeting
JPWO2004081047A1 (ja) モノクローナル抗体及びこれを産生するハイブリドーマ
KR20150041801A (ko) 복합체-특이적 항체 및 항체 단편 및 그의 용도
ES2661516T3 (es) Biomarcadores
CN113203858B (zh) 一种肿瘤检测试剂盒
JP2019510506A (ja) 抗ポドカリキシン様タンパク質前駆体サブタイプ２モノクローナル抗体とその生成方法及び使用
JP7654591B2 (ja) インターロイキン－１９を標的とする化合物および方法
EP2700650B1 (en) Monoclonal antibodies which specifically recognize human liver-carboxylesterase 1, hybridoma cell lines which produce monoclonal antibodies, and uses thereof
WO2019098133A1 (ja) モノクローナル抗体及びその使用
JP7053058B2 (ja) グリピカンエピトープおよびその使用
JP6861349B2 (ja) Ｃｄ８１ ｌｅｌ特異的モノクローナル抗体
JP2025506203A (ja) 抗ｈｅｒ２／ｎｅｕ抗体および使用方法
KR20120028603A (ko) 돼지인플루엔자 바이러스 헤마글루티닌 검출용 항체 및 이의 용도
KR20220055423A (ko) SARS-CoV-2 특이적 신규 항체를 이용한 SARS-CoV-2의 검출방법
US20210024644A1 (en) N-cadherin binding molecules and uses thereof
JP6099393B2 (ja) オートタキシンアイソフォーム特異的抗体および検出方法
CN108727493B (zh) 抗Stathmin单克隆抗体及其用途
JPWO2012008595A1 (ja) 抗ｌｒ１１抗体の製造法及び免疫学的測定用標準品
US20240309110A1 (en) Combotope Antibody Libraries
HK1238267B (en) Glypican epitopes and uses thereof
US10538580B2 (en) Anti-equol antibody composition and use therefor
WO2025043244A1 (en) Tandem antigen capture assays for measuring qsox1 isoforms
KR20230135087A (ko) 항-mcl1 항체를 투여하여 암을 모니터링하기 위한 물질및 방법
JP2017043595A (ja) モノクローナル抗ＡＧＥｓ抗体及びその製造方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15877381

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2973771

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 11201705808X

Country of ref document: SG

Ref document number: 15543877

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2017537906

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2015877381

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2015376851

Country of ref document: AU

Date of ref document: 20150116

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20177022547

Country of ref document: KR

Kind code of ref document: A