CA2973771C - Glypican epitopes and uses thereof - Google Patents

Abstract

The present invention relates to epitopes of glypican-1 (GPC-1) and uses thereof.

Description

WO 2016/112423 PCT/ A U2015/000019 GLYPICAN EPITOPES AND USES THEREOF Technical Field The present invention relates generally to the fields of immunology and medidne.

More specifically, the present invention relates to epitopes of glypican-1 (GPC>l) and uses thereof Background Prostate cancer is the most commonly occurring cancer in men of all races, and is second only to lung can.cer in tnortality among among white, black, American Indian/Alaska Native, and Hispanic men_ [n 201 l, 209,292 men in the United States of America \,Vere diaf,,J-Jlosed with prostate cancer and 27,970 of these died from the disease (U.S.

Cancer Statistics Working Group, "United Stauw Cancer Statistics: J 999---2011 Incidence and Mortali(v fVcb-based Report'', Atlanta (GA): Department of Health and Hun:mn Services, Centers for Disease Control and Prevt~ntion, and National Cancer Institute; 2014).

Treatment with surgery and/or radiotherapy is successful 111 many patients if prostate cancer is diagnosed early.

Hov,,rever, many patients with advanced disease and a sizeable proportion of all prostate cancer patients eventually develop metastatic disease following localised therapy.

A need thus exists for convenient, reliable and accurate tests for diabrnosing prostate cancer, especially during the early stages of the disease_ Glypican-1 (GPC-1) is a cell su.rfrice heparan sulfate proteoglycan with a core protein that is anchored to the cytoplasmic membrane via a glycosyl phosphatidylinoshol.

It is a member of a larger family of glypicans. GPC-1 has been reported to be overexpressed in some fom1S of cancer (e.g. pancreatic cancer, breast cancer), but expression does not significantly differ in others_ The present inventors have recently determined that GPC- l is overexpressed by prostate cancer cells, and can be used as a means of diag11osing the disease (US provisional patent application no. 61/928/776 entitled "Cell ,5wfhce Prostate Cancer Anr~gen .fbr Diagnosis··, \Valsh et al. - Ullpublished; PCT application number PCT/AU20l.4/000999 "Monoclonal A}./TJ-GPC-1 antibodies and uses thenxf", Walsh et al. - unpublished_).

In vievv of the association bet.,veen CiPC-l levels and. prostate cancer determined by the present inventors, a need exists fr)J the identification of epitopes within GPC-.1 that are advantageous for detecting and quantifying GPC-1 levels in persons undergoing d.iagnostic and/or prognostic tests.

Summary of the Invention The present inventors have detennined that a series of epitopes within the GPC-1 protein are preferably targekd by binding entities including, but not limited to, antibodies, Accordingly, the present invention reh~tes to at least the follo\ving embodiments: Embodiment 1: An epitope for an anti-glyJ)ican 1 (GPC-1) antibody located within a portion of the GPC-1 t1exihle loop defined by an amino acid sequence KVNPQGPGPEEK (SEQ ID NO: 1).

Embodiment 2: TI1e epitope according to embodiment ! , ,vherein the epitope comprises a first segment comprising an amino add sequence selected from: (i) VNPQGPGPEEK (SEQ ID NO: 2)~ or (ii) a variant of VNPQGPGPEEK (SEQ ID NO: 2), wherein tbe variant comprises a substituted amino acid residue only at any one or more of positions 2, 3, 7, 8, 9, 10 and/or 1 t; or any one or more of positions 1, 2, 3, 4, 6, 8, ] 0 and/or 11; or any one or more of positions I, 2, 3, 4, 5, 8, l O and/or 11.

Embodiment 3: l11e epitope according to embodiment l \vherein the epitope comprises a first segment comprising an amino acid sequence selected from: (i) K VNPQGPGP (SEQ ID NO: 6); or (ii) a variant ofKVNPQGPGP (SEQ ID NO: 6) comprising a substituted amino acid residue only at any one or more of positions l, 3, 4, 8, and 9 of SEQ ID NO: 6.

Embodiment 4: l11e 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 5: The epitope according to embodiment 1, wherein the epitope comprises a first segmeut comprising an amino acid sequence selected from: (i) KVNPQGPGPE (SEQ ID NO: 5); or (ii) a variant of KVNPQGPGPE (SEQ ID NO: 5) comprising a substituted amino acid residue only at any one or more of positions J, 3, 4, 8, 9 and IO of SEQ ID NO: 5.

Embodiment 6: T11e variant according to embodiment 5, comprising a substituted amino acid residue only at any one or more of positions 8, 9 and l O of SEQ ID NO: 6, Embodiment 7: The variant according to enibodiment 5 or embodiment 6, \Vherein E (glu) at position 10 is substituted ivith any other amino acid. 2 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: position 1, \vherein K (lys) is substih1ted ,vith any one of W (trp), R (axg), L (lys), Y (tyr) or F (phe ); position 3, wherein N (asn) is substituted with any one ofH (his), P (pro) or D (asp); position 4, wherein P (pro) is substituted with any one of R (arg), K (lys), W (trp), S (ser), H (his) or N (asn); position 8, ,vherein G (gly) is substituted with any one of D (asp), E (glu\ N (asn), Q (gin), K (lys), R (arg) or A (ala); position 9, \Vherein P (pro) is substituted \Vith any one of M (met). A (ala), [ (ile), K (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 an amino acid sequence TQNARA (SEQ ID NO: 8).

Elnbodiment 10: The epitope according to any one of embodiment<; l to 8, comprising a second segment comprising an amino acid sequence TQNARAFRD (SEQ ID NO: 7).

Embodiment 11: The epitope according to embodiment I or embodiment 2, Yvherein the epitope comprises a first segment comprising an amino acid sequence selected from: (i) NPQGPGPEE (SEQ lD NO: 4); or (ii) a variant of NPQGPGPEE (SEQ JD NO: 4), wherein the variant comprises a substituted amino add residue only at any one or more of positions l, 2, 3, 5, 7 and 9 of SEQ ID NO: 4.

Embodiment 12; TI1e variant according to embodiment 11, 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: TI1e variant according to embodiment ll or embodiment 12, wherein the variant comprises a substitution at any one or more of position l, wherein N (asn) is substituted with H (his); position 2, wherein P (pro) is substituted with any other amino acid; position 3, wherein Q (gln) is substituted ,-vith a11y one of N (asn), M (1net), T (thr), S (ser), or R (arg); position 5, wherein P (pro) is substituted vvith A (ala); position 7, wherein P (pro) is substituted with any one of A (ala), D (asp), C (cys), E (glu). Z (glx), G (gly), H (his), K (lys), Jvl (met), F (phe), P (pro), S (ser), T (thr), W (trp), or Y (tyr); position 9, \.\'herein E (g!u) is substituted with any other amino acid. 3 Embodiment .l 4: The epitope according to any one of embodiments 11 to 13, wherein the epitope comprises a second segment comprising an amino acid sequeuce i\LSTASDDR (SEQ ID NO: 9).

Embodiment 15: T11e epitope according to embodiment I. or embodiment 2, ,vherein the epitope comprises a first segment comprising an amino acid sequence selected from: (i) VNPQGPGPEE (SEQ ID NO: 3); or (ii) a variant of VNPQGPGPEE (SEQ ID NO: 3), wherein the vadant comprises a substituted amino acid residue only at any 01.1e or more of positions 1, 2, \ 4, 5, 8 and 10 of SEQ IDN0: 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 JD NO: 3.

Embodiment 17: The variant according t,1 embodiment 15 or embodiment 16, wherein the variant comprises a substitution at any one or more of position I, wherein V (val) is substituted with any other amino acid; position 2, w·herein N (asn) is substituted with any other amino acid; position 3, wherein P (pro) is substituted with any other amino acid; position 4, wherein Q (gln) is substituted 1-vith any one of Y (tyr), A (ala), E (glu), V (val), M (met), F (phe), L (leu), I (ile), T (thr), or R (arg); position 5, wherein G (gly) is substituted with A (ala), S (ser), T (thr), H (his),. \V (trp), Y (tyr), F (phe), or M (met); position 8, wherein P (pro) is substituted with any other amino acid:, position 10, wherein E (glu) is substituted with Q (gln), D (asp), F (phe), H (his) or ]VT (met).

Embodiment 18: 111t~ ep.itope according to any one of embodiments 15 to 17, wherein the epitope comprises a second segment comprising: an amino add sequence PRERPP (SEQ ID NO: 10) or an amino acid sequence QDASDDGSGS (SEQ ID NO: 11 ).

Embodime.nt 19: The epitope according to any one of ernbodiments 15 to 17., wherein the epitope comprises a second segment comprising an amino add sequence PRERPP (SEQ 1D NO: IO) and a third segment comprising an amino acid sequence QDASDDGSGS (SEQ JD NO: l lJ.

Embodiment 20: The epitope according to any one of embodiments 1 to l 0, comprising the amino acid sequence CGELYTQNARAFRDLCGNPKVNPQGPGPEEKRRRGC (SEQ ID NO: 12). 4 Embodiment 21 : The epitope according to any one of embodiments l. to 20, wherein the epitope is a linear epitope.

Embodiment 22: The epitope according to embodiment 19, w·herein the second segment and the third segment of the epitope are discontinuous.

Embodiment 23: The epitope according to any one of embodiments 9, IO, 14, 18 or 22 \Vherein the first st.~gment and the serond segmt!nt of the epitope are discontinuous.

Embodiment 24: An epitope for an anti-glypictm l (GPC-1) antibody comp1ising an amh10 acid sequence selected from any one or a plurality of: TQNARA (SEQ lD NO: 8), ALSTASDDR (SEQ ID NO: 9), PRERPP (SEQ ID NO: IO), QDASDDGSGS (SEQ ID NO: 11), LGPECSRAVMK (SEQ ID NO: 13\ and TQNARAFRD (SEQ 1D NO~ 7).

Embodiment 25: TI1e epitope according to any 011e of embodiments I to 24, wherein the epitope is an isolated poly1)eptide 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 acc()rding to any one of embodiments l to 25; each said epitope is an epitope according to Table l; or the combination of epitopes is any one or more of the combinations set out in Table 3.

Embodiment 27: An atrangement of epitopes comprising a combination of t1,vo or more distinct epitopes, wherein the arrangement comprises a first epitope according to any one of embodiments 1 to 10, and a second epitope accoriling to any one of embodiments 11 to 14.

Embodiment 28; An an-angement of epitopes comprising a combination of two or more distinct epitopes, wherein the arrangement comprises a first epitope according to any one of embodiments l to 10, and a second epitope acconling to any one of embodiments l 5 to 19.

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 phannaceuticaHy acceptable cmrier or exc1pieni.

Etnbodin1ent 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 enzyn1e-linked irnmunosorbent assay (ELIS.A).

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 embodirnent 32, Embodiment 34: A host cell comprising the vector according to embodiment 33.

Embodiment 35: An isolated binding entity 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 emity capable of specifically binding to an epitope according to any one of embodiments l to 25, wherein the binding entity is an antibody and with the proviso that the antibody is not a: l\UL38 antibody (CBA20140026), rabbit anti-GPC-l po1yclonal antibody (abl37604, abeam), mouse anti-glyvicm1 monoclonal antibody 2600 clone 4Dl (Millipore), or goat anti-glj1)ican 1 antibody (AA. 24-530).

Embodiment 37; A method for detecting prostate cancer in a subject, the rnethod comprising obtaining a biological sample from the subject, detecting the presence of an epitope according to any one of embodiments l to 25 in the sample, and determining that the subject has prostate cancer or an increased likelihood of developing prostate cancer based on an1ou.nt 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 l to 25.

Embodiment 39: The method according to embodiment 38, \Vherein the binding entity is a population of antibodies, Embodiment 40; The method according to embodiment 39, wherein the population of antibodies comprises any one or more of: MIL38 antibody (CBA20140026), rabbit anti-GPC- 1 polyclona1 antibody (abl37604, abeam), mouse anti-glypican monodonal antibody 2600 clone 401 (Milliporet or goat anti-glypican 1 a.ntibody (AA 24-530).

Etnbodit-nent 41: The method according to embodiment 39, wherein the population of antibodies does not contain any of lVllL38 antibody (CBA20140026), rabbit anti-GPC-! polydonal antibody (ab!37604, abeam), mouse anti-glypicru1 monocloual antibody 2600 clone 4D l (Millipore), or 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 epitupe prest:nt in the biological sample with an arnuunt 6 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 ,ktected in the sample is increased by more than 50% over the amount of epitope detected in the control san1ple.

Embodiment 44: The method according to any one of embodirnents 37 to 41, wherein detecting the presence of the epitope comprises contacting the sample with a population of MIL38 antibodies as deposited at Cellbank Australia under accession number CBA20140026.

Embodiment 45: TI1e method according to any one of embodiments 37 to 43, wherein detecting the presence of the t~pitope comprises contacting the sample with a population of antibodies that does not comprise an antibody comprising a light chain variable region comprising: a complementarity determining region l (CDRl) comprising or consisting of an amino acid sequence defined by positious 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 detem1ini11g region 3 (CDR3) comprising or consisting of an amino acid sequence defined by positions l 13-12! of SEQ JD NO: 20.

Embodiment 46: The rnethod according to any one of embodiments 37 to 45, fwther comprising determining the level of prostate-specific antigen (PSA) in the biological sample and comparing the level dete,:ted to that of the control sample.

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 methotl acCQrding to any one of embodiments 37 to 46, \vhere.in the biological sample is a tissue sample.

Embodiment 49: A fusion protein compnsmg the epitope according to any one of embodiments l to 25.

Etnbodin1ent 50: Use of the epitope according to any one of embodiments l to 25, the arrangement of epitopes according to any one of embodiments 26 to 28, or the fusion protein according to embodiment 48, as a positive control element in a method for detecting GPC- l.

Embodiment 51: The use according to embodiment 50, \Vherein the method is the method for detecting prostate cancer according to any one of embodiments 3 7 to 48_ 7 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 \¾·holly validate that the method or assay is capable of fimctioning con-ecdy.

Brief Description of the Figures Preferred ernbodiments of the present invention wfll now be described, by \Nay of example only, \vith reference to the accompanying figures wherein: Figure 1 shows a rendering of chain B as present in Protein Data Bank identifier (PBD 1D) 4AD7 (http://wwv,r.ebi.ac.uk/pdbsum/4AD7); Figure 2 shov>'s box plot graphs of raw data of i\,1IL38-AM4 antibody screening.

The bottom and top of the boxes are the 25th and 75th percentile of the data.

The band near the n1iddle of the box is the 50th percentile (the median).

The "vhiskers am at 1.5 the interquantile range, an indication of statistical outliers ,vithin the dataset (Mcgill et al., The American Statistician, 32: l 2-16, 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 }tg/ml (top trace) or 10 p.g/ml of MlL38-AM4 (bottom trace); Figure 4 shows rendering of chain B from Protein Data Bank identifier (PBD ID) 4AD7 (http://w-ww.ehi.ac.uk/pdbsum/4AD7) with sequence TQNARAFRDLYS (SEQ ID NO: 21) in dark-coloured spheres, and residues K347, V348, G362, and K363 in lightcoloured spheres.

The loop cm:mecting V348 to G362 is not resolved from the X- ray diffraction; Figure 5 shows box plot graphs of rm.v data of polyckmal and monoclonal anti-GPC-1 and MIL38-AM3 antibody screening. T11e 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 l .5 the inter-quantile range, an indication of statistic.al outliers ,vithin the dataset (Mcgill et al_, TI1e American Statistician, 32: 12-16, 1978); Figure 6 shows plots of the peptide number versus the recorded intensity obtained for three commercially avai.lable anti-glypican l prnparations tested on the array used to map MIL38-AM4, which is shO\vn for comparison; Figure 7 shows rendering of chain B from Protein Data Bank identifier (PBD ID) 4AD7 (http://\vww.ebi.ac.uk/pdbsum/4AD7).

Figure 7A shows rendering of chain B ,vith sequence PRERPP (SEQ ID NO: 10) in light-shaded spheres, and residues K347, V348, 8 G362, and K363 in dark-shaded spheres.

The loop connecting V348 to G362 is not resolved from the X-rny diffraction.

These epitopes are recognized by rabbit anti-GP(>.!.

An extra epitope, QDASDDGSGS (SEQ ID NO: 11 ), is not resolved in d1e coordinate file ..

Figure 7B shmvs 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 shmvs box plot _b}faphs of raw data of antibody scret~ning.

The bottom and top of the boxes are the 25th and 75th percenlile of the tfata.

The band near the middle of the box is the 50th percentile (the rnedian).

The whiskers are at 1.5 the inter-quantile range, an indication of statistical outliers within the dataset (Mcgill et al., The American Statistician, 32: 12-16, 1978); Figure 9 shows a lettei:plot representation of rabbit polydona! Ab l37604 probed on the substitution analysis of set 3.

The central/horizontal bar represents the mean fotensity recorded for the base sequence.

Individual mut11tions are drav..n in the rnlumn corresponding to the position of the mutation, and symbols a.re plotted at the height con-esponding to the recorded intensity; F'igure 10 shows a letterplot representation of goat polyclonal allti-GPC-1 probed on the substitution analysis of set 2 (Example 3); Figure 11 sho'-VS a letterplot representation of MIL38~AM4 probed on the substitution analysis of set .3 (Example 3); Figure 12 shows a heatma_p representation of the data obtained for MIL38-AJvt4 probed on the peptides of set l (Example 3 ).

Sig-nal intensity runs from black (baseline) to ,vh1te (mean) to grey (max); 'X' = x-axis; x-axis peptide sequences depicted in colunms X1-X33 are as follov/s: X-1 peptide is residues 344 - 353 of SEQ ID NO: 1; X-2 peptide is residues 348 - 357 of SEQ ID NO: l; X-3 peptide is residues 352 - 361 of SEQ ID NO; l; X-4 peptide is residues 356 - 365 of SEQ JD NO: 1; X-5 peptide is residues 344 - 354 of SEQ ID NO: l; X-6 pt!ptide is residues 348 - 358 of SEQ ID NO: 1; X-7 peptide is residues 352 - 362 of SEQ ID NO: l; X-8 peptide is residues 356 - 366 of SEQ ID NO: l; X-9 peptide is residues 344 - 355 of SEQ ID NO: l; X-10 peptide is residues 348 - 359 of SEQ ID NO: 1; X-11 peptide is residues 352 - 363 of SEQ 1D NO: 1; X-12 peptide is residues 344 - 356 of SEQ ID NO: l; X-13 peptide is residues 348 - 360 of SEQ JU NO: l; X-14 peptide is residues 352 - 364 of SEQ ID NO: 1:_ X-15 peptide is residues 344 - 357 of SEQ ID NO: l; X-16 peptide is residues 348 - 361 of SEQ ID NO: l; X-17 peptide is residues 352 - 365 of SEQ lD NO: 1; X-18 peptide ts residues 344 - 358 of SEQ ID NO: l; X-19 peptide is residues 348 - 362 of SEQ ID NO: 1; X-20 peptide is residues: 352 - 366 of SEQ lD NO: l; X-2 l peptide is residues 344 - 359 of SEQ ID NO: l; X-22 peptide is residues 348 - 363 of 9 SEQ ID NO: 1~ X-23 peptide is residues 344 - 360 of SEQ ID NO: 1; X-24 peptide is residues 348 • 364 of SEQ JU NO: I; X-25 peptide is residues 344 - 361 of SEQ ID NO: J; X-26 peptide is residues 348 - 365 of SEQ lD NO: l; X-27 peptide is residues 344 - 362 of SEQ ID NO: l; X-28 peptide is residues 348 - 366 of SEQ JD NO: 1; X-29 peptide is residues 344 - 363 of SEQ ID NO: 1; X-30 peptide is residues 344 ~ 364 of SEQ ID NO: 1; X-31 peptide is n$idues 344 - 365 of SEQ ID NO: 1; X-32 peptide is residues 344 - 366 of SEQ ID NO: 1, X-33 peptide is a scramble/random sequence; 'Y' '" y-axis~ y-axis peptide sequences depicted in columns Yl-Y19 are as fi:.)llov;rs_ Y-1 peptide is residues 131 - 140 of SEQ lD NO: l; Y-2 peptide is residues 135 - 144 of SEQ ID NO: l; Y-3 peptide is residues 139 - 148 of SEQ 1D NO: 1; Y-4 peptide is residues 131 - 141 of SEQ ID NO: 1; Y-5 peptide is residues 135 - 145 of SEQ lD NO: I; Y-6 peptide is residues 139 - .149 of SEQ ID NO: I; Y-7 peptide is residues 131 -142 of SEQ lD NO: 1; Y-8 peptide is residues 135 - !46 of SEQ ID NO: 1; Y-9 peptide is residues 131 - 143 of SEQ ID NO: l; Y-10 peptide is residuesl35 - 147 of SEQ ID NO: l; Y-11 peptide is residues Bl - 144 of SEQ ID NO: l; Y-12 peptide is residues 135 - 148 of SEQ JD 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.: l; Y-16 peptide is residues 131 - 147 of SEQ ID NO: 1; Y-17 peptide is residues 13 l - I 48 of SEQ JD NO: 1; Y-18 peptide is residues 131 - 149 of SEQ ID NO: 1 ~ Y - l 9 peptide is a scramble/random sequence.

Figure 13 is a scatter plot matrix of all results obtained on the peptides of set l; Figure 14 shows results of 2D electrophoresis and western blotting which demonsu·ate that MIL38 and anti-GPC-l antibodies show overlapping reactivity on 2D gel western blot; Figure 15 shows western blots of immunoprecipitations conducted on DU-145 prostate cancer or C3 (MIL38 negative) cell membnme protein extracts with either MIL38 or antiGPC- 1 antibodies; and Figure 16 shows western blots of immunoprecipitations demonstrating that MIL38 antibody can detect glypican-1 in the plasma (Figure 16A) of prostate cancer patients and in extracts of prostate cancers (Figure 168).

Figure 16A lanes are: 046 IP NT- IP from prostate cancer plasrna; 046 IP Hep!- TP fronl prostate cancer plasma treated vvith heparinase; 042 IP NT- IP :from normal control plasma; 042 JP Hepl- [P from normal control plasma treated vvith heparinase~ Magic Mark-commercial protein marker as molecular weight standard.

Definitions As used in this applicatiotL the singular form "a", ''an'" and "the" include plural references unless the context dearly dictates othen.vise.

For exarnple, the phrase "an antibody" also includes multiple antibodies.

As used herein, the term "comprising" means ''including." Variations of the word '\:omprisin.g", such as "'comprise" and '\:omprises," have com.~. spondingly varied meanings.

Thus, for ex~unple, a sample "comprising" antibody A ma.y consist exclusively of antibody A or may include one or more additional components (e.g. antibody B) As used herein the terms "multiple" and "plurality" mean more than one.

In ce11ain 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.

The term "epiwpe"' as used berein refers to the specific portion(s) of an antigen which interact (e.g, bind) with one or rnore binding entities such as, for example, a protein, ligand, antibody, antibody frag;inent, or antibody derivative.

As used herein, the terms "antibody'" and "antibodies" include IgG (iltduding IgG l, IgG2, !gG3, and IgG4), IgA (including IgAl and IgA2), IgD, IgE, lgM, and fgY, whole antibodies, il1eluding 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, disulfide-linked Fvs (sdFvJ and fragments comprising either a VL or VH domain, The antibodies may be from any animal origin or appropriate productiou host.

Antigen-binding antibody fragments, including singlechain antibodies, may comprise the variable region.ls alone or in combination ,vith the entire or panial of the following: hinge region, CHl, CH2, and CH3 domains, Also induded are any combinations of variable region/s and hinge region, CH 1, CH2, and CH3 domains.

Antibodies may be monoclonal, po1yclonal, chimeric, multispecific, humanised, and human monoclonal and polydonal antibodies which specifically bind the biological moleCllk, The antibody may be a bi-specific antlbody, avibody, diabody, tribody, tetrabody, nanobody, single domain antibody, VHH domain, human antibody, fully huma11ized antibody, partially humanized antibody, anticalin, adnectin, or af:fibody.

As used herein the tem1 "monoclonal antibody" refers to an antibody that reco!:,inises a single antigenic epitope, and that is obtained from a population of substantially homogeneous 11 ant1bod1es 1,vhich bind specifically to the same antigenic epitope, and are identical with the potential exception of naturally occurring mutation/s that may be present in minor amounts.

As used herein,. the term "humanised antibody" refers to forms of antibodies that contain sequences from human antibodies as ,ve1l as non-human antibodies (e.g. murine antibodies).

For example, a humanised antibody can comprise substantially all of at least one and typically t\vo vadabk domains, in which all/substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all/substantially all of the FR regions are from the human immunoglobulin sequence.

The humanised antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fe) which may typically be that of a human immunoglobulin.

As used herein, the term "chimeric antibody" refers to an antibody which exhibits a desired biological ,Ktiv.ity, 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.is is/are identical to or homologous with corresponding sequences in antibodies derived frorn another different species.

For example, 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 fm example by genetic engineering from inununoglohnlin gene segments belonging to different species.

As used herein, the tenn "hybridoma" refers to a cell produced by the fusion of an immortal cell (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.

As used herein, the tenns "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.

For example, if the antibody, antibody variant, antibody derivative, or antigen binding fragment ("molecule A") is capable of "binding specifica.lly" or "specifically binding" to a given target molecule ("molecule B"), 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 ,vill selectively bind to molecule B and other alternative potential binding partners ,viU remain substantially unbound by molecule A. [n general, molecule A \.Viii preferentially bind to molecule B at least 10-fold, p1eferahly 50- 12 fold, more prefornbly l 00-fold, and most preferably greater than 100-fold more frequently than other potential binding partners.

Molecule A tnay be capable of binding to molecules that are not molecu]e B at a ,veak, 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.

As used herein, the tt.~m1 "subject" includes any animal of economic:, social or researd1 importimce including bovine, equine., ovine, prilnate, avian and rodent species.

Hence, a "subject" may be a mammal such as, for example, a human or a non-human mammal.

As used herein, the term "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.

As used herein, the terms "protein'', ''peptide" and "polypeptide" each refer to a polymer made up of arnino acids liuked together by peptide bonds and a.re used interchangeably.

For the purposes of the present invention a "polypeptide" may constitute a full length protein or a portion of a fuU length protein, and there is no intended difference in the meaning of a "peptide'' and a "polypeptide".

As used herein a "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.

Non-limiting examples of conservative amino add substitutions include: A (Ala) substituted ,vith S (Ser); R (arg) substituted with K l}ys); N (asn) substituted ,vith Q (gin) or H (his); D (asp) substituted ,vith E (glu); Q (gin) substituted with N (asn); C (cys) substituted with S (ser); E (glu) substituted with D (asp); G (gly) substituted with P (pro} As used herein, the term "polynndeotide" refers to a single- or double-stranded polymer of deoxyribonucleoti<le bast"!s, ribonucleotide bases,, known analogues or natmal nucleotides, or mixtures thereof As used herein the tenn "binding entity" encompasses any molecule capable of binding specifically to a GPC-1 epitope or mimic thereof as described herein.

Non-limiting examples ofhimiing entities include polypeptides such as, for example, antibodies.

As used herein, the tenn ·'kif' refers to any delivery system for delivering materials.

Such delivery systems include systems that a1h:.1\v 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, \Vritten instrnct.ions for performing ,Ui assay etc,) from one location to another.

For example, kits may include 13 one or more enclosures, such as boxes, containing the relevant reaction reagents and/or supporting materials.

As used herein, the term "positive 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.

It will be understood that use of the. term "between" herein when referring to a range of numerical values encompasses the numerical values at each endpoint of the range.

For example, 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.

Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art.

Detailed Description The present inventors have identified specific epitopes within glypican-1 (GPC- 1) advantageous for detecting and quantifying GPC-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.

Accordingly, 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.

Glypican-1 (GPC-1) The present invention anses from a senes of epitopes m glypican-1 heparin sulfate proteoglycan (GPC-1) which are targeted by specific binding entities (e.g. antibodies).

The epitopes may be present in a mammalian GPC-1 protein such as, for example, bovine, equine, ovine, primate or rodent species.

Hence, the epitopes may be present in a 14 Date Reyue/Date Received 2021-04-20 mammalian GPC-1 protein such as, for example, a human or a non-human mammal (e.g. a dog GPC- l protein).

Additionally or alternatively, the epitopes may he present in a non"'.1nammalian GPC-1 protein such as, for example, an avian GPC-1 protein.

The epitopes may be present in a human GPC-l protein (e.g. as defined by a sequence set forth in any one of: NCBJ reference sequence accession no. NP ... 002072.2, GenBank accession no, AAH51279,1, GenBank accession no. AAA98132.l, GenBank ,lccession no.

EAW7 l 184.1, UniProtKB/Swiss-Prot accession no. P35052.2, and/or SEQ ID NO: 14).

It will be also understood that the epitopes may be present in a GPC-1 variant (e.g. a GPC-1 isofonn, splice variant, or allotype).

The epitopes may be present in cell-surface bound and/or secreted fom1s of GPC-1.

EpitOJ)CS G(rpican-I epitopes The present invention provides GPC-l epitopes and components thereot: and also indudes combinations of said epitopes and/or epitope components.

In some embodiments, 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 I below.

Table /.'.

Non-limiting t:1:amples of (iPC-1 epitopes according to tire prt•sent inventfon KVNPQGPGPEEK (or a variant or fnrnmcm thereof) 2 VNPQGPGPEEK (or a variant or fra<•ment thereof) I -····························-·············································-·····························-····· O •••••••••••••••••••••••••• ······-·····························-················-·························< 3 VNPQGPGPEE (or a variant or frn rmcnt thereof) I 4 NPQGPGPEE ( or a variant or frai,,rtncnt thereof) 5 KVNPQGPGPE (or a variant or fra,, ucnt thereof) 6 KVNPQGPGP (or a variant or fragment thereof) 7 T NARAFRD or a variant or frar1mcnt thereof) 8 TQNARA (or a variant or fragment thereot) I --nnnnnnnnnnnn-- nnnn, •,nnnnnnnn,, nnnnnnn,nn,nnnnnnnn_,b,_nnn-nnnnnnnnnn- • nnnnnnnnn-nn-nnnnnn-nnnnnnnn-nnnnnnnn-nnnnnnnnnnnn,l 9 ALSTASDDR (or a variant or fraorncnt thereof) I lO PRERPP (m a variant or fraument thereof) 11 QDASDDGSGS (or a variallt or fragment thereof) 12 CGELYTQNARAFRDLCGNPKVNPQGPG.PEEKRRRGC (or a variant or fragment thereof) 13 LGPECSRAVMK (or a variant m fragment thereof) In certain embodiments an epitope of the present invention comprises or consists of a plurality of segments.

These cpitopes may be linear or discontinuous.

Non-limiting examples of epitopes comprising a plurality of segments are set out in Table 2 below, Tablf! 2: Further mm-limiting exmnpk~- of fiPC-1 epitope~, accor<ling ta the present invention I ···········-t l 7 1st scgm.cnt K VNPQGPGPEEK (or a variant or fragment thereof) ......... 2ndsegment:TQNARAFRD (or a variant or fragment thereof) 6 1st segment K VNPQGPGP (or a variant or fragment thereof) I __ 7 ----------·--·----· __ 2nd. sc·gment: .TQ NARAFRD. (or .a variant. or. fragment. thereof) ······--··--···········--··--·······--··--··--·······_j 4 1st segment; NPQGPGPEE (or a variant or fragment thcreot) I 7 2ndscgrncnt: TQ:NARAFRD(or.avariantorfra(11nentthereof) , 3 7 1st segment VNPQGPGPEE (or a variant or fragment thereo!) 2nd segment: TQNARAFRD (or a variant or fragment thereof) 2 l st segment VNPQGPGPERK (or a variant or fragment thereof) i 7 2nd segment: TQNARAFRD (or a variant or fragment thereof) i ........................ ......................................................................................................................................................................................................... 1 5 l st segment KVNPQGPGPE (or a variant (1r fragment thereof) 1 1 . 7 2nd se ment: TQNARAFRD (or a variant or frm:,,mcnt thereof.) , l 8 6 8 4 8 3 8 2 8 I st segment K VNPQGPGPEEK (or a variant or fragment thereof) 2nd sc<rrncnt: TQNARA (or a variant or fnvmcnt thereof) 1st segment KVNPQGPGP (ora vammt or fragment thereof) 2nd semnent: TQNARA (or a variant or frn<•ment thereof) I st segment NPQGPGPEE (or a variant or fragment thereof) 2nd segment: TQNARA (or a variant or fragment thereof) l st segment VNPQGPGPEE (or a: variant or fragment thereof) 2nd sc-nncnt: TQNARA (or a variant or fra1~1cnt thereof) I st segment: VNPQGPGPEEK { or a variant or fragment therco:t) 2nd sehrmcnt: TQNARA ( or a variant or frairment thereof) I st segment KVNPQGPGPE (or a variant or fragment thereat) 16 8 2nd segment: TQNARA (or a variant or fragment thereof) I 1st segment KVNPQGPGPEEK (or a variant or fragment thereof) 9 2nd segment: ALSTASDDR (or a vnriant or frrrn:rnent thereof) 6 !st segment KVNPQGPGP (or a variant or fragment thereof) 9 2nd segment: ALSTASDDR (or a variant or fragrnent thereof) 4 l st segment: NPQGPGPEE (or a variant or fragment thereo±) 9 2nd SCj,'Tilent: ALSTASDDR (or a variant or fragment thereof) 3 l st segment: VNPQGPGPEE (or a variant or fragment thereof) 9 2nd sehrment: ALST ASDDR (or a variant or frmm1cnt thereof) 2 l st segment: VNPQGPGPEEK (or a variant or fragment tbereot) 9 2nd segment: .ALSTASDDR (or a variant or fragment thereof} s I st segment: KVNP()GPGPE (or a variant or fragment thereof) 9 2nd segment: ALSTASDDR (or a variant or frn,gment thereof} l 1st segment KVNPQGPGPEEK for a variant or fra1,'f11cnt thereof} 11 2nd segment: QDASDDGSGS for a variant or fraument thereof) 6 .l st segment: KVNPQGPGP (or a variant or fragment thercot) 11 2nd segment: QDASDDGSGS (or a variant or fragment thereof) 4 I st segment: NPQGPGPEE (or a varian.t or fragment thereof) 11 2nd segment: QDASDD(lSGS {or a variant or frn1-,m1ent thereot) 3 .I st segim-. . nt: VNPQGPGPEE (or a variant or fragment thereof) 11 2nd segment: QDASDDGSGS (or a variant or fragment thereot) 2 l st segment: VNPQGPGPEEK (or a variant or fragment thereof) 11 2nd segment: QDASDDGSGS (or a variant or fragment thereat) 5 J st segment: KVNPQGPGPE ( OT a variant or fragment thereof) 11 2nd segment: QDASDDGSGS (or a variant or fraw11ent thereof) 1 1st segment KVNPQGPGPEEK ( or a variant or fragment thereot) 13 2nd segment: LGPECSRAVMK (or a variant or frmrmcut thereof) 6 l st segment: KVNPQGPGP (or a variant or fragment thereof) 13 2nd segment: LGPECSRAV~'lK (or a variant or fragment thereof) 4 1st segment NPQGPGPEE (or a variailt or fragment thereof) 13 2nd segment: LGPECSRAV~·1K (or a variant or fragment thereof) 17 3 I st t.egmc11t VNPQG:PGPEE (or a variant or fragment thereof) 13 2nd segment: LGPECSRA VtvIK (or a variant or fragment thereof) 2 I st segment VNPQGPGPEEK (or a variant or fragment thereof) 13 2nd segment: LGPECSRA VlVlK (or a variant or fragment thereof) 5 l st segment: K VNPQGPGPE (or a varim1t or fragment thereof) 13 2nd segment: LGPECSRA VMK {or a variant or framnent thereof) 1 1st segment KVNPQGPGPEEK (or a variant or fragment thereof) 10 2nd segment: PRERPP ( or a variant or fragment thereofJ 6 l st se!,,1111c-nt: KVNP(>GPGP (or a variant or fragment thereof) 10 2nd segment: PRERPP (or a variant or fragment thereof) 4 l st segment: NPQGPGPEE (or a variant or fragment thereof) IO 2nd segment: PRERPP (or a variant or fragincnt thereat) 3 l st segment VNPQGPGPEE (or a variant or fragment thereof) HJ 211d segment: PRERPP (or a variant or fragme11t thereof) 2 l st segment: VNPQGPGPEEK (or a variant or fragment thereof) 10 211d segment: PRERPP (or a variant or fraumrnt thereof) 5 1st segment: KVNP(__)GPGPE (or a variant or fragi11ent thereof) 10 2nd segment: PRERPP (or a variant or fragment thereof) ~ 11 I st segment: QDASDDGSGS (or a va_riant or fragment thereof) 13 2nd segment: LGPECSRAVMK (or a variant or fragment thereof) lO l st segment: PRERPP (or ;_1 variant or fragment thcn:ot) 13 2nd segment: LGPECSRA VMK (or a variant or fragment there0f) 9 l st segment: ALSTASDDR (or a variant or fragment thereof) 13 2nd segment: LGPECSRA VMK (or a variant or fragment thereof) 8 1st segment: TQNARA (or a variant or fragme11t thereof) 13 2nd segment: LGPECSRA VfvfK (or a variant or fragment thereof) 7 1st segment TQNARAFRD (or a variant or fragment thereof) 13 2nd segment: LGPECSRA Vl'vfK (or a variant or framnent thereof) I 7 l st segment: TQNARAFRD (or a variant or fragment thereot) 9 2nd segn1em: ALSTASDDR (or a variant or frafc.>n1cnt thereof) 8 1st segment: TQNARA (or a variant or fragment thereof) ~ 18 9 2nd segment: ALSTASDDR (or a variant or fragment thereof) I to lst segment: PRERPP (or a variant or fragment thereof) 9 2nd segment: ALST.ASDDR (or a variant or fragment thereof) I 11 I st segment: QDASDDGSGS (or a variant or fragment thereof) 9 2nd segment: ALSTASDDR ( or a variant or fragment thereof) I 7 l st segment: TQNARAFRD (or a variant or fragment thereof) I 10 2nd SCj,YJ11Cnt: PRERPP (or a 'Variant or fra~nent thereof) 8 l st segment: TQNARA (or a variant or fragment thereof) IO 2nd schrrn.cnt: PRERPP (or a variant or :fragment thereof) ~ 11 l st segment: QDASDDGSGS (or a variant or fragment thereof) 10 2nd segment: PRERPP ( or a variant or fragment thereof) ! ! 7 lst segment TQNARAFRD (or a variant or fragment thereof) 11 2nd segment: QDASDDGSGS (or a variant or fragrncnt thcn:-of) 8 1st segment TQNARA (or a variant or fragment thereof) 11 2nd segment: QDASDDGSGS (or a variant or fra£Q1:1cnt thereof) 1 1st segment: KVNPQGPGPEEK (or a variant or fragment thereof) to 2nd segment: PRERPP (or a variant or fragment thereof) 11 3rd segment: QDASDDGSGS (or a variant or fragment thereof) 6 .I st segim-. . nt: KVNPQGPGP (or a variant or fragment thereof) lO 2nd segment: PRERPP (or a variant or fragment thereof) 11 3rd segment: QDASDDGSGS (or a variant or fragment thereof) 4 l st segment: NPQGPGPEE (or a vaiiant or fragment thereof) 10 2nd segment: PRERPP (or a variant or fragment thereof) 11 3rd segment: QDASDDGSGS (or a variant or framncnt thereof) 3 l st segment: VNP()GPGPEE (or a variant or fragment thereof) IO 2nd segment: PRERPP (or a variant or fragment thereof) 11 3rd segment: QDASDDGSGS (or a variant or fragment thereof) 2 1st segment VNP(_)GPGPEEK {or a variant or fragme11t thereof) 10 2nd segment: PRERPP (or a variant or fragment thereof) 11 3rd segment: QDASDDGSGS (or a variant or fragtnent thereof) 5 .l st segment: KVNPQGPGPE (or a variant or ftagmcm thereof) I 19 to 2nd segment: PRERPP (or a variant or fragment thereof) 11 3rd segment: QDASDDGSGS (or a variant or fragment thereof) 1 I st segment K VNPQGPGPEEK (or a variant or fragment thereof) 10 2nd segment: PRERPP (or a variant or fragment thereof) 11 3rd segment: QDASDDGSGS (or a variant or fra!$ncnt thereof) In other embodiments, the present invention provides combinations of distinct epitopes that comprise or consist of a plurality of discrete epitopes.

These discrete epitopes may be linear or discontinuous.

Non-limiting examples of epitope combinations compnsmg a plurality of distinct epitopes are set out in TMble 3 below.

Table 3: Non-limiting e.i.:amples of GPC-1 epitope combim1tilms accor<fin.g to the presem ilmmtimt l ] st cpitopc: K VNP(_)GPGPEEK (or a variant or fragment r.hcreoi) 7 2nd e :iito 1e: TQNARAfRD {or a variant of frag:ment thereof) 6 l st epitopc: KVNPQGPGP (or a variant or fragment thereof) 7 2nd e ito .c: TQNAR.AFRD (or a variant or fragment thereof) 4 1st epitope: NPQGPGPEE (or a variant or fragment thereot) 1~7 __i ,_2nd cpito_pc:_TQNARAFRD (or a vi:u-iant_or_fra:hrrncnt_thcrcof).'-----------1 3 1st epitope: VNPQGPGPEE (or a variant or fragment thereof) 7 2nd c ito e: TONARAFRD (or a variant or fragment thereof) 2 1st epitopc: VNPQGPGPEEK (or a variant or fragment thereof) 7 2nd cpito e: TQNARAFRD (or a variant or fragment thercot) 5 1st cpitopc: KVNPQGPGPE (or a variant or fragment thereof) 7 2nd c ito e: TONARAFRD (or a variant or fragment thereof) l 8 6 8 4 8 3 1st epitopc: KVNPQGPGPEEK (or a variant or fragmcnr rhcreof) 2nd epitopc: TQNARA ( or a variant or fra ,mcnt thereof) 1st cpitopc: KVNPQGPGP (or a variant or fragment thereof) 2nd c ito e: TQNARA (or a variant or frm4mcnt thereof) 1st cpitope: NPQGPGPEE (or a variant or frabll11cnt Lhcreo:t) 2nd c ito e: TQNARA (or a variant or fragment thereof) 1st cJito1e; VN )GPGPEE (or a variant or frn m1cnt thcreol) 8 2nd epitopc: TQNARA (or a variant or fragment thereof) I 2 lst cpitopc: VNPQGPGPEEK (or a variant or frngmcnt thereof) 8 2nd epitopc: TQNARA ( or a variant or fragment thereof) 5 I st cpitopc: K VNPQGPGPE (ma variant or fra_b>rnent therco(l 8 2nd cpitopc: TQNARA (or a variant or fragment thereof) I I l st epitopc: KVNPQGPGPEEK (or a variant or fragment thereof) 9 2nd cpitopc: ALSTASDDR (or a variant or fragment thereof) 6 l st cpitopc: KVNPQGPGP (or a variant or fra1:,•me11t thereof) 9 2nd cpitopc: i\LSTASDDR for a variant or frnJtment thereof} 4 l st epitope: NPQGPGPEE (or a vnria11t or fragment thereof) 9 2nd epitope: ALSTASDDR (or a variant or frngment thereof) 3 I st cpitopc: VNPQGPGPEE (or a variant or fragment thereof) 9 2nd epitopc: ALSTASDDR (or a variant or fragment thereof} 2 lst epitope: VNPQGPGPEEK (or a variant or fragment thereof) 9 2nd cpitope: ALSTASDDR (or a variant or fragment thereof} 5 1st cpitope: K VNPQGPGPE (or a vm-iant or fra1,w1ent thereof) 9 2nd epitopc: ALSTASDDR (or a variant or fragment thereof) I 1 I st cpitopc: KVNPQGPGPEEK (or a variant or fragment thereof) 11 2nd cpitope: QDASDDGSGS (or a variant or fragment thereof) 6 .I st r..-pitopc: KVNPQGPGP (or a variant or frag;ncnt thereof) 11 2nd cpitopc: QDASDDGSGS (or a variant or frn.~mcnt t.hcrcoi) 4 l st epitopc: NPQGPGPEE (or a variant or fragment thereof) 11 2nd cpitopc: QDASDDGSGS (or a variant or fr,1gmcnt thereof) 3 J st epitopc: VNPQGPGPEE (or a variant or fragment thereof) 11 2nd epitopc: QDASDDGSGS (or a variant or fragment thereof) 2 1st cpitopc: VNPQGPGPEEK (or a variant or fragment thereof) ll 2nd cpitopc: QDASDDGSGS (or a ,,,ariant or fi,tttmcnt thereof) 5 Ist cpitopc: KVNPQGPGPE (or a variant or fragment thereof) 11 2nd cpitopc: QDASDDGSGS (or a variant or fragment thereof) 1 I st cpitope: K VNPQGPGPEEK (or a vartant or fri.igrncnt thereof) 13 2nd enitope: LGPECSRA VMK (or a variant or frag1nent thereot) 21 6 I st epitopc: KVNPQGPGP (or a variant or fragment thereof) 13 2nd cpitope: LGPECSRA V.MK (or a variant or fragment thereof) 4 I st cpitope: NPQGPGPEE (or a variant or fragment thereof) 13 2nd epitopc: LGPECSRA V.MK (or a variant or fragment thereof) I 3 1st epitope: VNPQGPGPEE ( or a variant or fragment thereot) 13 2nd cpitope: LGPECSRA VMK (or a variant or fragment thereof) I 2 I st cpitopc: VNPQGPGPEEK ( or a Yariant or frngment thereof) 13 2nd cpitope: LGPECSRAVMK (or a variant or fragment thereof) 5 1st epitope: KVNPQGPGPE (or a variant or fragment thereot) 13 2nd epitope: LGPECSRAVMK (or a , .. ariant or frag1nent thereof) ! l 1st epitope: KVNPQGPGPEEK (or a variant or fragment thereof} IO 2nd epitope: PRERPP (or a variant or framnc:nt thcn.-of) 6 1st epitope: KVNP(~GPGP for a variant or fragment thereof) HJ 211d cpitope: PRERPP (or a variant or framnc:nt therec,t) 4 1st cpitopc: NPQGPGPEE (or a variant or fragment thereof) 10 211d cpitopc: PRERPP (or a variant or fragment thereof) 3 I st cpitope; VNPQGPGPEE (or a variant or fragment thereof) 10 2nd cpitopc: PRERPP (or a variant or fra~mcnt thereof) 2 I st epitope: VNPQGPGPEEK (or a variant or fragment thereof) to 2nd cpitopc: PRERPP (or a variant or fral!mcut thereof) 5 1st epitope KVNPQGPGPE (or a variant or fragment thereof) 10 2nd epitope: PRERPP (or a variant or fowment thereof) I 11 1st epitopc: QDASDDGSGS (or a variant or fragment thereof) 13 2nd cpitope: LGPECSRAVMK (or a variant or fragment thereof) to 1st epitope: PRERPP (or a variatlt or fragment thereof) 13 2nd cpitope: LGPECSRAVMK (or a variant or fragment thereof) 9 I st epitopc; ALST ASDDR (or a variant or fragment thereof) 13 2nd cpitope: LGPECSRAVi\1K (or a variant or fragment thereof) 8 l st epitopc: TQNARA (or a variant or fragment thereof) 13 2nd cpitope: LGPECSRAVMK for a variant or fragment thereof) 7 1st cpitopc; TQNARAFRD (or a variant or fragment thereof) 13 2nd epitopc: LGPECSRA VMK for a variant or fra~mcnt thcrcot) I 7 lst cpitopc: TQNARAFRD (or a variant or fragment thereof) 9 2nd cpitopc: ALSTASDDR (or a variant or fragment thcrcot) 8 I st cpitopc: TQNARA (or a variant or frU,!;.rment thereof) 9 2nd cpitopc: ALSTASDDR (or a variant or fragment thcrcot) 10 1st epitopc: PRERPP (or a variant or fragment thereof) 9 2nd eoitope: ALSTASDDR for a variant or fowmcnt thereof) 11 1st cpitope: QDASDDGSGS (or a variant or fragment thereof) 9 2nd epitope: ALSTASDDR (or a variant or fragment thereof) 7 l st epitope: TQNARAFRD (or a variant f'lr fragment thereof) 10 2nd epitope: PRERPP ( or a variant or fragment thereof) 8 I st cpitopc: TQNARA (or a variant or fragment thereof) IO 2nd epitopc: PRERPP ( or a variant or fral~me.m thereof) 11 lst cpitopc: QDASDDGSGS (or a variant or fragment thereof) lO 2nd epitope: PRERPP ( or a variant or fragment thereof) I 7 .l st epitope: TQNARAFRD (or a variant or fragment thereof) 11 2nd epitope: QDASDDGSGS (or a variant or fragment thereof) 8 I st cpitope: TQNARA. (or a variant or fragment thereof) 11 2nd epitope: QDASDDGSGS (or a variant or fragment thereof) l I st epitope: KVNPQGPGPEEK (or a variant or fr%'111Cnt thereof) 10 2nd epitopc: PRERPP (or a variant or fragment thereof) 11 3rd cpitope: QDASDDGSOS (or a variant or frngment thereof) 6 J st epitopc: KVNPQGPGP (or a variant or fragment thereof) IO 2nd epitope: PRERPP (or a variant or fragrnent thereof) 11 3rd epitope: QDASDDGSGS (or a variant or fragment thereof) 4 I s:t epitope: NPQGPGPEE (or a variant or fragment thereof) 10 2nd epitope: PRERPP (or a variant or fragment thereof) tl 3rd eoitope: QDASDDGSGS (or a variant or fragment thereof) 3 1st epitopc: VNPQGPGPEE lor a variant or fragment thereof) 10 2nd epitopc: PRERPP (or a variant or fragment thereof) 11 3rd epitope: QDASDDGSGS (or a variant or fragment thereof) 23 2 I st epitope: VNP()GPGPEEK (or a variant or fragment thereof) HJ 2nd epitope: PRERPP (or a variant or fragment thereof) 11 3rd cpitopc: QDASDDGSGS (or a variant or frat,ry.nent thereof) 5 l st cpitope: KVNPQGPGPE (or a variant or fragment thereof) 10 2nd epitope: PRERPP ( or u variant or fragment thereof) l1 3rd cpitope: QDASDDGSGS (or a variant or fragment thereof) I lst cpitope: KVNPQGPGPEEK (or a variant or fn:.tgment thereof) I 10 2nd epitope: PRERPP (or a variant or fragment thereof) ...1..1... ................ }rd _epitopc:_ QDASDDGSGS_ _ (or _a _variant_ or. fragment. thereof) ................................................... ...J A combination of epitopes according to the present invention may comprise prostatespecific antigen (PSA), also know11 as gamma-seminoprotein or kallikrein-3 (KLK3 ).

Accordingly, a combination of epitopes according to the present invention may comprise any one of the epitopes listed in Table l or Table 2 in combination \Yith PSA, or any one of the epitope combinations listed in Table 3 further combined v,.:ith PSA.

Variants The present invention provides variants of the GPC-1 epitopes described herein.

The variants may comprise conservathe or non-conservative amino acid substitution(s),, as known to those of ordinary skill in the art.

In some embodiments, a variant of an epitope of the present invention 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~,.o, 45(J/ii, or 99~o sequence identity with the sequence of a GPC- l epitope described herein.

The variant may retain identical, substantially identicat or altered biological activity in comparison to the GPC-1 epitope sequence from which the variant arise&.

The variant rnay be a homologue GPC-1 epitope fmm 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).

Differences m sequence identity may ansc from ammo acid substitutions (e.g. conservative and/or non-conservative substitutions), insertions ~md/or deletions. A 24 conservative amino acid substitution refers to a substitution or replacement of one amino add for another a.m:ino acid with similar properties within a polypeptide cha:in, as ·well known to those of ordinary skill in the art For example, the substitution of the charged amino acid glutamic acid (Glu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution.

The percentage of stiquem.:e idt~ntity betwt~en t\vo sequences may be cktem1ined ,vithout difficulty using methods known to those of ordinary skill in 1he ~lft.

For example, the percentage of sequence identity bervveen tvvo 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 ali!;,.'11 the nvo sequences optimaily. A percentage of sequence identity may then be caJculated by detennining the number of positions at whicb the identical nucleic add base or amino acid residue occurs in both sequences to yield the nun1ber of matched positions,, dividing the number of matched positions by the total number of positions in the Vv'indow of comparison and multiplying the result by ] 00 to yield the percentage of sequence identlty, The level of sequence idenfay may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of \Visconsin Biotechnology Center, l 710 University Ave., Madison, Wis. 53 705).

This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

Other suitable examples computer sofh.vare for measuring the degree of sequence identity betwee11 tv,.·o or more sequences include, but are not limited to, CLUSTAL in the PC/Gene prngram (available from IntelHgenetics., Mountain Viev,·, California); the ALIGN program (Version LO) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Soft\vare Package, Version IO (available from Accelrys foe,, 9685 Scranton Road, San Diego, California, USA).

In relation to the above en:1bodiments,, including those set out in Tables 1-3 above, Table 4 below· provides suitable and non-limiting examples of the variants referred to.

N °' Variant l I \V, R, L Y.orF Variant 2 I any anrnm ·----------------------------r·--a--c--i-.d-- ------------- Variant 3 any ammo acid Variru1t l Variant 2 Variant 3 1::1111:111::1:1!:~,f~ Variant I Variant 2 Variant 3 H, P, or K K, W.

V D S, H. orN any H any amino amino acid ---m---c--i-d-- ------------- any any any amino ammo ammo ,1..:1d m:id acid H, P, or K K, V-l.

V D S, H. orN ,my H aJYJ 1lnJH10 mmno acid add :c1ny ,my any ammo amino acid H, P, nr R. K, W.

V D S,H,orN any H any amint1 ammo acid acid nny Hny any :mmm mnino amino acid add acid D, L N, Q, K, R. () G p or A N, M, T. S. G /1, G orR -------------------------------- ---·-.···.--, .----·-····1--······---·-··1•·--_, Y, A. E, V, A. S, 1 . H, P b ,vL F, L, I, T, w. Y, F, orR !v1 D. L N.

Q. K ' R.

Q G p or A N, M, T. S. { .. _) A G orR Y. A. E, y, I A; s, T, ri,, t p r G \L }, L, LI. \\, Y, I·, D, L N, Q, K. R.

Q G p or 1\ N, M, T, S, G l\ (i or R Y, !\, E, V. !\,S,T,H, p ( .. . 1 M, F, L, LT. W. Y. F, orR M any M, A, T, K. R. Q, amino S.T.orY acid A. D, C, E, Z, G, E H, K, M. F, P, S. _ T,_ \V, _m Y .................... any amino acid F :my M, A. l, K. R. Q, uminci S.T.mY add A. D, C, E, Z, G, E H. K, M, F, P, S, T, V•i. orY any amino acid ,my M, A. I, K R. Q, amino S, T, or Y ucid A, D, C, E. Z. G, E H. K, M, F, P. S, T, W.or Y any amino acid E. any amino acid any amino acid Q, D. F. 11, or 1\.-f any ammn acid any amino add Q, D, F, H,orM any ammo aci,J any amino add Q, D, .F, H,orM any amino acid :any a1n1no acid any ammo acid any amino acid any ammo -ac-id any ~ N .0. .. -°....-..'.. .N.. N I.,; "'d t""l ~ N .0. .. ~ 0 .0. .. 1,0 ~ ""_,'' w__,, ... ".0. .' _, I 0_ , ..I. "' N -.i ,,,,,,,,,,,,,,,,,,:,,,,,,,,,, ,,,,,r ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,u,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,:1,,,:,,,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,1,,, c,c,c,c,c,,c,c,c,c,c1,,,~, c,,c,,c,c,c,c,c,c,c,c1 ,,,,,,,,,,,,,,,,,,,,I,,,,,,,,,,,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,1,,,,,,,,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,1,,, ,c,c,c,c,c:,,,c,c1:,c8? Vafr.mt 2 Variant3 H. P, or D H any amino acid R, K. W, S, H, orN any amino u..:id any mnino acid ,,,,,,,,,,,,,.,,,,,,,,,,,,,,,,::,,:i:,,,,;,,,,, ,,,,,,,,c,,,,,,,a,,c,c,,c,«,,ac.-c-- Q I G N, \,L T, S, G orR Y, r\, E, V, M, F. L l, T, Qr R A,S,T, H, W, Y, F, M Ip A p Q, K, R, I. or A G M, A, L K, R, Q. s, T, (lf y A, D. C. E. Z, G.

H, K M, F, P, S, T. W,or Y any amino acid any amino acid E F .. ,. . ,. ,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,:,,,i,:,,:,;,,, ,,,,,,::,,,:,r,,,;;; ,,,,,,,,,,,,,,.,,,,,,,,,c,c,1,,, any amino acid any amino add Q. D. F, H,orM ,,., .. ,. ., .. ,. . ,.. , . ,.. ,.. ,., . ,. ., . ,.. ,.. ,. ,.. ,. . ,. ,. . ,. , ,,,i,c,,,., .. ,. ., ,,,,,,,,,,,,,,,,:,,,,,,,,,,,,,,,,,u,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,:1 ,,,:,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,:,,1,,,,,,,, ,,,,,c,c,c,,,,,,,,,,,,,,,c,,,,,:1 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,I,,,,,,,,,,,,,,,,c,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,:r,,, ,,,er, c,c,c,c,c,c,c,,c,c,c1c,,,,, Variant 1 Variant 2 Variam3 'W, R, L, Y, orF V any any amino amino acid ,icid any any mr1i110 amino acid :icid H. P. or D H any amino acid R, K. w, S. H, orN nny amim1 o.:id aT1y amir10 add _.._.._.._.._.._.._.._..,.,r-,:•'r, ,.,.,.,.,.,.,.,.,.,.,.,-,:•:•::•:~:•:-:n:·:':'"'·-·•·•·-·•·.-•·••·••.-,--i:,:-:!V:·:·:•:•:•:•:•:•·•:--:•:"·•·•·•·•·•·-·• Q N, .\.1, T, S, orR Y, A, E, V, .M, t, L !, 'I, (!I' R , ....... •,•.·:.·.··················· ..... ·.•.•, ( ., .J G A,S, T,H, \v, Y, F, M r A p D, E, N, Q, K, R, or/-\.

G G M, A, L K, R, Q.

S, T. or Y A, D, C. E, Z, G.

H, K. M, F, P, S, T. W,or Y a11y amino acid any amino acid E r ~, ,,,,,,,,,,,,,,,,,,,,i,.,,~~''"'''; ,,,,,,,,,,,,,,1,,,,,~, ,,,,,,,,,,,,,;,i,,,,,;,,,,,,,,;,,,,,,,,;,,,c,,,,; ,,,,,,,,,1, ,n,,cn,,,,,,,.,,,,,,::>.,,,,i ,,,,,,,,,,1 ,:,,,:,,,,,:,,,,,,,,,:,:,,,,,,,:,,,,,,,,,,,,,i:,c,::>. ,,,,,,,,,,,,,:,,,,,i ,,,,,,,,,,,,,,,,,,,,,.,,,,,,,,,.,,,,,,,,,,,,.c,l':c,>. ,,,,,,,,,,,,c,,,,,,.,,,,,,,.,,,,,,::>.c,,,,,,,1,,,,,,,,., ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,! ,,,,,,,,,,,,,,n,1 :,,,,,,,i,,,,,,.,,,,,,,,,.,,,,,,,,,., ,,,,,,,,,,,,1, Variant l Variant 2 Varkmt 3 ...........,====~• W. R, L Y. orF any amino acid al1,Y amino acid I_ Variant CON V ,my ,1mino acid any amino acid CON H, P. or R. K, W.

D S, H, orN H ,my amin<J acid any any amino mnino add acid CON CON D, E. N, Q, K, K M.. A. L K. R, Q, Q G p or A S. T. or Y N. M. T, S, G A G A, D, C. E, Z, G. mR H, K M, F. l\ S.

T. W,or Y Y, A, E, V, A, S. T. H, p G any amino acid ,vL F, LI, T, W, Y, F, ~,r R M CON CON icoN CON CON ,:cc,,,,,,,,:ci':r'ri'ic'Li:1:)'t, :'c'cl':'c'i:i:, ',':',',',',',',',',',',','',T',':!& :'c'c,'i'il',','ii;, ,','i','ic'/t,',Jt'rii', ',8i:>8,8,tt't,',',',t',','J,'ic',U', i8,'i',',','i',',',',t ,'c':','ci':'':'!c ,',':ltt't,t',t',','i' i,',',',t['c':'t, ,,,,,_.;;,,,,,,,,,,,,,,,,,,,,,@,,,,, ,,,,,,,,,,,,,,,,,tc:cce:'J: ,,,,,,,:,:,,,,,:,:;,:,,:,~ ,,,,,,,,,,,,,,,,,,,,,,,,,i ,,,,,,,,,,,,,r ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,1,:,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,I ,,,,,,,,,,,,,,1 ,,,,,,,,,,,,,i,,,,,,,,,,,::>.c,,,,,,,,,,,,,, ,,,,,,,,,1, ~ N -0 --~ .N.. .

N (,,,j -= r"'l s~ -0 u, -0 \0 ~ ""'' -.J w -.J -.J f-' "0 ' f-' -.J I -.J I f-' "' N QO i ariant i CON CON CON i CON CON CON i CON I CON Va:riant ,. ............................. ~ ...C...O....N... ........... 1-. CON··-~---........ •.. . -.. ···········---~··························'---··---~····'---········. . ···'--···············. . ········'--··'··································. . ·····'---'·····'··············'···'········. . ···········'-·······················' CON CON CON CON CON I CON CON CON CON 'A,ry amino acid'--· A {1.ll'ai, R (arg), N (ami, (mp), B (asxj, C (cys.j, E (,;;luj, Q (.v,ln), Z (1;/:r). G (g(r}. H (hL\), I (ile), !, ({eu), K (fr~), A{ (metj, F (phe), P (jwoj, S {,erj, T (thr), W (trpi. Y (tyri. or V /vcdi. 'CON'·- conservative amino acid sui>stiturion ~ N .0. .. <::", -........ .N. . N I.,; "'d t"'l ~ N .0. .. ~ 0 .0. .. 1,0 ~ 0 ""_'', w__,, .... "' 0 ._..,.

I 0_ , I .... "' WO 2016/112423 PCT/ AU2015/000019 Accordingly, a variant of KVNPOGPGPEEK (SEQ ID NO.: 1) may comprise V (val) at position 2, Q (gln) at position 5, G (gly) at position 6, and P (pro) at position 7.

The variant may fmtl1er comprise K (lys) at position l, and/or N (asn) at position 3, and/or P (pro) at position 4.

Additionally or altematively, the variant may further comprise G (gly) at position 8, and/or P (pro) at position 9, and/or E (g1u) at position 10.

AdditionaHy or alternatively, the variant may ,:omprise one, two, three. four, five, six, seven or eight substituted residues.

The substituted amino acid residue{s) may be at any one or more ofpositfons l, 3, 4, 8, 9, IO, 11, and/or t2 of SEQ lD NO; L Alternatively, a variant of KVNPQGPGP~EK (SEQ JD NO: 1) 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.

Additionally or alternatively, the variant may comprise one, tv,,ro, tluee, four, five, six, seven, eight or niue substituted residues.

The substituted amino acid residue(s) may be at any one or more ofpositions 1, 2, 3, 4, 5, 7, 9, 11, and/or 12 of SEQ ID NO: 1 Alternatively, a variant of KVNPQGPGP&EK (SEQ lD NO: 1) may comprise P (pro) at position 7, G (gly) at position 8, and E (glu) at position 10.

The vruiant may further comprise Q (gh1) at position 5, and/or G (gly) at position 6, and/or E (glu) at position l l.

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, l l, and/or 12 of SEQ ID NO: 1.

A variant of VNPQGPGPEEK (SEQ ID NO: 2) may comprise V (val) at position I, Q (g1n} at positjon 4, G (gly) at pos1tion 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 positio11 7, and/or P (pro) at position 8, and/or E (gJu) at position 9.

Additionally or alternatively, the variant may comprise one, tvvo, three, fow-, five, six, or seven substimted residues.

The substituted amino acid residue(s) may be at any one or more ofpositions 2, 3, 7, 8, 9, lO, andior 11 of SEQ ID NO: 2.

Alternatively, a variant of VNPQGPGPE_EK (SEQ JD NO: 2) may comprise G (gly) .at position 5, G (gly) at position 7,, and E (glu) at posit1on 9.

The variant may fi.nther comprise N (asn) at position 2, and/or Q (gln) at position 4, and/or P (pro) at position 6.

Additionally or alternatively, the variant may comprise one, two, three, fbur, five, s1x, 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, 8, 10, and/or 1 l of SEQ ID NO: 2. 29 WO 2016/112423 PCT/ AU2015/000019 Alternatively, a variant ofVNPQGPGP!;;,EK (SEQ ID NO: 2) may comprise P (pro) at position 6, G (gly) at position 7, and E (gill) at position 9.

The variant may further comprise Q (gin) at position 4, and/or G (gly) at position 5, and/or E (glu) at position I 0.

Additionally or alternatively, 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, 5, 8, l 0, and/or l lof SEQ ID NO: 2.

A variant ofVNPOGPGPEE (SEQ ID NO: 3) may comprise V (val) at position 1, Q (gln) at position 4, G (gl:y) at position 5, and P (pro) at position 6.

The variant may further comprise N (mm) at position 2, and/or P (pro) at position 3. A.dditionally 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, fi,1e or six substituted residues.

The substituted amino add residue(s) may be at any one or more of positions 2, 3, 7, 8, 9, and/or lO of SEQ ID NO; 3.

Alternatively, a variant of VNPQGPGPEE (SEQ ID NO: 3) 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 (gln) at position 4, and/or P (pro) at position 6.

Additionally or alternatively, the variant may comprise one, t\vo, three, four, five, six, or seven substituted residues.

The substituted amino acid residue(s) may be at any one or more ofpositions l, 2, 3, 4, 6, 8, and/or lO of SEQ ID NO: 3.

Alternatively, a variant of VNPQGPGPE_E (SEQ lD NO: 3) may comprise P (pro) at position 6, G (gly) at position 7, and E (glu) at position 9.

The variant may farther comprise Q (gln) at position 4, and/or G (gly) at position 5, an<lJor E (glu) at position I 0, Additiona11y or alternatively, the variaut may comprise one, two, three, four, five, six, or seven substituted residues.

The substituted amino acid residue(s) may he at any one or more of positions l, 2, 3, 4, 5, 8, and/or 10 of SEQ ID NO: 3.

A variant of NPOGPGPEE (SEQ lD NO: 4) may comprise Q (gln) at position 3, G (gly} at position 4, and P (pro) at position 5.

The variant may funher comprise N (aS11) at position l, and/or P (_pro) at position 2.

Additionally or alternatively, the variant may further comprise G (gly) at position 6, and/or P (prn) at position 7, and/or E (glu) at position 8.

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 l, 2, 6, 7, 8, and/or 9 of SEQ ID NO: 4.

Alternatively, a variant of NPQGPGPEF (SEQ ID NO: 4) may comprise G (gly) at position 4, G (gly) at position 6, and E (glu) at position 8.

The variant may fmther comprise WO 2016/112423 PCT/ AU2015/000019 N {asn) at position l, and/or Q (gln) at position 3, and/or P (pro) at position 5.

Additlonally or alternatively, tlie variant may c01nprise one, two, three, four, five, or six substituted residues.

The substituted amino acid residue(s) may be at any one or more of positions I, 2, 3, 5, 7, and/or 9 of SEQ ID NO: 4.

Alternatively, a variant of NPQGPGPE_E (SEQ ID NO: 4) may comprise P (pro) at position 5, G (gly) at position 6, and E (glu) at position 8.

The variant may further comprise Q (gln) at position 3, and/or G (gly) at position 4, and/or E (glu) at position 9.

Additionally or alternafrvely, the variant may co1nprise one, two, three, frmr, five, or six, substituted residues.

The substituted amino acid residue(si may l:>e at any one or more of positions I, 2, 3, 4, 7, and/or 9 of SEQ JD NO: 4.

A variant of KVNPOGPGPE (SEQ lD NO: 5) may comprise V (val} at position 2, Q (gl11) at position 5, G (gly) at position 6_, and P (pro) at position 7.

The variant may further coinprise K (lys) at position 1, a.nd/or N (asn) at position 3, and/or P (Pm) 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 va1iant may comprise one, t\vo, three, four, five or six substituted residues.

The substituted amino add residue(s) may be at any one or more of positions 1, 3, 4, 8, 9, and/or IO of SEQ ID NO: 5.

Alternatively, a variant of KVNPQGPGPE (SEQ ID NO: 5) may comprise G (gly) at positil)n 6, G (gly) at position 8,. and E (glu) at p(Jsition 10.

The variant may fu1ther comprise N (asn) at position 3, andlor Q (gln) at position 5, and/or P (pro) at position 7.

Additionally nr alternatively, the variant may comprise one, t\vo, three, four, five, six, or seven substituted residues, The substituted amino acid residue(s) may be at a.ny one or more of positions 1, 2, 3, 4, 5, 7, and/or 9 of SEQ TD NO: 5.

Alternatively, a variant of KVNPQGPGP;E (SEQ ID NO: 5) 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 (g1y) at position 6.

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 ,my one or more of positions l, 2, 3, 4, 5, 6. and/or 9 of SEQ ID NO: 5.

A variant of KVNPQGPGP (SEQ ID NO: 6) may comprise V (val) at position 2, Q (gln) 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, andior N (asn) at position 3, and/or P (pro) at position 4.

Additionally or alternatively, the variant may fun.her comprise G (gly) at position 8, and/or P (pro) at position 9.

Additionally or alternatively, the variant may comprise one, two, three, 31 WO 2016/112423 PCT/ AU2015/000019 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 SEQ ID NO: 6.

A variant of KVNPQGPGP (SEQ l.D NO: 6) 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, andior P (pro) at position 7.

Additionally or alternatively, the variant may comprise one, t\vo, three, four, five, six, or seven substituted residt1es. TI1e substitutt~d 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 N0:6.

A variant ofKVNPQGPGP (SEQ ID NO: 6) may comprise P (proJ at position 7, and G (gly) at position 8.

The variant may further comprise Q (gln) at position 5, and/or G (gly) at position 6.

Additionally or alternatively, the variant may comprise one, two, three, four, five, six, or seven substituted residues.

The substituted amino acid residne(s) may be at any ont~ 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 f01th in SEQ ID NOs: 7-13 may comprise an amino add substitution at any one or more position(s) of the epitope sequence.

The amino ac.id substitution may be a conservative amino-add s1,.1bstimtio11 or a non-conservative amino acid substitution, as are knO\vn to those of ordinary skill in the art The variants may c.omprise conservative substitntion(s) only, non-conservative substitution(s) only, or a mixture of conservative substiution(s) a:nd non-conservative substitution(s)_ Fragments The present invention provides fragments of the GPC -l epitopes and variants described herein. ln some embodiments, a fragment of an epitope of the present invention may comprise OI 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 lO and 20, between 10 and 15, between 7 and 15, between 7 and 13, bet\.veen 8 and 12, between 8 and I 0, beh:veen l t and 19., betw·een 12 and 18., or beiween t 3 and 17 amino adds in length.

Generally, a fragment of a foll GPC-1 epitope disclosed herein may possess similar or in some cases improved immunological properties compared to the fiill GPC-1 epitope_ In relation to the above embodiments, including those set out in Tables 1-4 above, Table 5 below provides suitable and non-limiting examples of the fragments referred to. 32 (,,; (,,; SEQ£DNO: 1 K VNPQGPGPEEK (or a 1,arianl therenf a!, disclosed herein) SEQlDNO: 2 VNPQGPGPEEK (or a variant thcn::llf as disclosed herein) SE:QIDNO: 3 VNPQGPGI'EE SEQ JD NO: 4 NPQGPGPEE (or a vari.mt thereof as disdt,sed herein) SRQJDNO: 5 KVNPQGPGPE (or a Vl!rimH thereof a, disclosed herein) SF.QJDNO: 6 KVNPQGPGP ( or a vmianl there~i fas disclosed herein) SEQlDN0:7 TQNARAFRD !---------1 ( or a variaru thereof as disclosed herein) SEQ TD NO: S TQNARA 1 _______ __,jor n variant thereof as disclosed herein) SEQ TD NO: 9 ALSTASIJDR t<:si<lm:, 1-11, l-! 0. l-9, 1-S. 2-12. 2- l I, 2-!0, 2-9, 2-8, 3-12, 3. J I. 3-10, 3-9, 3-8, 4-12, 4-11, 4- 10, 4-9. ()I' 4-g resi<luc, 1-10, 1,9, 1-8, 1-7, 2-11, 2-10, 2-9. 2-8, 2-7, 3-11, J-10, VJ. 3-8()r3-7 residl1es l-9, 1-8, 1-7, 2-10, 231. 2-8, 2-7, 3-10, J-9, 3-8 orJ-7 residues 1-8, 1-7, 1-6, 2-9, 2-8, 2-7. 2-6, 38, 3-8, or 3-7 residues 1-9, 1-8. 1-7, 2-Hl, 2-9. 2-8, 2-7, 3-10, '.l-9, 3-8 or 3-7 resklucs 1-8, 1-7, 1-6, 2-9, 2-8, 2-7. 2-6, 3-9, 3-8, or 3-7 residuc:s 1-8, 1-7. 1-6, 2-9, 2-8, 2-7, J-9, 3-8, or 3-7 re,iduc:~ l-5, l-4, 2-6, nr 2-5 rec,idue5 1-8, 1-7. 1-6. 2-9, 2-8. 2-7, J-9, 3-8, or J-7 ~ N .0. .. ~ -....-.... .N.. N (,,; ""d l"'l ~ N .0. .. u, .0. .. 1-C g 0 ""...J' w ...J ...J f-' "0 f-' ...J I ...J I f-' " (,,; ..... 0:w a variant ther<1of as disclos,,d herein) SEQlDNO; 10 PRERPP retiidues 1-5, 1-4, 2-6, N 2-5 (or a variant there{)f as disclosed herein) SEQlDNO: II ()DASDDGSGS residues 1-9, J .g, 1-7. 2-10, 2-9, 2-8, 3-10, 3-!l, M 3-8 (or u variant th.-r~'<Jf as disclosed !wrein) SEQ ID NO: 12 CGELYTQNARAFRDLCGNPKVNPQGJ>GPEEKR I r~&idues 1-35, 1-H, 1-33, l-32, 1-31. l-30, 1-29, 1-2&. l-27, 1-26, 1-25. 2-36, 2-35, 2-34, 2-:B, RRGC (or a niri,mt thercvfas disi:foscd herein) SEQ ID NO: B LOPFC.SRAVMK , ---------------------·-------······---L { or_ a_ rnrjant th<..'tl!of as_ diBclos<'d herdn) --·---·---·---···· 2-32, 2-3 L 2-30,, 2-29, 2-28. 2-27, 2-26, 2-25. 3-36, 3-35, 3-34, 3-33, 3-32, 3-:ll, 3-30,. 3-29, 3- 28, 3-27, 3-26, 3-25, 4-36, 4-35, 4-34. 4-33, 4-32, 4-31, 4-3(), 4-29. 4-28, 4-27, 4-2<\, 4-25, 5-36. 5-35, 5-34, 5-33, 5-32, 5-31, 5-30. 5-29, 5-28. S-27, 5-26, S-25, 6-36, 6-35, 6-34, 6-33, 6-32, 6- 31, 6-30, 6-29. &-28, li-27, 6-26, 6-25, 7-36. 7-35, 7-34, 7.33, 7-32, 7-J L 7-Jo, 7-29, 7-28, 7-27, 7-26, 7-25, 8-36. 8-35, 8-34, 8-33, 8-32, S--31, 8-30. 8-29, 8-28, 8-27. 8-26, 8-25, 9-36, 9-35, 9- 34, 9-33, 9-32. 9-31, 9-30, 9-29, 9-28, 9-27, 9-26, 9-25. 10-36. 10-35, !0-34, !0-33. 10-:\2, 10- 31, l 0-30,Jl)-29, 10-28.10-27, l Q-1_6, ()t 10-25 residue~ 1-10. 1-9, 1-t-t. 1-7, 2-1 L 2-10. 2-9, 2-8. 2-7, 3-11, 3- Hl, 3-9, 3-8 nr 1-7 ~ N .0. .. ~ -....-.... .N... .

N (,,; '"d l"'l ~ N .0. .. u, .0. .. \,C g 0 ""' -.J w -.J -.J f-' "0 f-' -.J I -.J I f-' " WO 2016/112423 PCT/ A U2015/000019 Fusion po~vpeptides Certain embodiments of the present invention provide GPC-1 epitopes in tbe form of fusion polypeptides.

For example, two or more full epiwpes selected from Table t, two or rnore epitope segments selected from Table 2, a combination of two or more full s 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 fom1 the fusion polypeptides, The fusion polypeptides can be prepared using standard techniques known to those of ordinary skill 111 the art including, for example, chemical conjugation. T11e fusion rn polypeptides may also be expressed as a recombinant polypeptide in an expression system.

For example, DNA sequences encoding pol:wepdde components of the fusion polypeptide may be assembled separately, and ligated into an appropriate expression vector.

The 3' tem1inus 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 1s 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 pol'.11)eptide components of the fusion protein by a distance sufficient to ensure that each polypeptide 20 component folds into appropriate secondary and/or tertiary stmctures.

Non-limiting examples of suitable linkers include peptides/polypeptides, alkyl chains or other convenient spacer molecules as know11 to those of ordinary skill in the art.

Suitable peptide linker sequences may be selected according to factors including an ability to adopt a tlexible extended confonnation, an inability to adopt a secondary strncture that 2:; 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 \.vith functional epitope/s in polypeptide component/s of the fusion polypeptide.

By way of non-1.imiting example only, the peptide linker sequences may contain Gly, Asn and/or Ser residues, Additionally or alternatively, other near neutral amino acids 30 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; Musphy et al., Proc.

Natl.

Acad.

Sci. USA 83:8258•8262, 1986; and l\1aratea ct al., Gene 40:39-46, 1985).

The linker sequence may generally be from l to about 50 amino acids in length.

Accordingly. the 3s linker sequences may be 5, lO, l 5, 20, 30, 40, bet\veen 10 and 50, between 10 and 40, between 10 and 30, between 20 and 30, between I and 5, or between 5 and 10 amino acids in length.

Fusion polypeptides according to the present invention may not require a !hiker sequence if the first aud second polypeptide components of the fusion polypeptide have non-essential N-terminal amino acid regions that can be used to separate the s fonct:ioual domains and prevent steric interforence.

Pofynuc!eofidt~,", vectot:') and host eel/:,, The present invention also provides polynucleotides encoding GPC-1 epitope/s of the present invention, segments of the OPC-1 epitopes, and fusion proteins comprising rn GPC- l epitopes and/or seginents thereof.

The polynucleotides may be cloned into a vector.

The vector may cmnprise, for example, a DNA, RNA or complementary DNA (cDNA) sequence encoding the GPC-1 epitopes, GPC-1 epitope scgment/s, and/or fusion proteins.

The vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of forei!:,.m 1s sequences, their introduction into cells and the expression of the introduced sequences. T11Jically the vector is m1 expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals and transcription tem1ination sequences.

The invention also contemplates host cells transformed by such vectors, For 20 example, the polynudeotides of the invention may be cloned i11to a vector which is transforrned into a bacterial host cell, for example/;'. coli.

Methods for the construction of vectors and their transformation into host cells are genera11y knovm in the art, and described in, for example,, Molecular Cloning: A Laboratory .Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New 25 York, and, At1subel F, M. et al, (Eds) Currem Protocols in Afolecular Biology (2007), John ·wiley and Sons, Inc.

Produc!hm GPC-1 epitopes of the present invention, segments of the GPC-1 epitopes, and 30 fusion proteins comprising the GPC-I epitopes and/or segments thereof can be manufactured according to standard methodologies well known to persons of ordinary ski L1 in the art.

The epitopes, segments and fusion proteins may be prepared using any of a variety of .. vell-known synthetic and/or recombinant techniques.

Polypeptides may, for example, ,s be generated by synthetic means using methodologies well known to those of ordinary 36 skill in the art.

In one non-limiting example, commercially available solid-phase techniques ( e.g. the Ivlerrifield solid-phase synthesis method) may be used in which amino adds are sequentially added to a growing amino acid chain (~forrifield, J.

Am.

Chem.

Soc. 85:2149-21.46, 1963).

Equipment for automated synthesis of the epitopes, s segments and fusion proteins disclosed herein is commercially available (e.g.

Perkin Elmer/Applied BioSysterns Division (Foster City, Calif.), and may be utilised according to the manufacturer's instructions.

Additionally or alternatively, the epitopes, segme.nts and fusion proteins may be produced by any other method available to one of skill in the rut For example, rn recombinant means may be used in \Vhich nucleic acids encoding selected epit<.)pe/s, segment/sand/or fusion prntein/s may be inserted into an expression vector using at1y of a v;,uiety of prncedures known in the an (e.g.

Sambrook et al., 1989, Molecular Cloni11g, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory P.rcss, Cold Spring Harbor, N.Y.; Sambrook and Russell, Molecular Cloning: A Laboratory Jvianua], 3rd edition (Jan. 1s 15, 2001) Cold Spring Harbor Laboratory Press, lSBN: 0879695765; Ammbel et al., Current Protocols in Molecular Biology, Green Publishing: Associates and Wiley lnterscience, NY ( 1989) ).

Construction of suitable expression vectors containing: nucleic adds e11coding selected epitope/s, segrnent/s and/or fosion protein/s employs standard ligation techniques know· to those of ordinary skill in the art. 20 The ligated nucleic acid sequences can be operably linked to suitable transcriptional or translational regulatory elements that facilitate expression of the epitopeis, segment.ls and/or fusion protei.n/s.

Re!;,>iilatory elements responsible for the expression of prnteins may be located 5' to the coding region for the polypeptide.

Stop codons that end tra11slation and transcription tennfoation signals may be present 3' to the nucleic acid 25 sequence encoding the epitope/s, segment/s and/or fusion protein/s.

After construction of a nucleic acid encoding the polypepiide/s of interest with the operably linked regulatory elements, the resultimt expression cassette can be introduced into a host cell m1d the encoded polypeptide/scan be expressed.

In accordance with the present invention, GPC-1 epitopes, segments of the GPC-l 30 epitopes, and fusion proteins comprising the GPC- l epitopes and/or segments thereof, may be "isol.ak:d".

An ''isolated" polypeptide is one rhat is removed from its original environment.

For example, a natmally~occurring pmiein or polypeptide referred to herein is considered to be isolated .if lt is separated from some or all of the co-existing materials in the natural system. [solated polypeptides reterred to herein may also be purified.

For example, the isolated polypeptides may be at least about 90%1 pure, at least about: 950,,,; pure or at I.east about 99~·{> pure.

Polynucleotides and nucleic acids according to the present invention are generally known in the art, and are described, for example, in Ausubel F. M. et al. (Eds) Current s Protocols in j'vfolecular Biology (2007), John Wiley and Sons, Inc; Sambrook 111 al. (1989) Molecular Cloning: A Lahoratmy Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York; and Maniatis el al.

Molecular Cloning ( 1982 ), 280-281.

Polynudeotides may be prepared, for example, by chemical synthesis techniques, for example, the phosphodiester and phosphotriester methods (see for rn example Narang S. A, et af. ( 1979) Meth, EnzymoL 68;90; Brown, E. L. (1979) et at, Meth.

Enzymol. 68: 109; and U.S.

Patent No. 4356270), the diethylphosphoramidite melhod (see Beaucage S .L et al. ( 198 l) Teimhedron Letters, 22: 1859-1862). A method for synthesizing ollgonuclcotides on a modified solid support is described in U.S.

Patent No. 4458066. .Supports Epitopes and fusion polypeptides according to the present invention may be attached to a suppoit TI1e 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 20 mixture.

Suitable supports for immobilizing or localizing epitopes and fusion polypeptides are well know·n 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 microassays, plastic and glass plates with individual wells, as well as other materials compatible with the reaction conditions.

In certain 25 preferred embodiments, the support can be a plastic material such as plastic beads or wafers, or that of the well or tube iu which a particular assay (e.g. an ELISA) is conducted.

Binding Entities 30 The present invention provides binding entities capable of binding specifically to the GPC-1 epitopes described herein.

The bim1iug entity may be any molecule capable of binding specifically to a GPC-1 epitope as described herein.

Non-limiting examples of binding entities include polypeptides, antibodies, antibody fragments, molecular imprints, lectins, and capture ,s compounds.

The binding entity may be an agent that can bind to a GPC-1 epitope of the 38 present invention or alternatively a different region ofGPC-1, 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-l s epitopes of the present invention can hind to a given target epit:ope, combination of epitope segments, or epitope cmnbination preferentially over other non-target molecules.

For example, if the binding entity ("molecule A") is capable of "binding specifically" or "specifically binding" to a given target GPC-1 epitope ("molecule B''), mo1ecule A has the capacity to discriminate betvv'een molecule B and any other number of potential rn alternative binding partners.

Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners,. molecule A. will selectively hind to molecule B and other alternative poienL1al bind.ing partners ,.vill remain substantially unbound by molecule A. ln general, molecule A will prcforentially bind to molecule B at least 10-fold, preferably 50-fold, more preferably 1004o1d, and most 15 preferably greater than 100-fold more frequently than other potential binding partners.

Molecule A may be capablt~ of binding to molecules that an.~ not molecule B at a weak, yet detectable level.

This is commonly known as background binding and is readily discernible from molecule B-specifk binding, for example, by use of an appropriate contro1, 20 In the knuwledge of the specific GPC- l epitopes provided. herein, persons of ordinary skill in the art can generate binding entities vvithout inventive effort.

For example, polyclonal and monoclonal antibody preparations that bind specifically to GPC- 1 epitope/s of the present invention can be prepared using known techniques.

Any technique that provides for the production of antibody molecules by 25 continuous cell lines in culture may be med in the preparation of monoclonal antibodies directed toward a target GPC-1 epitope.

General methodology is described in Harlow and Laue (eds.), O 988), "An1ibodies-A La.boratmy 1vfanual ", Cold Spring Harbor Laboratory, N:Y.

Specific methodologies include the hybridoma technique originally developed by Kohler et al., ( t975), ''Contimt(WS cultures t?{fused cells secreting antibody rdjm:'lieflned 30 spec[j}city", Nature, 256:495-497, as well as the trioma technique, the human B-cell hybridoma techniqlle (Kozbor et al., (1983), "The Production of'llfonoc!onal Antibodies From Human Lymphm..ytes", fmmunology Today, 4:72-79), a.nd the EBV-hybridorna technique to produce human monoclonal antibodies (Cole et al., (1985), in '\l..:fonoclonal Antibodies and Cancer Therapy", pp. 77- 96,.

Alan R.

Liss, Inc,).

Immortal, antibody- ,s producing cell lines can be created by techniques other than fusion, such as direct 39 transformation of B lymphocytes wil:11 oncoge11ic DNA, or hansfoction with Epstein-Barr virus (see, for example, M.

Schreier el al., ( 1980), "H.vbridoma techniques", Cold Spring Harbor Laboratory; Hammerling e1 al., (1981), "Monoclonal Antibodies and T-ce!l Hybridomas", Elsevier/North-Holland Biochemical Press, Amsterdam; Kennett et al., s (1980), "1Wonoctonal Antibodies", Plenum Press). 1n brief, a means of producing a hybridoma from which the rnonoclonal antibody is produced, a myeloma or other self-perpetuating cel1 line can be fused with 1ymphoc)1es obtained from the spleen of a mammal hyperimnmnised 1,vith a recognition factor-binding ponion thereoi~ or recognition factor, or an origin-specific DNA-binding portion thereof_ rn Hybridomas producing a monoclonal antibody capable of binding specifically to a GPC-1 epitope of the present invention are identified by their ability to immunoreact •..-vith the epilope/s presenLed.

A monoclonal antibody useful in practicing the invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a 1s hybridoma that secretes antibody molecules of the appropriate antigen specificity. T11e culture is maintained under conditions ai1d 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. 20 Similarly, there are various procedures known in the art which may be used for the pmduction of polyclonal amlbodfos.

For the production of polydonal antibodies against a given ctJmbination of GPC-1 epitopes, various host animals can be immunised by injection \-..·ith the epitopes, including, but not limited to, rabbits, chickens, mice, rats, sheep, goats, etc.

Further, the target molecule can be conjugated to an immunogenic 25 carrier (e.g., bovine sernm albumin (BSA) or keyhole limpet bemocya11in (KLH)).

Also, various adjuva.nts may be used to increase the inmumological response, including, hut 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 1impet hemocya.nins, dinitrophenol, and potentially 30 useful buman adjm:ants such as BCG (bacille Calmette-Guerin) and Corynebacterium panmm.

Screeuing for the desired antibody can be accomplished by a variety of techniques known in the art.

Suitable assays for immunospedfic binding of antibodies include, but are not limited to, rndioimn:mnoassays, ELISAs (enzyme-linked hmmmosorbent assay), ,s sandv,-·ich immunoassays, immunoradiometric assays., gel diffusion precipitation reactions, immunodfffi.1sim1 assays, in situ immunoassay:s, Western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays, and the like (see, for example, Ausubel ef al., (1994), "Ouren! Protocols in Molecular Biology", VoL l, John Wiley & Sons, s Inc., Ne\.v York).

Antibody binding may be detected by virtue of a detectable label on the primary antibody.

Alternatively, the antibody may be detected by virtue of its binding with a secondary antibody or reagent which is appropriately labelled. A variety of methods for the detection of bindit1g in an immunoassay are known in the art and are included in the scope of the present invention. rn The antibodies (or fragments thereof) raised against specific GPC-1 epitope/s of i.nterest have binding affinity for the epitope/s.

Preferably, the antibodies (or fragments thereof) have binding affinity or avidity g,reater than about 105 ?vr\ more preferably greater than about 106 i\f1 , still more preferably greater than about l07 rvrt and most preferably greater than about 108M-1 . 1s ln terms of obtaining a suitable amount of an antibody according to the present invention, one n::iay manufacttirl~ the antibody(s) using batch fermentation 'With serum free medium.

After fennentation 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 20 with s:olvent/detergent to inactivate any lipid enveloped viruses.

Further purification, typically by anion and cation exchange chromatography rnay 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 fommlated bulk preparation may then be sterilised and viral filtered and dispensed. 25 Non-limiting examples of antibodies capable to binding to one or multiple GPC-1 epitopes of the present invention include: (i) MlL38 antibody as deposited under the terms of the Budapest Treaty at Cellbank Australia at 214 Hawkesbury Road, \Vestmead, NS\V 2145, Australia on 22nd August 20 l 4 under accession number CBA20 140026; 30 (ii) anti-Glypican 1/GPCI antibody (abl37604): a rabbit polyclonal (IgG) specific for human GPC-1 and co1mnercially available from abcam:it (see: http://www.abcam.com/glypican-l-g,pc 1-antibody-ab 137604.html); (iii) !V[AB2600!Anti-Glypican-l Antibody, clone 4DI: a mouse monoclonal (lgGk) specific for human and rat GPC-l and commercially availabl.e from Millipore (see: 41 http://www.emdrnill:ipore.com/AlJ/entprodnct/,"1VfM_NF-MAB2600?cid=BI-XX-BRC-ABl{ JC>ANTI-B033-l308J; (iv) goat anti-glypican 1 antibody (AA 24-530): a goat polyclonal specific for human GPC-1 and c.:onunercially available from LifeSpan BioSciences, Inc. (see s https: / iw,,.,,w. ls bio .com/ anti bodi es/anti-gpc J -autib ody-gl ypic an-antibody-aa24-5 3 0-iccw ·b-\vestern-flm.v-ls-c330760/34 l l 04 ).

Compositions and Kits Compositions and kits according to the present invention may comprise: rn - one or more GPC-1 epitope/s, variant/s or thlb':mentls thereof as described herein (e.g.

Table l); and/ox - one or more GPC-1 epitope sef:,rrnent/s, variant/s or fragment/s thereof as described herein (e.g.

Table 2); and/or - one or more GPC-1 epitope combinations as described herein (e.g.

Table 3); andlor 1s - one or more fusion proteins comprising GPC-1 epitope/s, segment1s of GPC-1 epitopes, and/or variant/s or fragment/s thereof~ as described herein (see subsection above entitled "Fusion polypeptides''); and/or - oi1e or more binding entities as described herein (see section above entitled "Rinding enlities "). 20 The compositions may additionally comprise an acceptable carrier, adjuvant and/or diluent. TI1e carriers, diluents and adjuvants may be "'acceptable" in tenns of being compatible with the other ingredients of the composition, and/or ·'pharmaceutically acceptable" in generally being not deleterious to any recipient thereof Suitable carriers, diluents and adjuvants are ,vell k110\Vt1 to those of ordinary skrn in the rut (see, for 25 example, Gilman et al, (eds,) Goodman and Gilman's: the pharmacological basis of therapeutics, 8tl1 Ed., Pergamon Press (1990); Remington's Phannaceutica] Sciences, Mack Publishing Co.: Eastcm, Pa., 17th Ed. (1985)).

The compositions may, in some embodiments, be used for diagnostic or research purposes.

The kits may be fragmented or combined kits. A "fragmented kit" refers to a 30 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 rnntainers that each contain a snb ponion of the total kit components are included ,vithjn the meaning of the term. fragmented kit A ·'combined kit" refers to a delivery system containing all of the kit components in a single container (e.g. in a single box housing 42 each of t11e des1red components).

The kits may, m some embodiments, be used for diagnostic or research purposes.

Diagnostic ~-fethods s GPC-1 epitopes of the present invention, segments of the GPC-1 epitopes, and fusion proteins comprising the GPC- l epitopes and/or segments thereof.. can be used in diagnostic methods.

Specifically, the present invent.ors. hm'e discovered that glypican-1 is a new marker for prostate cancer (US provisional patent application no. 6 l/928/776 entitled '"('ell S1afi1ce Pros tare Cancer Antigen jar Diagnosis'', \\'alsh el of. - rn unpublished).

Accordingly, tl1e present invention provides for methods for the diagnosis, prognosis, or likelihood of developing prostate cancer in subjects based on the detection of GPC-1 ep-itopc/s, GPC-1 epitopc segments, and/or variants and fragments of the epltopes/epitope segments as described herein.

Additionally or alternatively, variants and 1s fragments ofthe GPC-l epitopes and/or GPC~l epitope fragments may be detected in the diagnostic methods.

Generally, the methods comprise determining the level of GPC-l epitope/s and/or GPC-1 epitope segments in a biological samp'le fron:1 a subject to be tested.

Addltiona1ly or alternatively, the level of variant/s and fragmentls of the GPC-1 epitopes and/or 20 segments thereof in the biological sample may also be detennined when carrying out the methods.

Non-limiting examples of GPC-1 epitopes, GPC-1 epitope segments, and variants and fragments incllide those referred to in the section entitled "Epitopes" (see in particular Tables 1-3 and associated description of varim1ts and fragments).

The methods may additionally comprise determining levels of prostate-specific 25 antigen (PSA), also known as gamma-seminoprotein or kallikrein-3 (KLK3), in the biological sample, and optionally comparing the level of PSA detected ,vilh that of a control.

In smne embodiments, the level of GPC-l epitope/s, GPC-1 epitope segments, and/or variants and fragrnents of the epitopes/epitope segments detected in the subject's 30 biological sample may be compared to levels determined to be present in a control sample.

The levels in the control sample may be detennined before, during or after detem1ining the level present in the subject's biological sample.

By way of non-limiting example, the levels present in the control sampl.e may be detennined based on those present in an equivalent biological sample from an indiYidual or based on mean levels ,s present in biological samples from a population of individuals.

The individual or 43 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 indicative that the subject has prostate cancer, or an increased likelihood. of developing prostate cancer.

For example, an increase 5 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 10 prostate cancer.

In some embodiments, GPC-1 epitope/s, GPC-1 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 assay to confirm that the assay as performed is capable of detecting the GPC-1 epitope/s, 15 GPC-1 epitope segments, and/or variants and fragments.

In some embodiments, the diagnostic methods of the present invention utilise fusion proteins of the present invention.

Non-limiting examples of 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. 20 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 25 rhabdomyosarcoma.

Without limitation to specific detection methods and by way of non-limiting example only, the detection of the GPC-I epitopes, GPC-1 epitope segments, variants and fragments may be by way of standard assays known to those of ordinary skill in the art including, but not limited to Western blot analysis, enzyme-linked immunosorbent assays (ELlSAs), 30 fluorescent activated cell sorting (PACS), a biofilm test, or an affinity ring test (see for example; US application 2013/016, 736). A binding entity according to the present invention may be used the assays.

In some embodiments, the binding entity is an antibody.

In other embodiments, the binding entity is not an antibody.

In some embodiments, the antibody is selected from any 35 one or more of 44 Date Reyue/Date Received 2021-04-20 (i) MIL38 antibody as deposited under the terms of the Budapest Treaty at CeHbank Australia at 214 Hawkesbury Road, Westmead, NSW 2145, Australia on 22nd August 2014 under accession number C BA20140026; (ii) anti-Glypican 1/GPCI antibody (abl37604): a rabbit polydonal (lgG) s specific for human GPC- l and commercially available from abcamit' (see: http://www.abcam.com/glypicau-1-gpc I-antibody-ab 137604 .. html); (iii) MAB2600 Auti-Glypican-1 Antibody, done 4D1: a mouse monoclonal (lgGk) specific for human and rat GPC- l and commercially available from Millipore (see: http://www.emdmillipore.convAU/en/producti,MM~Nf-MAB26009cid=BI-XX-BRC-Arn BIOC-ANTI-B03J-1308)~ (iv) goat anti-glypican l antibody (AA 24-530): a goat polyclonal specific for human GPC-1 and commercially available from LifoSpan Biosciences, lnc. (see https: //www .ls bio .com/antibodi es/anti-gpc 1-anti body-gl yp i c an-antibody-aa2 4-5 3 O-icc ·wb-westem-flmv-ls-c330760/341104). 1s ln some embodiments, the antibody is not selected from any one or more of: (i) :ti.UL38 antibody as deposited mtdt.~r the terms of the Bt1dapest Treaty at Ce11bank Australia at 214 Hav,·kesbury Road, Westmead, NSW 2145, Australia on 22nd August2014 under accession number CBA20140026: (ii) anti-Glypican I iGPC1 antibody (ab 137604): a rabbit polydonal (IgG) 20 specific for human GPC-1 and commercially available from abeam~' {see: http://-ww'>v.abeam.com/ glypican-1-gpc I -antibody-ab 13 7604 .html); (iii) MAB2600 Anti-Glypican- l Antibody, clone 401: a mouse monoclonal (IgGk) specific for human and rat GPC-1 and commercially available from Millipore (see: http://www.emdmillipore.com/AU/en/product/, Mrvl_NF-iv1AB2600?cid=B[-XX-BRC-A- 25 BIOC-ANTI-B033--l308t (iv) goat anti-glypican l antibody (AA 24-530); a goal polydona! specific for human GPC-1 and commercially available from LifeSpan BioSciences, Inc. (see https://www.lsbio.com/antibodies/anti-gpc l-antibody-glypican-antibody-aa24-530-kcv{ b-westem-flow-ls-c330760!341 l04). 30 In some embodiments, the binding entity is an antibody population comprising MlL38 antibody (CBA20140026) and not comprising an anti-glypican l (GPC-l) antibody capable of binding 1o au epitope comprising ,m amino acid sequeuce selected from any one or a plurality of: TQNARA (SEQ ID NO: 8), ALST ASDDR (SEQ ID NO: 9), PRERPP (SEQ ID NO: 10), QDASDDGSGS (SEQ lD NO: ll), LGPECSRAVlvlK ,s (SEQ lD NO: 13), and TQNARAFRD (SEQ ID NO: 7) It will he appreciated by persons of ordinary skill in the art that numerous variations and/or modifications can be made to the present invention as disclosed in the specific embodiments without departing from the spirit or scope of the present invention as broadly described.

The present embodiments are, therefore, to be considered in all s respects as illustrative and not restrictive. rn Examples The present invention will now be described with reference to specific Examples, which should not be construed as in any way limiting.

Example 1: identificatfon and characterisation of a glypican-l epitope bound by murinc ~mti-human glypican l (MIL38-AM4) l.1 1Uaterillls tmdMetlwtls 15 Empirical sets spanning the reference sequence ,vith different confonnatimial constraints were made, and the ,rntibody \Vas probed on the peptide array.

Antibodies 7'vfurine anti human glypican 1 monoclonal antibody MIL38-AM4 was provided as 20 set out in Table 6 below. 1,ible 6: d£'!'il'r-iption ,~fanti/Jmly used ~ ~~~~ 1.

MlL38-AM4.* .......................

Monse .............................. 4.6mgiml(l OOiiD ................. -20 cc.73 ............... OK ............... , * produced .hy hybridomo cells as dqiosired at Ceffbank Australia under acces.,ion number (BA20140026 Peptides The following human glypican-1 (GPC-1) sequence was used as a basis to generate a Hbrnry of structmed peptides. 30 P35052 (GPCJ. HUMAN) 1 lYJELRARGt·VWL LCAP"'A..½L\tAC ARGDPASKSR SCGE\iRQIYG AKGFSLSD~lP 'SO 51 QAEISGEHLR ICPQGY'.I'CCT Sfil-1E:::NLANR SHAELETA::..R DSSRVLQAML 100 101 ATQLRSFDDH FQELLNDSER TLQATF:PGAF GELYTQNJtRA FRDLYSELRL 150 151 YYRGANLHLE E'I'LAEFWARL LERLE'KQLHP QLLLPI:JDY:::,D CLGKQAEALR 200 35 201 PFGEAPRELR LRATRAFVAA R.SFVQGLGVA sD,r'lRKVAQV PLGPECSRAV 250 46 251 MI(LVYC:AHC:::., GVPGARPCPD YCRNVLKGCL ANQADLDAEW RN::.,LDSMVLI 300 301 TDKFWGTSGV ESVIGSVETW LP,EAIN,l\.LQD NRDTLTAK\/I QGCGNPKVNF 350 351 QGPGPEEKRR RG.KLAPRERP PSGTLEKLVS EA.KAQLRDVQ DFWISLPGTL 400 !JOl CSEY1'1ALSTI-\. SDDRCWNGMA RGRYLPEVMG DGLANQINNP EVEVDITKPD 450 5 451 MTIRQQir.1JQL KIMTNRLRSA YNGNDVDYQD P,SDDGSGSGS GDGCLDDLCS 500 !:)01 RIS::VSRKSSSS RTPI,THALPG LSEGEGQKTS A.ASCPQPPTF LLPLLLFLAL 5!::,0 551 TVARPRWR 5 5 8 (SEQ ID NO: 14) w Figure lA shows the rendering of chain B as present under Protein Data Bank identifier (PRD TD) 4A D7 (http://v,,cww.ebLac.uk/pdbsum/4AD7), Chemical~}' UnkedPeprides on Scaffold/; (CLIPS) technology 111e following provides description of general principles of the CLIPS technology 15 utilised.

CLIPS technology strncturally fixes peptides into defined three-dimensional structures.

This results in functional mimks of even the most complex binding sites.

CLIPS technology is now routinely used to shape peptide libraries into single, double or triple looped stnictures as v.rell as sheet- and heUx-like folds. 21, The CLIPS reaction takes place between bromo groups of the CLIPS scatrold and thiol sidechains of cysteines.

The reaction is fast and specific under tnild conditions.

Using this chemistry, native protein sequences are transformed into CLIPS constructs 'vvith a range t1f structures including single T2 loops, T3 double loops, conjugated T2+T3 loops, stabilized beta sheets, and stahilized alpha hellxes (Timmerman et al., J, MoL 25 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.

On a solid carrier. a manix of linear peptides is synthesized, "vhich are subsequently shaped into spatially defined CLIPS constructs.

Constructs representing 31, both pa.its of the discontinuous epitope in the correct conformation bind the antibody with high affinity, which is detected and quantifie~l Constructs presenting the focomplete epitope bind the antibody ,vith 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, 35 The target protein coutaining a discontinuous conformational epitope 1s converted into a matrix library, Combinatorial peptides are synthesized on a proprietary minicard 47 and chemically converted into spatially defined CLIPS constrncts.

Binding of antibodies is quantified.

Pq.11ide .\ynthesis s To rnconslrnct discontinuous epitopes of the target molecule a library of structured peptides was synthesized.

This was done using Chemically Linked Peptides on Scaffolds {CLIPS) technology, CLIPS ted,nology allmved the generation of structured peptides n1 single loops, double-loops, triple loops, sheet-like folds, helix-like folds and combinations thereof. CLIPS templates were coupled to cysteine residues.

The side-chains of multiple rn cysteines in the peptides ,vere coupled to one or two CUPS templates.

For example, a 0.5 mM solution of the T2 CLIPS template l,3-bis (bromornethyl) benzene was dissolved in amn10niurn bicarbonate (20 mM, pH 7.9)/acetonitrile (l:l(v/v)).

This solution was added onto the peptide arrays, The CLIPS template bound to side-chains of two cystcines as present in the solid•phase bot1nd peptides of the peptide•arrays (455 wells plate \:1.rith 3 μI 15 wells).

The peptide arrays were gently shaken :in the solution for 30 to 60 minutes while completely covered in solution.

Finally, the peptide an-ays were washed t~x:tensively w1tl1 excess of H2.0 and sonicated in disrupt-buffer containing 1 percent SDS/0.1 percent betamercaptoethanol in PBS (pH 7.2) at 70"C for 30 minutes, follmNed by sonkation in H2O for another 45 minutes.

The T3 CLIPS carryi11g peptides were made in a similar way but 20 with three cysteines.

Linear and CLIPS pepddes were chemkall sy1lthesized according to the following designs: Set 1 2s Label Mimk Type Description w Set2 Label Mimic Type Description lin Linear peptides Linear peptides oflensith 15 ,vith an overlap of 14 that cover reforence sequence (SEQ JD NO: 14). loop Loop shaped peptides Peptides of length 17, with cyste.ine residues at pos.itions 1 and 1 7, Positions 2 - 16 contain the linear l 5mers of Set 1 in 'Which cysteines are replaced by alanine.

After synthesis, 48 WO 2016/112423 Set 3 s Label rn Mirnic T)'JJe Description 1s Set 4 Label Mimic Type Descriptio11 2s Set 5 Label Mimic Type Description PCT/AU2015/000019 peptides are constrained by a P2 CLIPSTM linker to c011strain the shape. hel i3 Helical peptides Peptides of length 15, with an overlap of 14 derived from the reference sequence (SEQ ID NO: 14).

Cysteine residues were replaced by Alanine.

Then positions 3 and 7 were replaced by Cysteine, which were connected by a P2 CUPSn1,.i This i, i+4 connection ('stapling') induced helix t1ucleaii011 in sequences that were prone lo helical fi.)lding. hel i7 Helical peptides Peptides of length 15,. \vith an overlap of 14 derived froni the reference sequence (SEQ 1D NO: 14).

Cysteine residues were replaced by Alanine.

Then positions 3 and 11 \\·ere replaced by Cysteine, which were connected by a P2 CLIPSTM_ This i, i+ 7 connection ('stapling') induced helix nucleation in sequences that ,;vere prone to helical folding. sheet Sheet shaped peptides Peptides of length 15, with an overlap of 14 derived from the reference sequence (SEQ lD NO: 14).

Cysteine residues were replaced by Alanine.

Then positions 6 and 9 were replaced by Cysteine, \.Vhkh were connected by a P2 CUPS TM. [n peptides prone to p sheet folding this shape was stabilized. 49 ELISA screening "l11e binding of antibody to each of the synthesized peptides was tested in a PEPSCAN-bas.ed EUSA ..

The peptide amrys were incubated ,vith primary antibody solution (o·vernight at 4"C).

After ,vitshing, the peptide arrays \Vere incubated with a 5 1/1000 dilution of an antibody peroxidase conjugate (SBA, cat.m. 2010-05) for one hour at 25°C After v.-asbing, the peroxidase substrate 2,2 '-azino-di-3-ethylbenztbiazoline sulfonate (ABTS) and 2 J,tl/ml of 3 percent HzO2 \vere added.

After one hour, the color development was measured. TI1e color development was quantified \Vith a charge coupled device (CCD) - camera and a11 image processing system. w Data processing The values obtained from the CCD camera ranged from 0 to 3000 nt_-'\U. similar to a standard 96-well plate ELISA-reader.

The results were quantified and stored in the Peplab database.

Occasionally a weU contained an air-bubble resulting in a false-positive 15 value.

The cards were marnmUy inspected and any values caused by an air-bubble were scored as 0. ,~vnthesis quulity coniro! To verify the quality of the synthesized peptides, a separate set of positive and 20 negative control peptides was synthesized in parallel.

These were screened with antibody 57.9 (rel Posthumus et al., J.

Virology, 1990, 64:3304-3309).

Screening deiails Table 7 summarises antibody binding conditions.

For the Pepscan .Buffer and Pre- 2s conditioning (SQ\ the numbens indicaLe the relative amount of competing protein (a combination of horse serum a.nd ovalbumin). 1'uhle 7: scree11it1g condition.I I l\lIL38-k'H4* l ~tg!ml 50%SQ 50%SQ ! [ l\UL38-Al\14*. . ..... IOpg:ml . . _50%S9 _ . . . . 50o/~SQ _ .. ! * produced by hybridoma cells as deposited at Cd/bank Australia muter ac-cessi'on nu.mber CBA.20140026 50 JJrimw:.v experimental results and signal to noise ratio determination A graphical overvit~\V of the complete dataset of ra,;v ELISA results generated by s the screening is showu in Figure 2.

Here a box plot depicts each dataset and indicates the average ELJSA signal, the distribution and the outliers within each dataset.

Depe11diug on experiment conditi.ons (amount of antibody, blocking strength etc) different distributions of ELISA data were obtained.

The data enabled identification of an epitope, The arrny was incubated with Mll38-AM4 at dilutions of l ~1g/ml and 10 ~tg/ml, rn under nomnil stringency conditions. A concentration dependent response was observed (Figures 3A and 3B).

At l 0 μg/rnl saturation was not observed, indicating that none of the peptides represented the ful1 epitope.

Two clusters of responses ihat are not adjacent in the primary structure were identified.

Two common cores emerged: usTQNARA.FRDw, (SEQ ID NO: 7) and JisVNPQGPGPEEK.~ss (SEQ ID NO: 2), with 15 the latter stretch clearly dom1na11t v,rith respect to the fonner.

In t11e three-dimensional (3D) model derived from Protein Data Ba11k identifier (PBD ID) 4AD7 (http://wv.rw.ebtac.ukipdbsum/4AD7) (Figure 4), the stretch nsVNPQGPGPEEK35g (SEQ ID NO: 2) was not resolved, nor was it in another publicly available pdb coordinate file (4ACR, 4BWE), indicating that it probably is flexible.

The 20 stretch us TQNARAFRD14, (SEQ ID NO: 7) lies adjacent to this missing loop, as is evident from the positions of V348 and 0362, ,vllich are resolved in tlle 3D model. 1t is commo11 to see binding to incomplete mimics that partially fulfill antibody requirements similar to the binding that we observe for MIL38-AM4 to the peptides 111 this array.

We postulate a discontinuous epitope, consisting of major contributions from 25 the flexible loop .l4sVNPQGPGPEEK35~ (SEQ lD NO: 2), and minor contributions from (residues of) tJ1e helix 1.1sTQNARAFRD143 (SEQ ID NO: 7). 1.3 ,\'mnmm-y aml J)iscus:;i:tm This study aimed to map the epitope of murine anti human glypican I (MIL38- 30 AM4).

Empirical sets spanning the referenc.e sequence with different conformational constraints were made, and the antibody was probed on the peptide array.

Sibmificant binding ir1dicated a discontinuous epitope, consisting of major contributions from the flexible loop .,,rnVNPQGPGPEEKm (SEQ ID NO: 2), and minor co.ntt"ibutions from (residues of) the helix 135TQNARAFRDw (SEQ ID NO: 7). 51 Example 2: identification and characterisation of additional glypican-1 epitopes bound by other anti-glypic~n-1 antibodies 1. l J.fateril1l!I· and Mc,71/wtls s Table 8 below provides information on the antibodies used i.n this study.

The first three antibodies listed are commercially available.

Tahlii ,'{: dt'Si.'T'lj1fi<m ,if 1mtihadies w,cd - '' ' ' ' "'"~!~.~- ! Anti-Gtytlirnn 1 (GPCl) (AA 24-530)" gnal I mglml 4 "Ci l I OK I f Anti Glypictm l/GPC'l antibody rabbit 0.97mg/ml -20 'Ti 73 OK i ! (ab137(i04)* I t__::~;:~1~:~:r~k.:~:1:~~::c:Dl) ..................... mouse ................... l mgi1nl ..................... 4 _"C/ .l l ................. OK .................. ...1 ! MILJ8-AM3~ mouse I .25mgirnl -W <•Ci 73 used I ~ ..... , ........ , ................................... , ............................................ , ............................................ , ............................................ , ......................................... ' in -~ ( ·ommen:fa!{r available from.· amfb()dir/s-online (htip;i;i.;,1,.,w.amibodicw-online. com); Lf!esp,m Biosciences, inc. (hftps:.:/ww1-1.·.lsbio.cam; * f"t:,mmercially awiilablefrmn t1bca11/; thnp://1v1vw.abcam.com) 'Cm11merciallvr.1vcd!ablefrom Aferck Adillij11we thttp:i>www.emdmitlipore.6mtJ § Orw (?f two dtstinCI anlihodv populations produced l~v mixed h_rbridoma deposited al flw American nss1.1,! 15 Type Cul ti.we Colieclimr (A [CCj under accession number 1/B J 1785 Peptides The human glypican-1 (GPC-1) sequence defined by SEQ ID NO: 14 \.vas used as a basis to generate a Library of strnctured peptides. flgure IA shows the rendering of chain B as present under Protein Data Bank identifier (PBD ID) 4AD7 (http://www.ebi.ac.uk/pdbsum/4AD7).

Chemical(v Linked Peptides on Sci:rflhld£ ((1_! P/-,') technology The general principles of the CLIPS technology utilised il1 these experiments is set om in Example 1 above.

Pepiide -~vnthesis Peptide synthesis \Vas pe1f01111ed usmg the methods referred in Example 1. '.in Chemically synthesized linear and CLIPS peptides were synthesized. according to the desigt1s shmvn in Example l (sets 1-5). 52 ELISA screening "l11e binding of antibody to each of the synthesized peptides was tested m a PEPS CAN -based EUSA, as set out in Example l _ Data processing and synthesi.,; quali(v control Data processing and synthesis quality control was performed as per Example l _ Screening de toils rn Table 9 summarises antibody binding conditions, For the Peps.can 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 1educ.e stringency of binding. 15 Table 9: .w::Peeniltg cmu!itions 2.2 Re.mlt!,, Primary experimental results and signal Jo noise ratio determination 20 A graphical overview of the complete dataset of rav,' ELISA results generated by the screening is shown in Figure .5.

Here 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 ELIS,,\ data are obtained_ The data enabled identification of an epitope for the 2s monoclonal and polyclonal sera, but not for MJL38-AtvB_ 53 !'v/ll,38-A 113 Even when tested at high concentrations ( l Oμg/ml) and at reduced stringency (PBSTween) IV1IL38-Al'vl3 could not be detected.

The sample was also tried with a different secondary antibody (Shet~p Anti Mouse lgG HRP; GE Healthcare, NXA931), but again s no result was obtained.

As a confirmation we tried a direct ELISA, in which l'vfIL38-AM3 was coated onto the plate.

Using Rabbit Anti Mouse [gG - HRP (Southern Biotech) the antibody could not be detected. rn Rabbit Anti G-(J'picanl/GPCI an1ihody ahl37604 Rabbit anti GPCl yields 3 main signals, as can be seen in the corresponding panel in Figure 6.

These signals correspond to Lhe stretches 348 VNPQGPGPEEK,ss (SEQ lD NO: 2}, :16r,PR.ERFPm (SEQ ID NO: 10), andnQDASDDGSGS,i.iq(S.EQ ID NO: 11). 15 Mouse mAb 2600 (MiJ/ipore) Mouse antibody 2600 recognizes one clear peak in all peptide sets, sharing the common core '.i:-1.2LGPECSRA, VMK252 (SEQ m .1\0: 13).

This can be seen in the corresponding panel in Figure 6. 20 Goat anti-CiPC-f ((WC!) Goat antl-GPC-1 y1elds 2 main signals, as can be seen in the corresponding panel in Figurt 6.

These s.ignals correspond to the stretches 348VNPQGPGPEEK3ss (SEQ lD NO: 2), and 4otsALSTASDDR414 (SEQ ID NO: 9}. 2s 2.3 Sum11Utl)' and /)iscussion This study aimed to profile three commercially available anti-Glypican l antibodies and an extra mAb (MIL38-AM3) on the same arrays, The antibodies were probed on an existing peptide array as set out in Example L The commercially available anti GPC-1 antibodies could all be mapped using these arrays.

Antibody l\UL38-AM3 proved 30 refractory to mapping, either due to the discontinuous nature of the epitope or to sample degradation, and did not yield signal 011 any of the arrays.

Rabbit anti GPC-1 recogmzes at least three stTetcbes in glypican l, .rn,VNPQGPGPEEK35,1 (SEQ m NO: 2), ,6c,PRERPP:rn (SEQ ID NO: 10), and 411,QDASDDGSGS487 (SEQ ID NO: 11 ).

Two of these stretches are resolved in ,s coordinate file 4AD7.pbd, as depicted in Figure 7A.

Since this is a polydonal antibody 54 preparation, it cannot be assessed whether the epit:opes are linear or are pan of a complex: epitope.

Mouse anti g!ypican mab 2600 recognizes the stretch 242LGPECSRA vrvtK:m (SEQ ID NO: 13) in al1 peptide sets of the array.

The localization of epitope in coordinate file s 4AD7.pdb is depicted in Figure 78.

Goat anii glypican recobrn1zes at least two stretches in glypican l, Hx VNPQGPGPEEK3si! (SEQ lD NO: 2), and 4lw<\.LST/tSDDR4t4 (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.

Neitl1er stretch is resolved in the available rn coordinate file.

The rabbit and goat polyclonal preparations both recognize a stt·etcll ,48VNPQGPGPEEK,s8 (SEQ JD NO: 2) in Glypican l, which also fonns 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 1s epitopes form a discontinuous epitope, or are manifestations of the polyclonal nature of the sample.

Neither pAb recognizes the stretch mTQNARAw,, which is thought to contribute to !v1IL38-AM4 hinding- l\.fouse 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 gJypican-1 epitopes bound by other auti-glypican-1 antibodies 3.1 1Uateriaf.,, and liiethods 25 Table 10 below provides information on the antibodies used in this study.

Three a.nti-glypica.n l antibodies were used, each having been used in earlier experiments as described i.n Example 1 and/or Example 2 above.

Ta hie W: description of antibfJdies U'ietf ~~101$~■■m-•~■~~•··■••■■"■•■~•m•~-•~• •~~•~•)1 fft¾••,~■~-~~~~~1''~1fff®,,1:?1~'s~1::~-··•~1'1¾•·····~ I Anti-Glytlicait 1 (GPCI) (AA 24-530)" goat l mg/ml 4 "Ci l I OK I t Anti Glypk-an l/GPCl antibody rnbbil 0.97mg/Jul -20 '''C/ Tl OK I I (abl37604)" I t "''-'-•"'""""""•''-'•'>'-> .'n'>>'-''-'-. i • ·•"'-""""""""'"""'n'>'>'>'>',,'>'>'n',,'>'>'>'>>'>'>"'>'>'>'>'•"""'""""""""""'''"'-'•'n',,',',,'>',,'>'>'n\, ..... ,. ... ,.,.,.,,_,_,.,,.,.,,.,,.,,.,,,.,.,_,.,.,_ '"'"""'""''"""""• ........ ,.,,,,.,.,,_,._,.,.,_,,_,,.,,,.,.,.,,,,,.,,_,.,_,.,._,,_,_,.,.,._.,,._,,. _ "''"'"'""""'""""""""""'"'" ~ i M.ILl8-AM4 mouse 4.6 mμ/rnt -20 C'(! 7'.I OK I .l" -""'"""'"""""""""""'"'''""''""'"'"""'"'"""""""""'"'''""''""'''"""'''"''"""""""'"'''""'" "-··,,,· """"""""'""""""'"""'""'""""'"""""'"""""""'""''; 30 ·'' Commercfal(r mmilctble Ji-r;m: antibudies-onli,w (http::'.IH•ww,anribodies-online. com): l,t/c',1pan Biosciences, Ifie. fhflps:.,/wwl-v,lsbio.com) * Commercialfr amilaMefi'om ahcam'" {htip:tlH'Wh',abcam,com) 1 Produced by !rybridoma cells as deposited ar Cellbank Atwral!a under acce:s.ston number CliA20140026 55 Peptides The human glypican-1 (GPC- J) sequence on which this study \.vas based is defined in SEQ m NO: 14.

The follmving seqnences of residues were used: GPCl residues #344-366 GNPKVNPQGPGPEEKRRRGKLAP (SEQ ID NO: 15) GPCl residues #131-149 GELYTQNARAFRDLYSELR (SEQ ro NO: 16) Peplide synthesis 10 Peptide synthesis ,vas performed usmg the methods refeJTed in Exmnple 1.

Chemically synthesized linear and CLIPS peptides were synthesized according to the designs shown belm'I.': Set 1 Mimic Type Label Discontinuous epitope mimics MAT.A,MAT.B Description Constrained peptide mimics of varying len!:,>ih.

From the two sta1ting sequences (SEQ lD NO: 15 and SEQ lD NO: 16) all 10 to 22, and 10 to 18 mer peptides with stepsize 4 were made, and these were been paired.

At the tcm1ini and hl between the t\vo peptides cystcincs \Vere placed.

These were linked by a T3 CUJ-'S. 15 ~ Mimic Type Linear peptides Label RN .PKVNPQGPGPEEKRR (SEQ ID NO: J 7) Description Substitution analysis, starting from the base sequence PKVNPQGPGPEEKRR (SEQ lD NO: 18), aLI individual amino acids ,:vere replaced by all naturally occurring amino acids, except cysteine.

Set 3 Mimic Type Constrained peptides.

Label RN.PKVNPQGPGPEEKRR ___ L0OP (SEQ ID NO: 17), 56 RN.F.LCTQNCRAFRDLYS_he1i3 (SEQ ID NO: 19} RN.ELCTQNCRAFRDLYS_LOOP (SEQ ID NO: 19) Description Substitution analyses, starting from the base sequences as indicated in the label names, aH individual amino acids were replaced by all naturally occuning ami110 acids, except cysteine.

EUSA screening The binding of antibody to e-ach of the synthesizt!d peptides was tested in a PEPSCAN-based ELISA, as set out in Example l. s Data processing and -~vmhesis qualizr control Data processing and synthesis quality control was perfonned as per Example l.

Hem map analysis A brief overview of the heat map methodology used is set out below. m A heat map is a graphical representation of data vvhere the values taken by a variable in a two-dimensional map are represented as colours.

For double-looped CLIPS peptides, such a two-dimensional map can be derived from the independent sequences of the fast and second loops.

For example, the sequences of a given series of 16 CLIPS peptides are effectively permutations of 4 unique sub-sequences in loop I and 4 unique J5 sub-sequences in loop 2.

Thus, 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 sequeuce of the second loop.

To further facilitate the visualization, ELISA values can be replaced with a continuous gradient of shading, [n this case, extremely lov,, values an~ light coloured, :xi extremely high values are darker coloured, and average values are black coloured.

When this gradient map :is applied to a data matrix, a shaded heat map is obtained.

Screening details Table 11 summarises antibody bindiug cond1tfons.

For the Pepscan Buffer and Pre- 2s conditioning (SQ), the numbers indicate the relative amount of competing protein (a combinati011 of horse serum and ovalbumin). 57 3.2 Results PritnaJ)' experimental results and signal to noise ratio determination 5 A graphical overview of the complete dataset of raw ELISA results generated by the screening is shown in Figure 8.

Here a box plot depicts each dataset aitd indicates the ave:ragc EUSA signal, tho distribution and the outliers within each dataset.

Depending on experiment conditions (amom1t of antibody, blocking strength etc) different disuibutions of ELISA data are obtained.

Rabbit po~vdrmal Ab! 37604 Jn Example 2 the stretch 348 VNPQGPGPEEK35g (SEQ ID NO: 2) was found to suffice for binding some IgG from rabbit polyclona1 Ab 137604.

In this study all constructs containing _\,rnVNPQGPGPEEm (SEQ lD NO: 3) again were bound by IgG 15 from this sample at 1/2500 dilution. in the matrices of Sctl there is no augmentation of signal in specific constructs.

From the substitution analysis in Figure 9 it can he seen that residues P353, G354, and E356 are esse11tiat Limited substitutio11s are aI!o'wed at positions Q35 l, G352, and E357, Goat polyclonal antiGPC-1 In Example 2 the stretch 343\INPQGPGPEEK,ss (SEQ ID NO: 2) also sufficed to bind some Is>G from goat polyclonal antiGPC-l.

This antibody recognizes the same loop as the rabbit polydonal, but does so with a slightly different fine specificity. fn the 2s Letterp.1ot of Figure 10 it can be see.n that the most critical residues are G352, G354, and FJ56, and that limited substitution is allowed on positions N349, Q35 L and P353.

In the matrices of Set I augmentation of signal is seen.

Although 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. 58 Antihm~v Mll38-AM4 :In Examples I and 2 it was established that M IL38 AM4 binds glyt1ican on stretch J4HVNPQGPGPEEK:Nl (SEQ ID NO: 2), and also binds to the stretch mTQNARA140 (SEQ ID NO: 8), which was taken as an indication for a discontinuous epitope. s The looped constructs containing the main stretch pinpoint the residues that are most critical to binding.

From Figure 10 it can be seen that residues V348, Q351, G352, and P353 do not tolemte replacement, with significant requirement for K347, N349 and P350, and to a lesser extent from G354, P355, and E356.

Optimization of a mimic for recognition by MIL38-AM4 by adding residues from rn the range 1 35 - 143 to the main loop matrices of Set I.

The requirement for V348 and surrounding residues was again evident in these series.

There was additional binding in lhe matrix sets culminating in optimal signal for T3 constrained CGELYTQNARA.FRDLCGNPKVNPQGPGPEEKRRRGC (SEQ ID NO: 12) (Figure 12), 1s Similarities and dissimilarities The antibodies bind to or not bind to similar constructs, as can be seen in the scatter plots of Figure 13.

HmveYer, many constrncts are exclusively seen by one of the preparations (points along the axes, or in the lower right hand and upper left hand comers).

Conclusions 1n follow-up to Examples 1 and 2, the confom1ationaJ epitope of antibody MIL 38- AM4 was profiled.

Polyclonal antibodies AB 137604 (Rabbit) and anti GPC-1 24-530 (Goat) were found to recognize a similar epitope.

These were contrasted and compared on :~ the same arrays.

The two leads obtained in Example 1 that point to a discontinuous epitope for MIL 38 - AM4 were used to generate a matrix array in which the loops have different lengths. 111 addition, full substitution analyses of the individual lead sequences v.,-ere made. AU arrays were probed with the three antibodies listed above. 3n For recognition of glypican 1, all antibodies investie,rated in this study bind to au ep1tope that exclusively or mainly consists of the flexible loop between residues 34 7 and 358.

However their fine specificities differ, which may have implications for their functional properties, which in turn may influence selectivity in vivo or applicability in discrimitmtive tests. 59 The epitope of (some lgG spec:ies prese11t in) rabbit polydonal Ab 137604 is the linear stretch ,w,VNPQGPGPEEm (SEQ ID NO: 3). ·mere is no indication for the presence of antibodies that recog11ize a conformational epitope.

The same flexible loop is also seen by (some lgG species in) goat pol.yclonal anti s GPC-1.

There is some preference for structured mimics, a.lthough this is not major.

It may w·ell be that not all antibodies in this preparation see the flexible loop in the same manner.

Monoclonal antibody MIL38-Mv14 mainly binds glypican 1 on the loop between residues 347 ~ 355, but this antibody clearly benefits from the addition of residues from the range 135 - 143 to the peptide.

The mimics that are produced a1e still suboptimal, which is rn reflected in the fact that 1000-fold higher concentrations of antibody are needed to obtain similar signal intensities as are recorded for rabbit Ab 137604, further demonstrating the additional requirements posed on the binding substrate.

This does not have implications for the affinity towards tllc target protein, which is to be detem1ined by quantitative methods (e.g.

Biacore).

In fact exquisite selectivity 1s hallmarks antibodies tlmt can be used in 1•ivo without causing side effects.

X I R11bbit Anti Gl;t'1Jica11 34t VNPQGPGPEE"' I 11GPC1 antibodv (SEQ ID NO: 3) P353, G354, E356 N I (ab137604) • Based on the results presented above, Table 13 below represents a summary of w substitutions tolerated in the epitope sequences analysed. 60 -O', Tubl;: J 3: S11mmary• of tvle.r-atcJ :mb,;tihtlhms fo epiL,ipe :;rqm·m:es ;tlli~~~:i:i:iilliii::f:llii::iiii:~: Tolerated substitutions in Mll38-AM4 Ab Tolerated substitution~ i11 j G11al Anti• GIYflican 1 (GPCI) (AA 24- I 530 ► Ah et)itorie Tolerated substitutions in Rabbit Anti Glypican l/GPC1 1mtibody (ahl37604) WRLYF v I v' I X HPD I RKWSHN X v I H I fl' NMTSR I/ I v I v I YAEVMlLITR t,/ Means that :rnv amino :3C\iLsubstitution .:::rn he lok-r:i!ed at this JXlsition X Means that 110 amino aci~ substituti011 can he tokrat.cd at this position X X DENQKRA MAIKRQSTY ., ., V I X I A I X I NOT RNQVIL I X I v I t,/ I I ASTH WY FM. I X I X I v' ! X I QUHIM I I/ A (ala), R (arg). N (asn), (asp), B (asxj. C (cys), E (,.:!11), Q (gin)., Z {glx). G (glv), H (his), 1 (ilej. L (leu/, K {ly,), M (met), F (phe), P (pm}, S lwr), T (thr). W (trp), Y (tvr}, or V (val) ~ N -0 --~ .N.. .

N (,,,j -= r"'l ~ s -0 u, -0 \0 ~ ""'' -.J w -.J -.J f-' "0 ' f-' -.J I -.J I f-' "' Example 4.

MlL38 and anti-glypican-1 (anti-GPC-1) antibodies show ovedapping reacth'ity on 20 western blots.

Rabbit anti-GPC-1 antibody abl37604 showed reactivity with the glypican-t core protein at a molecular weight of approximately 60 kDa - the same nmlecnlar \Veight as s detected by l'vlIL38.

To confirm that MIL38 recog;li:zed glypican-1, prostate cancer DU- 145 M.PEK extracts "vere subjected to 2D electrophoresis and western blotting.

Membrn11e protein extracts (MPEK) ofDU-!45 prostate cancer cells were separated on 2D gel (pl gradient-horizontal, and molecular mass vertical). \Vestem blots using M1L38 antibody and commercial rGPC-1 rabbit polyclonal antibodies shm:v overlapping 10 reactivity marking a 60Kd protein (circled in Figure 14).

Lane D (Figure 14) is a one dimension separation for DU-145 extracts as a control.

Lane M (Figure 14) is a one dimension separation lane for molecular size markers as controls.

As shown in Figure 14, l\tlL38 antibody and the anti-GPC-1 antibodies shO\'lled overlapping reactivity detecti11g a protein with 60 kDa molecular weigl1t and isoelectric JS points ranging from 5 to 7.

Example 5. l\UL38 is detected in auti-GPC-l inmmnoprecipitates and vice versa.

MIL38 or rabbit anti-GPC-l antibodies were used to imnrnnoprecip.itate their respective antigens from DU-145 or C3 (MTL38 negative) MPEK extracts.

The 20 inum:mopredpitates (IPs) were western blotted with either MIL38 or anti-GPC-1 antibody (Figure 15).

A 60kDa GPC-1 reactive band was detected in MIL38 IPs blotted with anti-GPC-1, while a 60 kDa Iv1IL38 reactive band was detected in anti-GPC-l lPs blotted with MIL38.

No reactivity was detected ,vith the secondary only controls_ Furthermore, 2s imnmnoprecipitating vvith MIL38 antibody resulted in almost complete depletion of both rvHL38 and anti-GPC-1 antigens, strongly suggesting that the MIL38 antigen is glypicanL MIL38 and rabbit anti-GPC-1 antibodies ,:,,,ere each used to immunoprecipitate their antigens from DU-] 45 prostate cancer or C3 (t,,,•1IL38 negative) cell membrane protei11 30 extracts.

Shov,•'n are the western blots of tl1e immunoprecipitations detected with either MIUS or anti-GPC-l antibody.

Figure 15A depicts GPC'.1 detection of MIL38 immunopredpitates (left) and MIL38 detection of GPC l imrnunoprecipitates (right}.

Figure lSB depicts MIL38 detection of MTL38 immunoprecipitates as a cmHrol.

Lanes are: Magic Mark-commercial protein marker as control; DU 145 1\-JPEK- prostate cancer membrane protein extract {not immunoprecipitated); DU145 FT- prostate cancer flow through from iimmme precipitation; DU145 IP- immunoprecipitate using antibody; C3 MPEK- (fi-·1IL38 negative) control membra11e protein extract (not immunoprecipitated); CJ FT- flow through from immune precipitation; C3 IP elu1ion- (MIL.38 negative) s imnmnopredpitate using antibody. MIL38 can detect the immunoprecipitate from rGPCl antibody and vice versa. MIL3 8 can also bind to all controls including Dli 145 MPEK and to IP conducted by MlL38.

Example 6. GPC-1 can he detected by M1L38 in prostate cancer plasma samples and 10 in membrane extracts from prostate cancer patients.

Plasma samples from one nonnal patient (04:2) and one prostate cancer patient (046) ,vere immunoprecipitated with MlL38 antibody and the IP sample western blotted with MJL38 and ant.i-GPC-1 antibodies (Figure 16A).

Both antibodies detected specific bands of approx. 70kDa in both plasma samples.

JS The signals \Vere markedly higher (darker bands) for both MIL38 and anti-GPC-1 antibodies in the prostate cancer patient plasma compared to the normal patient plasma, suggesting that this soluble form of glypican-1 may be elevated in prostate cancer patients.

To detennine if i\t!IL38 antigen could be detected in membrane protein extrncts 211 from nonnal prostate and prostate cancer, Otle sample of each was obtained from Novus Bio.

Eqt1ivalent amounts of protein ,:vere western blotted using MIL38 antibody (Figure l6B).

The prostate cancer extract demonstrated much higher expression of the MlL38 antigen than the normal prostate sample. 25 Example 7.

Detection of MlL38 antigen in patient urine.

MIL38 can detect cells in the urine of prostate cancer patients.

To test the sensitivity and specificity of this detection method, 125 age-matched urine samples ,vere obtained.

Cells ,vere spun dmvn from the urine and analyzed by the MIL.38 indirect immunofluorescence assay. A total of 47 healthy controls, 3 7 benign prostatic .w hypertrophy (BPH) an<l 41 biopsy-confirmed prostate cancers were analyzed The MJL38 immunofluorescence assay (!FA) test demonstrated a sensitivity of 71 % 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). 63 1'11hle l .:J: Sen.1·itivity am/ specificity culculations flfprosfate c,m,·er detectfon in patient ur.ine.

Sensitivity and Specificity Cakula.tions Trne Positive False Positive 29 12 False Negative True Ne.,ative ~ 23 61 St•nsitivity and Specificity Calculations for BPH only True Positive False Positive 29 .1 .., L false Negative True Negative 9 28 I I I 64

Claims (31)

1. CLAIMS 1. An isolated epitope or epitope segment 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 N 0: 1 ), wherein the isolated epitope or epitope segment consists of: (i) KVNPQGPGPEEK (SEQ ID NO: 1); (ii) a fragment of KVNPQGPGPEEK (SEQ ID NO: 1) consisting of VNPQGPGPEEK (SEQ ID NO: 2), VNPQGPGPEE (SEQ ID NO: 3), NPQGPGPEE (SEQ ID NO: 4), KVNPQGPGPE (SEQ ID NO: 5) or KVNPQGPGP (SEQ ID NO: 6); (iii) a variant of SEQ ID NO: 3 with a substitution at any one of: position 1, wherein V (val) is substituted with any other amino acid, position 2, wherein N (asn) is substituted with any other amino acid, position 3, wherein P (pro) is substituted with any other amino acid, position 4, wherein Q (gln) 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), position 5, wherein G (gly) is substituted with A (ala), S (ser), T (thr), H (his), W (trp), Y (tyr), F (phe), or M (met), position 8, wherein P (pro) is substituted with any other amino acid, position 10, wherein E (glu) is substituted with Q (gln), D (asp), F (phe), H (his) or M (met), (iv) a variant of SEQ ID NO: 4 with a substitution at any one of: position 1, wherein N (asn) is substituted with H (his), position 2, wherein P (pro) is substituted with any other amino acid, position 3, wherein Q (gln) is substituted with any one ofN (asn), M (met), T (thr), S (ser), or R (arg), position 5, wherein P (pro) is substituted with A (ala), position 7, wherein 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), position 9, wherein E (glu) is substituted with any other amino acid; or (v) a variant of SEQ ID NO: 5 or SEQ ID NO: 6 with a substitution only at any one of: 65 Date Rer;ue/Date Received 2024-02-21 position 1, wherein K (lys) is substituted with any one ofW (trp ), R (arg), L (lys), Y (tyr) orF (phe); position 3, wherein N (asn) is substituted with any one ofH (his), P (pro) or D (asp); position 4, whereinP (pro) is substituted with anyone ofR (arg), K (lys), W (trp), S (ser), H (his) or N (asn); position 8, wherein G (gly) is substituted with any one of D (asp), E (glu), N (asn), Q (gin), K (lys), R (arg) or A (ala); position 9, whereinP (pro) is substituted with anyone ofM (met), A (ala), I (ile), K (lys), R (arg), Q (gin), S (ser), T (thr), or Y (tyr).
2. 2. An isolated epitope consisting of a first segment according to claim 1 and a second segment, wherein: (i) the first segment is: the fragment of KVNPQGPGPEEK (SEQ ID NO: 1) consisting ofVNPQGPGPEEK (SEQ ID NO: 2), KVNPQGPGPE (SEQ ID NO: 5) or KVNPQGPGP (SEQ ID NO: 6); the variant of VNPQGPGPEEK (SEQ ID NO: 2); the variant of KVNPQGPGPE (SEQ ID NO: 5); or the variant of KVNPQGPGP (SEQ ID NO: 6); and (ii) the second segment consists ofan amino acid sequence TQNARA (SEQ ID NO: 8).
3. 3. An isolated epitope consisting of a first segment according to claim 1 and a second segment, wherein: (i) the first segment is: the fragment ofKVNPQGPGPEEK (SEQ ID NO: 1) consisting ofVNPQGPGPEEK (SEQ ID NO: 2), KVNPQGPGPE (SEQ ID NO: 5) or KVNPQGPGP (SEQ ID NO: 6); the variant of VNPQGPGPEEK (SEQ ID NO: 2); the variant of KVNPQGPGPE (SEQ ID NO: 5); or the variant of KVNPQGPGP (SEQ ID NO: 6); and ( ii) the second segment consists of an amino acid sequence TQNARAFRD (SEQ ID NO: 7).
4. 4. An isolated epitope consisting of a first segment according to claim 1, and a second segment, wherein: (i) the first segment is: the fragment of KVNPQGPGPEEK (SEQ ID NO: 1) consisting ofNPQGPGPEE (SEQ ID NO: 4); or the variant ofNPQGPGPEE (SEQ ID NO: 4); and 66 Date Rer;ue/Date Received 2024-02-21 (ii) the second segment consists of an amino acid sequence ALSTASDDR (SEQ ID NO: 9).
5. 5. An isolated epi tope consisting of a first segment according to claim 1, and a second segment, wherein: (i) the first segment is: the fragment of KVNPQGPGPEEK (SEQ ID NO: 1) consisting ofVNPQGPGPEE (SEQ ID NO: 3); and (ii) the epitope second segment comprises: an amino acid sequence PRERPP (SEQ ID NO: 10) or an amino acid sequence QDASDDGSGS (SEQ ID NO: 11).
6. 6. An isolated epitope consisting of a first segment according to claim 1, a second segment, and a third segment wherein: (i) the first segment is: the fragment of KVNPQGPGPEEK (SEQ ID NO: 1) consisting ofVNPQGPGPEE (SEQ ID NO: 3); and (ii) the second segment consists of an amino acid sequence PRERPP (SEQ ID NO: 10), and the third segment consists ofan amino acid sequence QDASDDGSGS (SEQ ID NO: 11).
7. 7. An isolated epitope comprising the epitope segment according to claim 1 and consisting of the amino acid sequence CGELYTQNARAFRDLCGNPKVNPQGPGPEEKRRRGC (SEQ ID NO: 12).
8. 8. The epitope according to claim 1, wherein the epitope is a linear epitope.
9. 9. An isolated epitope consisting of a first segment according to claim 1, and a second segment consisting of an amino acid sequence selected from any one or a plurality of: TQNARA (SEQ ID NO: 8), ALSTASDDR (SEQ ID NO: 9), PRERPP (SEQ ID NO: 10), QDASDDGSGS (SEQ ID NO: 11), LGPECSRAVMK (SEQ ID NO: 13), and TQNARAFRD (SEQ ID NO: 7).
10. 10. The epitope or epitope segment according to any one of claims 1 to 9, wherein the epitope is a synthetic polypeptide. 67 Date Rer;ue/Date Received 2024-02-21
11. 11. A composition comprising an epitope according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier or excipient.
12. 12. An assembly comprising an epitope according to any one of claims 1 to 10, bound to one or more soluble or insoluble supports.
13. 13. The assembly of claim 12, wherein the assembly is a component of an enzymelinked immunosorbent assay (ELISA).
14. 14. A nucleic acid encoding the epitope according to any one of claims 1 to 10.
15. 15. A vector comprising the nucleic acid according to claim 14.
16. 16. A host cell comprising the vector according to claim 15.
17. 17. An isolated binding entity capable of specifically binding to an epitope according to any one of claims 1 to 10, wherein the binding entity is an antibody fragment.
18. 18. An isolated binding entity capable of specifically binding to an epitope according to any one of claims 1 to 10, wherein the binding entity is an antibody and with the proviso that the antibody is not a: MIL38 antibody (CBA20140026), rabbit anti-GPC-1 polyclonal antibody (abl37604, abeam), mouse anti-glypican monoclonal antibody 2600 clone 4Dl (Millipore), or goat anti-glypican 1 antibody (AA 24-530).
19. 19. A method for detecting prostate cancer in a subject, the method comprising detecting the presence of an epitope according to any one of claims 1 to 10 in a biological sample obtained from the subject, 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, wherein the method comprises comparing the amount of epitope present in the biological sample with an amount of epitope present in a control sample, and wherein the detection of an increased amount of epitope in the biological 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. 68 Date Rer;ue/Date Received 2024-02-21
20. 20. The method according to claim 19, wherein detecting the presence of the epitope in the sample comprises contacting the sample with a population of antibodies capable of specifically binding to an epitope according to any one of claims 1 to 10.
21. 21. The method according to claim 20, wherein the population of antibodies comprises any one or more of: MIL38 antibody (CBA20140026), rabbitanti-GPC-1 polyclonal antibody (ab137604, abeam), mouse anti-glypican monoclonal antibody 2600 clone 4Dl (Millipore), or goat anti-glypican 1 antibody (AA 24-530).
22. 22. The method according to claim 20, wherein the population of antibodies does not contain any of: MIL38 antibody (CBA20140026), rabbit anti-GPC-1 polyclonal antibody (ab137604, abeam), mouse anti-glypican monoclonal antibody 2600 clone 4Dl (Millipore), or goat anti-glypican 1 antibody (AA 24-530).
23. 23. The method according to claim 19, 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.
24. 24. The method according to any one of claims 19 to 21 or 23, wherein detecting the presence of the epitope comprises contacting the sample with a population of MIL38 antibodies as deposited at Cellbank Australia under accession number CBA20140026.
25. 25. The method according to any one of claims 19 to 23, wherein detecting the presence of the epitope comprises contacting the sample with a population of anti-glypican 1 antibodies that does not comprise an antibody comprising a light chain variable region comprising: a complementarity determining region 1 (CDRl) 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; a complementarity determining region 3 (CDR3) comprising or consisting of an amino acid sequence defined by positions 113-121 of SEQ ID NO: 20. 69 Date Rer;ue/Date Received 2024-02-21
26. 26. The method according to any one of claims 19 to 25, further comprising determining the level of prostate-specific antigen (PSA) in the biological sample and comparing the level detected to that of the control sample.
27. 27. The method according to any one of claims 19 to 26, wherein the biological sample is a body fluid sample.
28. 28. The method according to any one of claims 19 to 26, wherein the biological sample is a tissue sample.
29. 29. A fusion protein comprising the epitope according to any one of claims 1 to 10.
30. 30. Use of the epitope according to any one of claims 1 to 10, or the fusion protein according to claim 29, as a positive control element in a method for detecting GPC-1.
31. 31. The use according to claim 30, wherein the method is the method for detecting prostate cancer according to any one of claims 19 to 28. 70 Date Rer;ue/Date Received 2024-02-21