U.S. patent application number 12/217191 was filed with the patent office on 2010-07-01 for laminin receptor 1 precursor protein (37lrp) epitope delineated by an hepatocellular carcinoma specific antibody.
This patent application is currently assigned to Arius Research Inc.. Invention is credited to Susan E. Hahn, Michelle Kelleher, Fortunata McConkey, Andrea Warner, David S. F. Young.
Application Number | 20100166652 12/217191 |
Document ID | / |
Family ID | 35056187 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100166652 |
Kind Code |
A1 |
Young; David S. F. ; et
al. |
July 1, 2010 |
Laminin receptor 1 precursor protein (37LRP) epitope delineated by
an hepatocellular carcinoma specific antibody
Abstract
This invention relates to the diagnosis and treatment of
cancerous diseases, particularly to such diagnosis and treatment
which revolves around the ability of the 5LAC-23 monoclonal
antibody (or antigenic binding fragments derived therefrom) to bind
with the Laminin Receptor 1 Precursor Protein 37LRP; and most
particularly to diagnosis and treatment of Hepatocellular Carcinoma
by various means which rely upon direct binding of 5LAC-23 with the
particular antigenic moiety specifically recognized thereby and
generally overexpressed in Hepatocellular carcinoma cells. The
invention additionally relates to the treatment of such cells with
conjugated moieties effective to aid in differentiation, treatment
and diagnostic imaging thereof.
Inventors: |
Young; David S. F.;
(Toronto, CA) ; Hahn; Susan E.; (Toronto, CA)
; McConkey; Fortunata; (Shelburne, CA) ; Kelleher;
Michelle; (Wantage, GB) ; Warner; Andrea;
(Oxford, GB) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Arius Research Inc.
Oxford Biomedia UK Ltd.
|
Family ID: |
35056187 |
Appl. No.: |
12/217191 |
Filed: |
July 1, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11079969 |
Mar 14, 2005 |
7419792 |
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12217191 |
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10810163 |
Mar 26, 2004 |
7442776 |
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11079969 |
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10713642 |
Nov 13, 2003 |
7256272 |
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10810163 |
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09727361 |
Nov 29, 2000 |
6657048 |
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10713642 |
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09415278 |
Oct 8, 1999 |
6180357 |
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09727361 |
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Current U.S.
Class: |
424/1.49 ;
424/133.1; 424/134.1; 424/9.1; 435/325; 435/377; 435/7.21 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/30 20130101; G01N 33/57419 20130101; A61K 47/6851 20170801;
G01N 33/57476 20130101; G01N 33/6893 20130101; C07K 16/3069
20130101; A61P 43/00 20180101; G01N 33/57438 20130101; G01N
33/57415 20130101; G01N 33/57449 20130101; C07K 16/303
20130101 |
Class at
Publication: |
424/1.49 ;
435/325; 435/377; 435/7.21; 424/133.1; 424/134.1; 424/9.1 |
International
Class: |
A61K 51/10 20060101
A61K051/10; C12N 5/071 20100101 C12N005/071; G01N 33/567 20060101
G01N033/567; A61K 39/395 20060101 A61K039/395; A61K 49/00 20060101
A61K049/00; A61P 35/00 20060101 A61P035/00 |
Claims
1. A process for differentiation, treatment, or diagnostic imaging
of cells which express 37LRP comprising: providing a sample of said
cells; providing a conjugated moiety including an isolated
monoclonal antibody or antigen binding fragment thereof, said
isolated monoclonal antibody or antigen binding fragment thereof
being an isolated monoclonal antibody or antigen binding fragment
thereof which binds to an expressed 37LRP antigenic moiety, said
antigenic moiety characterized as being bound by the isolated
monoclonal antibody produced by the hybridoma deposited with the
ATCC as PTA-5690, said isolated monoclonal antibody or antigenic
binding fragment thereof being conjugated with at least one member
selected from the group consisting of drugs, toxins, enzymes, or
radioactive compounds; whereby binding of said conjugated moiety
with said cells results in differentiation, treatment, or
diagnostic imaging of said cells.
2. A method for treating a patient suffering from a cancerous
disease comprising: providing a conjugated moiety including an
isolated monoclonal antibody or antigen binding fragment thereof,
said isolated monoclonal antibody or antigen binding fragment
thereof being an isolated monoclonal antibody or antigen binding
fragment thereof which binds to an expressed 37LRP antigenic
moiety, said antigenic moiety characterized as being bound by the
isolated monoclonal antibody produced by the hybridoma deposited
with the ATCC as PTA-5690, said isolated monoclonal antibody or
antigenic binding fragment thereof being conjugated with at least
one member selected from the group consisting of drugs, toxins,
enzymes, or radioactive compounds; and administering said
conjugated moiety to said patient.
3. The method of claim 1 wherein said isolated monoclonal antibody
is a humanized version of the isolated monoclonal antibody produced
by the hybridoma deposited with the ATCC as PTA-5690 or antigenic
binding fragment thereof.
4. The method of claim 1 wherein said isolated monoclonal antibody
is a chimeric version of the isolated monoclonal antibody produced
by the hybridoma deposited with the ATCC as PTA-5690 or antigenic
binding fragment thereof.
5. The method of claim 2 wherein said isolated monoclonal antibody
is a humanized version of the isolated monoclonal antibody produced
by the hybridoma deposited with the ATCC as PTA-5690 or antigenic
binding fragment thereof.
6. The method of claim 1 wherein said isolated monoclonal antibody
is a chimeric version of the isolated monoclonal antibody produced
by the hybridoma deposited with the ATCC as PTA-5690 or antigenic
binding fragment thereof.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
11/079,969, filed Mar. 14, 2005, which is a continuation-in-part of
application Ser. No. 10/810,163, filed Mar. 26, 2004, which is a
continuation of application Ser. No. 10/713,642, filed Nov. 13,
2003, now U.S. Pat. No. 7,256,272, issued Aug. 14, 2007, which is a
continuation of application Ser. No. 09/727,361, filed Nov. 29,
2000, now U.S. Pat. No. 6,657,048, which is a continuation-in-part
of application Ser. No. 09/415,278, filed Oct. 8, 1999, now U.S.
Pat. No. 6,180,357 B1, the contents of each of which are herein
incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the diagnosis and treatment of
cancerous diseases, particularly to such diagnosis and treatment
which revolves around the ability of the 5LAC-23 monoclonal
antibody (or antigenic binding fragments derived therefrom) to bind
with the Laminin Receptor 1 Precursor Protein 37LRP; and most
particularly to diagnosis and treatment of Hepatocellular Carcinoma
by various means which rely upon direct binding of 5LAC-23 with the
particular antigenic moiety specifically recognized thereby and
generally overexpressed in Hepatocellular carcinoma cells.
BACKGROUND OF THE INVENTION
[0003] Tumor invasion and metastasis is characterized by a series
of processes that involve the cancer cells and the host extra
cellular matrix. Basement membranes are specialized extra cellular
structures that play an important role in organizing the cells that
rest on them. Metastases of tumor cells involve interaction of the
cells with the components of extracellular matrix (ECM). Laminin is
a major component of the basement membrane which promotes cellular
attachment, proliferation, growth, differentiation and migration
(Kleinman H K et al. J Cell Biochem 1985, 217:317-25. Martin G et
al, Annual Rev Cell Biol 1987, 3:57-85. BeckK et al, FASEB J 1990,
4:148-60). In vitro and in vivo models have shown tumor cell
binding to laminin to be associated with cancer invasion, migration
and the cability to metastasize (Terranova V P et al, Cancer Res
1982, 42:2265-2269. Varani J et al, Am J pathol 1983, 111:27-34.
Barsky S H et al, J Clin Invest 1984, 74:843-848. Malinoff H L, Int
J Cancer 1984, 33:651-655. Kanemoto K et al, Proc Natl Acad Sci USA
1990, 87:2279-2283). The 67 kD laminin receptor (67LR), is a
non-integrin high affinity laminin-binding protein whose expression
is significantly increased in cancer cells and interacts with
laminin among other cell surface proteins (Malinoff H L et al. Int
J Cnacer 1984, 33:651-655. Rao C N et al. Bicohem Biophys Res
Commun 1983, 111:804-808. Terranova V P et al. Proc Natl Acad Sci
USA 1983, 80:444-448. Malinoff H et al. J Cell Biol 1983,
96:1475-1480. Ruyman R B et al. J Cell Biol 1988, 107:1863-1871.
Albelda S M et al. FASEB J 1990, 4:2868-2880. Hail D E et al. J
Cell Biol 1990, 110:2175-2184). It has been shown that expression
of the 67LR is increased in cancers such as breast, colon, and
gastric carcinomas compared to normal tissues (Cioce V et al. J
Natl Cancer Inst 1991, 83:29-36. Castronovo V et al. Am J Pathol
1990, 137:1373-1381. D'Errico A et al. Mod Pathol 1991,
4:239-246).
[0004] The 67 kD laminin receptor (67LR; Table 1) was originally
isolated from cell membranes of murine melanoma (Rao et al, 1983.
Biochem Biophys Res. Commun. 111:804-808), fibrosarcoma cells
(Malinoff & Wicha, 1983. J Cell Biol 96: 1475-1479) and normal
bovine muscle cells (Lesot et al, 1983. EMBO J. 2: 861). Since then
it has been detected in a number of species and is present in a
broad range of human tissues (for review see Barsoum Rohrer and
Coggin 2000. Cell Mol Biol Lett. 5: 207-230; Menard et al, 1998 J.
Cell. Biochem. 67:155-165; Mecham 1991 Annu Rev Cell Biol
7:71-91).
TABLE-US-00001 TABLE 1 Synonyms for Laminin receptor 1 and
precursor Protein Names Laminin receptor 1 Laminin receptor 1
(LamR1/LAMR1/LR1) Laminin receptor (Laminin-R/LN-R/LR) Laminin
binding receptor (LAMBR/LBR) 67 kD Laminin receptor (67 kD LR/67LR)
Colon carcinoma laminin binding protein Laminin receptor 1 Laminin
receptor precursor (LRP); Precursor 37 kD Laminin receptor
precursor (37LRP) 37 kD Laminin binding protein (37LBP) Immature
Laminin receptor protein (iLRP): 32 kD Laminin binding protein (LBP
32)
[0005] Human cDNA for the 67LR was originally isolated from a
malignant colon carcinoma (hence the protein is also known as the
Colon Carcinoma Laminin Binding Protein; Yow et al, 1988. 85:
6394-6398) and is smaller than first anticipated. The cDNA
predicted sequence encodes for a 295 amino acid protein with a
calculated mass of 32 kD. However, it normally runs at 37-44 kD on
an SDS-PAGE gel, which may be due to the reduced electrophoretic
mobility of an acidic protein (calculated pI 4.83). A small number
(16) of hydrophobic amino acids are present towards the N-terminus
(Castronovo, Taraboletti & Sobel, 1991. J. Biol. Chem. 266:
20440-20446) that may span the cell membrane. The 37 kD protein and
the 67 kD protein were shown to be antigenically related (Rao et
al, 1989; Biochemistry 28: 7476-7486) while pulse chase experiments
performed on melanoma cells indicated that the 37 kD protein
generated was chased into the 67 kD product (Castronovo et al,
1991. Biochem. Biophys. Res. Commun. 177:177-183). These results
suggested that there was a direct precursor-product relationship
between the two proteins. Hence, the 37 kD protein is referred to
as the 37 kD laminin receptor precursor (37LRP; Table 1). The pulse
chase experiments did not reveal the presence of any intermediate
forms between the precursor and the final 67LR (Castronovo et al,
1991), although a 50 kD degradation product was detected. The 37 kD
polypeptide may have a multifunctional role in the cytoplasm and
the membrane and may be the ligand binding component of the 67LR
(Elias Campo et al. Am J pathol 1992, 141:No. 5 1073-1083). In
cancer cells it has been shown that antibodies to the 67 kD protein
bind to both the cell surface and cytoplasm (Wever U M et al.
Cancer Res 1987, 47:5691-5698).
[0006] It is not fully understood how the final receptor is made by
cells to achieve the 67 kD receptor, but acylation by the fatty
acids palmitate, oleate and stearate may be involved (Landowski,
Dratz, & Starkey, 1995. 34: 11276-87; Buto et al, 1998 J. Cell
Biochem. 69: 244-251). Extensive glycosylation is not involved. The
predicted cDNA sequence does not contain a consensus sequence site
for N-linked glycosylation and despite the presence of serine and
threonine residues, there is no evidence of O-linked glycosyl
groups (Castronovo et al, 1991. Biochem. Biophys. Res. Commun.
177:177-183; Landowski, Dratz, & Starkey, 1995. 34: 11276-87).
However, Castronovo (Castronovo, 1993 Invasion Metastasis 13:1-30)
suggested that the 67LR expresses epitopes that cross-react with
.beta.-galactosidase-binding lectins. The 67LR may comprise of a
dimer of the precursor polypeptide linked by lipids (Landowski,
Dratz & Starkey, 1998). It has also been suggested that
heterodimerisation may occur with a lectin-like protein or
galectin-3 (Castronovo et al, 1991; Buto et al, 1998).
Anti-galectin-3 antibodies recognized not only galectin-3 but also
the 67LR (Buto et al, 1998). The final structure of the receptor
remains to be elucidated.
[0007] The 67LR, when shed from the surface of cells in culture,
retains its capacity to bind to laminin (Karpatova et al, 1996. J.
Cell. Biochem. 60:226-503). It remains uncertain how the 67LR is
attached to the cell membrane. Although the receptor has 16
hydrophobic amino acids towards its' N-terminal end, it is possible
that it interacts with associated molecules rather than existing as
an integral membrane protein. However, it has been established that
the amino-terminal of the polypeptide is inaccessible in
non-permeabilised cells suggesting that indeed this region
interacts with other molecules (Castronovo et al, 1991. J. Biol.
Chem. 30 20440-20446; Wewer et al, 1987 Cancer Res.
47:5691-5698).
[0008] It has also been suggested that accessory factors may be
associated with the 67LR, or that it acts as an accessory molecule
itself. Such properties may assist in transport to the cell surface
and/or laminin binding. It has been noted that co-expression of
67LR and .alpha..sub.6.beta..sub.1 in small cell lung cancer cell
lines directly correlated with cell adhesion to laminin
(Pellergrine et al, 1994. Int J Cancer Suppl 8: 116-120). When
human melanoma cells were treated with laminin both 67LR and
.alpha..sub.6.beta..sub.1 co-translocated to the plasma membrane
(Romanov et al, 1994. Cell Adhes Commun. 2:201-209). 67LR
associated with .alpha..sub.6.beta..sub.1 mediated high-avidity
adherence of a population of human memory T cells to laminin
(Clanfield and Khakoo, 1999. J. Immunol. 163: 3430-3440). Ardini et
al, 1997 noted that the 67LR and .alpha..sub.6.beta..sub.4 not only
co-localised but were co-regulated, via physical interactions
between the 67LR and .alpha..sub.6 subunit (Ardini et al, 1997; J.
Biol. Chem. 272:2342-2345). However, in ovarian carcinomas
expression of 37LRP mRNA and protein is independent of the
.alpha..sub.6 subunit (Givant-Horwitz, 2003 Clin. Exp. Metastasis
20:599-609; Skubitz et al, 1996. Am J pathol 148:1445-1461).
Together these results suggest that the 67LR may associate with
laminin-specific integrins (in particular the .alpha..sub.6
subunit) in the cytoplasm, arriving at the cell membrane as a
complex where both receptors participate in the recognition of
laminin and determining whether the interaction is one of high or
low affinity (Landowski, Dratz, Starkey, 1995).
[0009] The active human 37LRP gene maps to 3p21.3, a chromosomal
locus that is frequently involved in genetic alterations associated
with cancers (Jackers et al, 1996. Oncogene 13: 495-503). The
active gene contains seven exons and six introns (Jackers et al
1996. Oncogene 13: 495-503; avian gene Clausse et al, 1996 DNA Cell
Biol 15: 1009-1023). It does not contain a classical TATA box but
there may be multiple transcription start sites. There are four Sp1
sites present in the promoter region, six Sp1 sites in intron 1 and
two Alu sequences in intron 3 that may affect alternative splicing.
Intron 4 contains the sequence for the small nuclear RNA E2
(Jackers et al 1996. Oncogene 13: 495-503). At least 26 copies of
the gene are present in the human genome, all demonstrating high
homology with the functional gene (Jackers et al, 1996. Biochem
Biophys Acta. 1305:98-104). Nineteen of these copies were analysed
and were shown to be processed pseudogenes giving rise to
dysfunctional transcripts. It is thought that these pseudogenes
have most probably been generated by retropositional events
(Jackers et al, 1996. Biochem Biophys Acta. 1305:98-104). The cDNA
is highly conserved throughout evolution with at least 98.3%
homology among mouse, bovine and human sequences while the rat and
human sequences share 99% homology (For review see Menard et al,
1997. J Cell Biochem 67:155-165).
[0010] The 37LRP gene appears to give rise to a number of
functional proteins other than the 37LRP. The 37LRP protein shares
99% homology with the p40 ribosome-associated protein (p40
polypeptide; p40; ribosomal protein SA; RPSA) involved in the
translational machinery (Makrides et al, 1988. Nucleic Acid Res.
16: 2349; Tohgo et al, 1994. FEBS Lett. 340: 133-138; Rosenthal
& Wordeman 1995. J. Cell Sci. 108: 245-256). A positional
marker in the development of the embryonic eye is also encoded for
by a gene identical to the 37LRP cDNA (Rabacchi et al, 1990.
Development 109: 521-531; McCafferey, Neve and Drager, 1990. PNAS
87: 8570-8574).
[0011] The oncofetal antigen (OFA; 37-44 kD) is an immunogenic
glycoprotein expressed in rodent and human tumors and early
foetuses. The murine 37LRP shares up to 99.5% identity with OFA
(Coggin, Barsoum, Rohrer 1999. Anticancer Research 19: 5535-5542).
It has been referred to as the auto-immunogenic homologue of 37LRP.
OFA has been shown to stimulate T and B lymphocytes in both mice
and humans, and play an immunogenic role in cancers, in particular
in renal cancers (Zelle-Rieser et al, 2001. J. Urol. 165:1705-9;
Holt et al, 2002. Clin. Cancer Res. 8:3369-3376; Rohrer et al,
1992; J. Natl. Cancer Inst. (Bethesda) 84: 602-609; Rohrer et al,
1994. J. Immunol. 155: 755-764; Rohrer et al, 1995. J. Immunol.
155:5719-5727; Rohrer et al, 2001. Mod. Aspects Immunibiol. 1:
191-195; Rohrer et al, 1999. J. Immunol. 162: 6880-6892).
[0012] There is some evidence to suggest that isoforms or
homologues of the 37LRP and 67LR may exist. A 55 kD protein has
been identified in human and bovine endothelial cells sharing
identity with 37LRP (Ireland et al, 1998. Clin. Exp. Immunol.
112:255-261), and a number of isoforms have been found in murine
brain tissue (Simoneau et al, 2003. Biol. Chem. 384:243-246). These
proteins may arise from the 37LRP being post-translationally
modified in various ways and/or interacting with other molecules,
or may arise from other highly homologous genes.
[0013] Over-expression and abnormal surface distribution of the
67LR has been demonstrated in a broad range of tumors, detected by
various technologies at the mRNA and protein levels (For review see
Menard et al 1998; Barsoum et al, 2000). Change in levels of 37LRP
and/or 67LR have been shown to affect tumor biology in terms of
disease progression, invasiveness, metastasis, aggressiveness and
prognosis.
[0014] The over-expression of the 67LR has been associated with the
receptor playing a role in tumor progression, although the stage of
progression may be dependent on the tumor type (Campo et al, 1992.
Am J Pathol 41:1073-83; Demeter et al, 1992 Cancer Res.
52:1561-1567; Martignone et al, 1992. Clin. Exp. Metastasis
10:379-386: breast cancer; Vasso et al, 1993; Cancer 15: 455-461:
melanoma; Boukerche et al, 2004. Gene 343:191-201: melanoma;
Waltregny et al 1997. J. Natl. Caner Inst 89:1224-1227). An
increase in 37LRP mRNA in frozen colorectal tissues could be seen
in adenocarcinomas compared with adenomas, whereas levels were
constant between normal and adenoma tissues. These results suggest
that expression of 37LRP or 67LR correlated with a late event in
disease progression from adenoma to adenocarcinoma/Dukes C
carcinoma (Campo et al, 1992. Am J Pathol 41:1073-83). In contrast,
37LRP mRNA increased in adenomatous cervical lesions suggesting an
early event in disease progression (Demeter et al, 1992 Cancer Res.
52:1561-1567). 67LR has also been implicated as a
lineage-associated antigen in monocytic acute myeloid leukaemia
(AML; Montouri et al, 1999. Clin. Cancer Res. 5:1465-1472).
[0015] Other studies have shown that the 67LR may play a role in
invasiveness and metastasis, implying that it plays a significant
role in the acquisition of a metastatic phenotype in various types
of tumors (Wewer et al, 1987. Cancer Res 47: 5691-8; Castronovo
& Sobel 1990. Biochem Biophys Res Commun 68: 1110-1117; Cioce
et al, 1991. J Natl Cancer Inst 83: 29-36; Sobel, 1993 Semin.
Cancer Biol. 4: 311-317; Castronovo Invasion Metastasis 1993
13:1-30; You et al, 1988. PNAS 85: 6394-6398; Pelosi et al, 1997.
J. Pathol. 183:62-69; Boukerche et al, 2004. Gene 343:191-201). For
example, levels of mRNA have been shown to increase in human colon
cell lines and tissues with greater malignant potential (Kondah et
al, 1992. Cancer Res 52: 791-796). Inhibition of metastasis of a
human fibrosarcoma cell line occurred when cells were pre-treated
with an IgG fraction (P4G) of sera from rabbits immunised with a
37LRP-GST fusion protein (345 bp cDNA; 13kD; Narumi et al, 1999.
Jpn J. Cancer Res. 90: 425-431). The sera also reduced cell
attachment to laminin in a dose-dependent manner. Antisense RNA of
37LRP also inhibited invasiveness of a poorly differentiated human
colon carcinoma cell line in vitro (Mafune and Ravikumar, 1992. J.
Surg. Res. 52:340-346).
[0016] The increase in 67LR expression during metastasis is often
paralleled by the decrease in expression of another non-integrin
laminin binding protein, galectin-3 (van den Brule et al, 1994.
Eur. J. Cancer 32A:1598-1602; Xu et al, 1995. Am. J. Pathol.
147:815-822; Castronovo et al, 1995. J. Pathol. 179:43-48; Lotz et
al, 1993. PNAS 90: 3466-3470). These results suggest that these two
laminin receptors are inversely regulated and this may account for
changes in laminin-binding affinity depending on which receptor is
being used. In contrast, a direct correlation between increased
expression of galectin-3 and the malignancy of colon carcinomas has
been observed (Schoeppner et al, 1995 Cancer 75:2818-2826).
[0017] 67LR expression may also be a marker for aggressiveness of a
tumor since increased expression tends to be associated with
proliferation and marked tumor growth. Increased levels of 37LRP
mRNA were detected in human lung cancer tissues (Satoh et al, 1992.
Biochem. Biophys. Res. Commun 182: 746-752) and pancreatic
endocrine tumors (Pelosi et al, 1997. J. Pathol. 183: 62-69) that
were rapidly proliferating. In cervical neoplasms associated with
human papillomavirus, increased levels of 37LRP mRNA were
correlated with proliferative properties of the cells rather than
with the invasive properties of the cells (Demeter et al, 1992).
Introduction of antisense 37LRP RNA into murine lung cancer cell
line T11 prolonged their doubling time (Satoh et al, 1999. Br. J.
Cancer 80:1115-1122). These cells also displayed weaker
interactions with laminin and survival time in mice subcutaneously
inoculated with cells treated with antisense RNA was prolonged. The
67LR may also play a role in tumour aggressivenss since it may
enhance proteolytic cleavage of laminin-1, therefore assisting in
the degradation rate of the basement membrane (Ardini et al, 2002.
Cancer Res. 62: 1321-1325).
[0018] Over-expression of the 37LRP and/or 67LR may also be
associated with poor prognosis in several types of tumors (for
review see Barsoum Rohrer and Coggin 2000. Cell Mol Biol Lett. 5:
207-230; Menard et al, 1998 J. Cell. Biochem. 67:155-165; Menard,
Tagliabue and Colnaghi, 1998. Breast Cancer Res. Treatment 52:
137-145). Prognosis is unfavourable in breast carcinomas that are
also producing laminin (Martigone et al 1993. J. Natl. Cancer Inst.
85: 379-386; Pellegrini et al, 1985 Breast Cancer Res Treat 35:
195-199). In human lymphomas, 67LR was detectable on the surface of
CD30.sup.+ anaplastic large cell lymphomas and in small subsets of
high-grade B-cell non-Hodgkin's or Hodgkin's lymphomas (Carbone et
al, 1995. Hum. Pathol. 2: 541-546).
[0019] Recently, the 67LR has been implicated in biological
processes other than tumor biology. The receptor was found to be
up-regulated by cytokines, inflammatory reagents, interactions with
extracellular matrix proteins including laminin and steroids (for
review see Menard et al, 1998), suggesting that the 37LRP and 67LR
may be regulated under normal conditions. The receptor may play a
role in lymphocyte chemotaxis, adhesion and homing and/or in host
defence mechanisms. The 67LR has been found on the surface of a
population (10-15%) of human activated memory peripheral blood T
cells (both CD4.sup.+ and CD8.sup.+ single positives). It has also
been shown to be up-regulated in response to neuropeptides (Chen et
al, 2002 Nat. Med. 8:1421-1426). A study by Ferrarini et al, 1996
supports an immunological role for the receptor since
.gamma..delta..sup.+ lymphocytes localised in lung tumor sites were
capable of killing lung cancer cells, mediated by interactions with
67LR (Ferrarini et al, 1996. J. Natl. Cancer Inst. 88:436-441). The
killing was shown to be independent of natural killer (NK) cells,
lymphokine-activated (LAK) cells and the T cell receptor (TCR)
whereas laminin could provide a co-stimulatory signal.
[0020] The 67LR may also affect the growth, migration and
trafficking of other cell types. 67LR interacts with the .alpha.
(.alpha.GMR) and .beta. (.beta. GMR) subunits of the GM-CSF
receptor (Chen et al, 2003. PNAS 100: 14000-14005) and inhibits the
formation of the GM-CSF receptor complex. GM-CSF regulates the
growth, differentiation and maturation of myeloid precursor cells
and enhances the function of mature neutrophils, eosinophils and
mononuclear phagocytes. 67LR may inhibit these activities by
preventing GM-CSF complex formation. Secretory and endocytic roles
for the 67LR have also been implied since it has been found in the
brush border and in Paneth cell secretory granules (Shmakov et al,
2000. J. Pathol. 191:318-322).
[0021] The precise way in which 67LR interacts with laminin remains
undefined. Two peptide domains have been identified from the 67LR
as possible laminin binding sites. One of these, Peptide G, a
synthetic peptide derived from the sequence of 37LRP, contains the
palindromic sequence LMWWML (SEQ ID No: 1). It was shown to bind
laminin, to inhibit binding of tumor cells to endothelial cells,
and to increase the metastases of human melanoma cells in nude mice
(Castronovo et al, 1991 J. Biol. Chem. 266:20440-20446; Castronovo,
Taraboletti and Sobel 1991. Cancer Res. 51: 5672-5678; Taraboletti
et al, 1993. J. Natl. Cancer. Inst. 85: 235-240). It was discovered
that peptide G increases and stabilises the binding of laminin on
tumor cells (Magnifico et al, 1996. J. Biol. Chem. 271:
31179-31184). The second possible laminin binding domain was
predicted from the hydrophobicity of the C-terminal sequence of the
37LRP (a.a. 205-229; Landowski, Uthayakumar, Starkey, 1995. Clin.
Exp. Metastasis 13: 357-372). It is also possible a lectin domain
of the 67LR interacts with laminin since laminin recognition of the
receptor is dependent on lactose (Castronovo et al, 1991. Biochem.
Biophys. Res. Commun. 177:177-183).
[0022] The 67LR may bind to laminin residues YIGSR (SEQ ID NO: 2)
(a.a. 929-933; .beta.1 chain; Massi, Rao and Hubbell, 1993. J.
Biol. Chem. 268:8063-8059; Landowski, Uthayakumar, Starkey, 1995.
13: 357-72), IKVAV (SEQ ID NO: 3) (a.a. 2091-2108; .alpha. chain;
Kibbey et al, 1993. PNAS 90: 10150-10153) and LGTIPG (SEQ ID NO: 4)
(a.a. 442-446; (.beta.1 chain; Mecham et al 1989 J. Biol. Chem.
264: 16652-16657). The 67LR may also bind to the carbohydrate
components of laminin, in particular poly(lactosamino) structures,
Gal(.alpha.1,3)Gal linkages and terminal non-reducing
.beta.-galactosyl residues (Mecham, 1991 Annu. Rev. Cell. Biol.
7:71-91). Binding to the residues YIGSR (SEQ ID NO: 2) inhibits
metastasis (Iwamoto et al, 1996. Br. J. Cancer 73: 589-595), while
metastasis is stimulated by IKVAV (SEQ ID NO: 3) interactions
(Bresalier et al, 1995. Cancer Res. 55: 2476-2480). Since the
discovery of the first laminin receptor, 67LR, in 1983 at least 14
other laminin receptors have been described (Mecham 1991) and may
utilize the same binding sites on laminin.
[0023] The 67LR also interacts with other molecules in addition to
laminin. These include elastin (Grosso et al, 1991. Biochemistry
30: 3346-3350), fibronectin (FN), type IV collagen (Narasimhan et
al, 1994. PNAS 91:7440-7444; Iwabuchi et al, 1996 Blood 87:
365-372) and heparin (Guo et al, 1992. PNAS 89: 3040-3044). Other
studies have also shown that the 67LR serves as a receptor for
sindbis virus (Wang et al, 1992 J. Virol. 66:4992-5001), while the
37LRP allows uptake of prion proteins (Rieger et al, 1997. Nat.
Med. 3:1383-1387).
Liver Cancer
[0024] The most common primary malignant tumor of the liver is
hepatoceullar carcinoma (HCC). The incidence of HCC is increased in
populations who are at high risk for Hepatitis B and C. Patients
already suffering from chronic hepatitis, cirrhosis,
hemochromatosis, and the two congenital hepatic disorders,
alpha-1-antitrypsin deficiency and tyrosinema, are also at higher
risk of developing HCC. Certain toxins and chemicals may also cause
primary liver cancer, including aflatoxin, a product from mould
found in improperly stored peanuts in Africa. If HCC is
successfully removed by resection, recurrence and metastasis are
likely. A number of studies have revealed various prognostic
markers for primary liver cancer and metastatic recurrence (For
review see Qin and Tang, 2004. J Cancer Res Clin Oncol 130:
497-513).
[0025] The association of liver diseases with HCC suggests
architectural changes may increase the chance of developing primary
liver cancer. Under normal conditions hepatocytes are characterised
by the absence of a basement membrane (Schaffner and Popper 1963
Gastroenterology 44:230-242). Extracellular matrix proteins,
including laminin, can be produced as cirrhosis develops and are
deposited around sinusoids, forming a structured basement membrane.
Laminin 5 was found to be present in primary HCC nodules but not in
normal or peri-tumoral cirrhotic tissues (Giannelli et al, 2003.
Clin. Can. Research. 9:3684-3691). Laminin has also been shown to
induce the expression of cytokeratin 19 suggesting that laminin
deposits cause abnormal expression of other proteins (Su et al,
2003. World J Gastroenterol: 921-929). It may be that the
expression of laminin increases expression of any of its
receptors.
[0026] The 67LR was found to be expressed in hepatocytes in 1990
(Clement et al, 1990. J. Cell Biol. 110:185-192), although it was
not the only laminin receptor present. An increase in the number of
67LR positive cells was observed in neoplastic regions compared to
adjacent parenchyma in liver samples taken from patients with HCC
and cirrhosis (Grigioni et al (1991, Am J Pathol 138:647-654).
Another study (Ozaki et al, 1998. Gut 43: 837-842) detected weak
37LRP mRNA expression in normal liver tissues. mRNA levels
increased in non-cancerous liver tissue with chronic liver disease
and were elevated further in tumor regions. 37LRP translation and
expressed protein were not determined in this study (Ozaki et al,
1998. Gut 43: 837-842). Increased biosynthesis of the 67LR was
observed in the metastatic HCC tissues with a direct correlation
between increases in RNA and protein (Zheng et al, 1997. J Tongji
Medical University. 17:200-202). L-02 normal hepatic cells and the
cancer cell lines HepG2 and SMMC-7721 showed varied patterns of
37LRP mRNA and 67LR expression that did not correlate with the
tumor state of the cell line (Zheng et al, 2002. Chinese J Cancer
22: 248-252). However, the carcinoma cell line SMMC-7721 may
express higher laminin binding affinity than the other cell lines
although this cannot be attributed to the 67LR alone since whole
cells were used for the binding studies. However, a proteomics
study revealed that 67LR is up-regulated in the highly metastatic
cell line MHCC97-H, compared to the low metastatic counterpart
MHCC97-L (Li et al, 2001. World J Gastroenterol 7: 630-636; Ding et
al, 2004. Proteomics 4; 982-994). Whether 67LR plays a direct role
in HCC metastasis remains to be determined.
SUMMARY OF THE INVENTION
[0027] The instant inventors have previously been awarded U.S. Pat.
No. 6,180,357, entitled "Individualized Patient Specific
Anti-Cancer Antibodies" directed to a process for selecting
individually customized anti-cancer antibodies, which are useful in
treating a cancerous disease. For the purpose of this document, the
terms "antibody" and "monoclonal antibody" (mAb) may be used
interchangeably and refer to intact immunoglobulins produced by
hybridomas (e.g. murine or human), immunoconjugates and, as
appropriate, immunoglobulin fragments and recombinant proteins
derived from said immunoglobulins, such as chimeric and humanized
immunoglobulins, F(ab') and F(ab').sub.2 fragments, single-chain
antibodies, recombinant immunoglobulin variable regions (Fv)s,
fusion proteins etc., provided that they recognize the same
antigenic moiety. It is well recognized in the art that some amino
acid sequence can be varied in a polypeptide without significant
effect on the structure or function of the protein. In the
molecular rearrangement of antibodies, modifications in the nucleic
or amino acid sequence of the backbone region can generally be
tolerated. These include, but are not limited to, substitutions
(preferred are conservative substitutions), deletions or additions.
Furthermore, it is within the purview of this invention to
conjugate standard chemotherapeutic modalities, e.g. radionuclides,
with the cancerous disease modifying antibodies (CDMAB) of the
instant invention, thereby focusing the use of said
chemotherapeutics. The CDMAB can also be conjugated to toxins,
cytotoxic moieties, drugs, enzymes e.g. biotin conjugated enzymes,
or hematogenous cells, thereby forming antibody conjugates.
[0028] The use of tumor-associated monoclonal antibodies as
carriers for cytotoxic agents has received considerable attention
in the past several years. The objective of much of this work has
been to improve the efficacy of anticancer drugs while diminishing
the undesired and often times toxic side-effects of the conjugated
drug or toxin.
[0029] In order for this approach to be effective, it is necessary
that the antibody be highly tumor selective and that the drug be
delivered in an active, cytotoxic form. Cytotoxic drugs such as
Methotrexate, Daunomycin Mitomycin C (MMC) and Vinca have been
attached to antibodies and the derived conjugates have been
investigated for anti-tumor activities. In addition, biologicals
such as Pseudomonas Exotoxin and newer toxins such as Calicheamicin
and Auristatins have been employed to enhance the efficacy of
anti-CD33 and anti-CD30 antibodies, respectively. Many examples
exist in the art which illustrate linkage of antibodies to drugs by
means of relatively stable chemical bonds which undergo slow
non-specific release. Radionuclides such as Iodine131, Yttrium90,
or Indium111 can also be conjugated to the antibody for the
purposes of tumor destruction or for diagnostic imaging.
Irrespective of the approach, a primary goal is to destroy the
tumor: the specific approach can be determined by the particular
anti-37LR antibody which is utilized so that the available
approaches to targeting the cells expressing the 37LR antigen can
vary considerably.
[0030] This application utilizes the method for producing patient
specific anti-cancer antibodies as taught in the '357 patent for
isolating hybridoma cell lines which encode for cancerous disease
modifying monoclonal antibodies. These antibodies can be made
specifically for one tumor and thus make possible the customization
of cancer therapy. Within the context of this application,
anti-cancer antibodies having either cell-killing (cytotoxic) or
cell-growth inhibiting (cytostatic) properties will hereafter be
referred to as cytotoxic. These antibodies can be used in aid of
staging and diagnosis of a cancer, and can be used to treat tumor
metastases.
[0031] Having recognized that a significant difference between
cancerous and normal cells is that cancerous cells contain antigens
that are specific to transformed cells, the scientific community
has long held that monoclonal antibodies can be designed to
specifically target transformed cells by binding specifically to
these cancer antigens; thus giving rise to the belief that
monoclonal antibodies can serve as "Magic Bullets" to eliminate
cancer cells. However, it is now widely recognized that no single
monoclonal antibody can serve in all instances of cancer, and that
monoclonal antibodies can be deployed, as a class, as targeted
cancer treatments.
[0032] Historically, the use of polyclonal antibodies has met with
limited success in the treatment of human cancers. Lymphomas and
leukemias have been treated with human plasma, but there were few
prolonged remissions or responses. Furthermore, there was a lack of
reproducibility and no additional benefit compared to chemotherapy.
Solid tumors such as breast cancers, melanomas and renal cell
carcinomas have also been treated with human blood, chimpanzee
serum, human plasma and horse serum with correspondingly
unpredictable and ineffective results.
[0033] There have been many clinical trials of monoclonal
antibodies for solid tumors. In the 1980s there were at least 4
clinical trials for human breast cancer that produced only 1
responder from at least 47 patients using antibodies against
specific antigens or based on tissue selectivity. It was not until
1998 that there was a successful clinical trial using a humanized
anti-Her2/neu antibody (HERCEPTIN) in combination with CISPLATIN.
In this trial 37 patients were assessed for responses of which
about a quarter had a partial response rate and an additional
quarter had minor or stable disease progression. The median time to
progression among the responders was 8.4 months with median
response duration of 5.3 months.
[0034] HERCEPTIN was approved in 1998 for first line use in
combination with Taxol.RTM.. Clinical study results showed an
increase in the median time to disease progression for those who
received antibody therapy plus Taxol.RTM. (6.9 months) in
comparison to the group that received Taxol.RTM. alone (3.0
months). There was also a slight increase in median survival; 22
versus 18 months for the HERCEPTIN plus Taxol.RTM. treatment arm
versus the Taxol.RTM. treatment alone arm. In addition, there was
an increase in the number of both complete (8 versus 2 percent) and
partial responders (34 versus 15 percent) in the antibody plus
Taxol.RTM. combination group in comparison to Taxol.RTM. alone.
However, treatment with HERCEPTIN and Taxol.RTM. led to a higher
incidence of cardiotoxicity in comparison to Taxol.RTM. treatment
alone (13 versus 1 percent respectively). In clinical trials the
expression level of Her2/neu, as determined by
immunohistochemistry, predicted responses to HERCEPTIN therapy.
Among patients with metastatic breast cancer only those with
overexpression of Her2/neu, designated as 2-3+ on an pathology
scoring scale, benefited from antibody therapy. Approximately 25
percent of patients who have metastatic breast cancer overexpress
Her2/neu and could be treated with HERCEPTIN; those without
overexpression, and thus would not benefit, are not treated with
the antibody. Selection for HERCEPTIN therapy represents a method
of selecting patients suitable for treatment based on the
identification of molecular markers of disease and this method has
been approved as a diagnostic test by the U.S. F.D.A. However,
there is still a large unmet need for patients with breast cancer.
Even those who can benefit from HERCEPTIN treatment would still
require chemotherapy and consequently would still have to deal
with, at least to some degree, the side effects of this kind of
treatment.
[0035] The clinical trials investigating colorectal cancer involve
antibodies against both glycoprotein and glycolipid targets.
Antibodies such as 17-1A, which has some specificity for
adenocarcinomas, has undergone Phase 2 clinical trials in over 60
patients with only 1 patient having a partial response. In other
trials, use of 17-1A produced only 1 complete response and 2 minor
responses among 52 patients in protocols using additional
cyclophosphamide. To date, Phase III clinical trials of 17-1A have
not demonstrated improved efficacy as adjuvant therapy for stage
III colon cancer. The use of a humanized murine monoclonal antibody
initially approved for imaging also did not produce tumor
regression.
[0036] Only recently have there been any positive results from
colorectal cancer clinical studies with the use of monoclonal
antibodies. In 2004, ERBITUX was approved for the second line
treatment of patients with EGFR-expressing metastatic colorectal
cancer who are refractory to irinotecan-based chemotherapy. Results
from both a two-arm Phase II clinical study and a single arm study
showed that ERBITUX in combination with irinotecan had a response
rate of 23 and 15 percent respectively with a median time to
disease progression of 4.1 and 6.5 months respectively. Results
from the same two-arm Phase II clinical study and another single
arm study showed that treatment with ERBITUX alone resulted in an
11 and 9 percent response rate respectively with a median time to
disease progression of 1.5 and 4.2 months respectively.
[0037] Consequently in both Switzerland and the United States,
ERBITUX treatment in combination with irinotecan, and in the United
States, ERBITUX treatment alone, has been approved as a second line
treatment of colon cancer patients who have failed first line
irinotecan therapy. Therefore, like HERCEPTIN, treatment in
Switzerland is only approved as a combination of monoclonal
antibody and chemotherapy. In addition, treatment in both
Switzerland and the US is only approved for patients as a second
line therapy. Also, in 2004, AVASTIN was approved for use in
combination with intravenous 5-fluorouracil-based chemotherapy as a
first line treatment of metastatic colorectal cancer. Phase III
clinical study results demonstrated a prolongation in the median
survival of patients treated with AVASTIN plus 5-fluorouracil
compared to patients treated with 5-fluourouracil alone (20 months
versus 16 months respectively). However, again like HERCEPTIN and
ERBITUX, treatment is only approved as a combination of monoclonal
antibody and chemotherapy. In addition, there are U.S. F.D.A
approved diagnostic tests based on the HERCEPTIN and ERBITUX
antigenic targets for use in cancer diagnosis based on the
immunohistochemistry platform.
[0038] There continues to be poor results for lung, brain, ovarian,
pancreatic, prostate, stomach cancer, and hepatocellular carcinoma.
The most promising recent results for non-small cell lung cancer
came from a Phase II clinical trial where treatment involved a
monoclonal antibody (SGN-15; dox-BR96, anti-Sialyl-LeX) conjugated
to the cell-killing drug doxorubicin in combination with the
chemotherapeutic agent TAXOTERE. TAXOTERE is the only FDA approved
chemotherapy for the second line treatment of lung cancer. Initial
data indicate an improved overall survival compared to TAXOTERE
alone. Out of the 62 patients who were recruited for the study,
two-thirds received SGN-15 in combination with TAXOTERE while the
remaining one-third received TAXOTERE alone. For the patients
receiving SGN-15 in combination with TAXOTERE, median overall
survival was 7.3 months in comparison to 5.9 months for patients
receiving TAXOTERE alone. Overall survival at 1 year and 18 months
was 29 and 18 percent respectively for patients receiving SGN-15
plus TAXOTERE compared to 24 and 8 percent respectively for
patients receiving TAXOTERE alone. Further clinical trials are
planned for this drug.
[0039] Although SGN-15 has not been approved for marketing, several
other antibody conjugates for cancer have been approved by the U.S.
F.D.A. since 2000. These include MYLOTARG (gemtuzumab ozogamicin, a
humanised anti-CD33 MAb) for the treatment of relapsed acute
myeloid leukemia, Zevalin (ibritumomab tiuxetan, Yttrium conjugated
RITUXIMAB, humanised anti-CD20 MAb) for the treatment of
non-Hodgkin's lymphoma, BEXXAR (I-131 conjugated tositumomab,
anti-CD20 MAb) for the treatment of recurrent non-Hodgkin's
lymphoma. These antibodies were developed against cancer specific
molecules, which rendered them appropriate for conjugation to
either toxins or radio-isotopes. It is apparent that only
hematogenous diseases are currently successfully treated with
conjugated antibodies, and solid tumors, such as hepatocellular
carcinoma, are still in need of such therapies.
[0040] The discovery of new drugs to treat disease is hindered by
the lack of identification of relevant targets, among the products
of 30,000 known genes, that unambiguously contribute to disease
pathogenesis. In oncology research, potential drug targets are
often selected simply due to the fact that they are over-expressed
in tumor cells. Targets thus identified are then screened for
interaction with a multitude of compounds. In the case of potential
antibody therapies, these candidate compounds are usually derived
from traditional methods of monoclonal antibody generation
according to the fundamental principles laid down by Kohler and
Milstein (1975, Nature, 256, 495-497, Kohler and Milstein). Spleen
cells are collected from mice immunized with antigen (e.g. whole
cells, cell fractions, purified antigen) and fused with
immortalized hybridoma partners. The resulting hybridomas are
screened and selected for secretion of antibodies which bind most
avidly to the target. Many therapeutic and diagnostic antibodies
directed against cancer cells, including HERCEPTIN and RITUXIMAB,
have been produced using these methods and selected on the basis of
their affinity and specificity of the targets. Development of
antigen-specific reagents with cytotoxic effects on tumor cells,
that bind cells expressing the recognized antigen(s) and which by
themselves, or associated with other molecules such as toxins,
drugs, or radio-isotopes, have cellular and in vivo physiological
activity such that these reagents inhibit tumor cell growth,
progression and metastasis, without significant deleterious effects
on normal cell populations, would be extremely beneficial as a
potential therapeutic and or diagnostic tool.
[0041] In order to validate the 5LAC-23 epitope as a
cancer-associated target, the expression of 5LAC-23 antigen in
frozen normal human tissues was determined. By immunohistochemistry
the 5LAC-23 antigen was shown to have restricted expression in
normal tissues. This was confirmed by examining the expression of
the 5LAC-23 antigen in formalin-fixed paraffin-embedded normal
organs in tissue array slides. In all, there is weak staining by
5LAC-23 of normal tissues, indicating restricted and reduced
expression of the antigen in normal liver, stomach, brain and
kidney tissues compared to cancers such as HCC (see below). Such
expression was also sometimes confined to the cytoplasm, which is
generally inaccessible to intact antibodies, in vivo. In the same
tissue array the expression of the 5LAC-23 antigen was prominent in
HCC although there was also expression in gastric
adenocarcinoma.
[0042] A more extensive study of the target in HCC was carried out
to examine the prevalence of the 5LAC-23 antigen in this cancer by
immunohistochemistry. Surprisingly, 73% of 55 samples of liver
cancer expressed this target. In order to demonstrate utility as a
target for diagnostics, theranostics, prognostics or therapeutics,
a comparison of the distribution of the target in matched normal
livers and liver cancer was carried out. Clearly, 5LAC-23 stained
only the tumor sample and was specific to the central region that
represents the malignant tissue in these sections. The sections
were negative when stained with the isotype control, indicating
that the binding of 5LAC-23 was specific and lending support for
the cancer specificity of the 5LAC-23 antigen. Further, it has also
been shown that the 5LAC-23 antigen can be detected with a variety
of assays, some non-limiting embodiments of which are included by
the way of example, such as through Western blotting, FACS
analysis, and immunohistochemistry. Other assays that will be
apparent to those skilled in the art and are within the purview of
this invention include: ELISA, immunocytochemistry, immunoaffinity
based assays such as SELDI mass spectroscopy, surface plasmon
resonance determinations, radioimmunoassay and molecular diagnostic
assays. As outlined herein, additional biochemical data also
indicate that the antigen recognized by 5LAC-23 is an epitope of
37LRP. This was supported by identifying the 5LAC-23 antigen with
two-dimensional electrophoresis and Western blotting, and mass
spectroscopy. The identification of the target antigen was
confirmed by demonstrating co-localization of the target with
antibodies known to bind to 37LRP such as H-150 in both western
blots as well as immunohistochemistry studies of tissues and cells
that are known to express 37LRP or transfected with cDNA for 37LRP.
Importantly, 5LAC-23 demonstrated a unique binding pattern compared
to the other anti-37LRP antibody, H-150. This suggests that the
epitopes recognized by the two antibodies are different, with
5LAC-23 demonstrating more restricted binding. Furthermore, in
Example 9 H-150 detects the 37LRP in all of cell lines but the
expression of the antigenic epitope recognized by 5LAC-23 varied
across the different cell lysates and in some was not present. This
difference between antibodies was not due to lower affinity of
5LAC-23 for its antigen compared to H-150 since the binding of both
antibodies to the CHO cell lysates was similar. In support of the
difference in epitopes detected by 5LAC-23 and H-150, 5LAC-23
detected a unique smear of approximately 110 kD in the LS 174T
lysates under non-reducing conditions (FIG. 12B, Lane 12). This
smear disappeared under reducing conditions without an increase in
the band corresponding to 37LRP precursor. These results present
further evidence that the epitope recognized by 5LAC-23 on 37LRP is
unique, compared to the known anti-LRP antibody H-150. These IHC
and biochemical results demonstrate that 5LAC-23 is directed
against an unique 37LRP antigenic epitope.
[0043] In toto, this data demonstrates that the 5LAC-23 antigen is
a cancer associated antigen and is expressed in humans, and is a
pathologically relevant cancer target. Further, this data also
demonstrates the binding of the 5LAC-23 antibody to human cancer
tissues, and can be used appropriately for assays that can be
diagnostic, predictive of therapy, or prognostic. In addition, the
cell localization of this antigen is indicative of the cancer
status of the cell due to the lack of expression of the antigen in
most non-malignant cells, and this observation permits the use of
this antigen, its gene or derivatives, its protein or its variants
to be used for assays that can be diagnostic, predictive of
therapy, or prognostic.
[0044] In all, this invention teaches the use of the 5LAC-23
antigen as a target for diagnostics, theranostics, prognostics or
therapeutics. Furthermore, this invention also teaches the use of
detecting the 5LAC-23 antigen in cancerous cells that can be useful
for the diagnosis, prediction of therapy, and prognosis of mammals
bearing tumors that express this antigen.
[0045] The clinical utility of a cancer drug is based on the
benefit of the drug under an acceptable risk profile to the
patient. In cancer therapy survival has generally been the most
sought after benefit, however there are a number of other
well-recognized benefits in addition to prolonging life. These
other benefits, where treatment does not adversely affect survival,
include symptom palliation, protection against adverse events,
prolongation in time to recurrence or disease-free survival, and
prolongation in time to progression. These criteria are generally
accepted and regulatory bodies such as the U.S. Food and Drug
Administration (F.D.A.) approve drugs that produce these benefits
(Hirschfeld et al. Critical Reviews in Oncology/Hematolgy
42:137-143 2002). In addition to these criteria it is well
recognized that there are other endpoints that may presage these
types of benefits. In part, the accelerated approval process
granted by the U.S. F.D.A. acknowledges that there are surrogates
that will likely predict patient benefit. As of year-end (2003),
there have been sixteen drugs approved under this process, and of
these, four have gone on to full approval, i.e., follow-up studies
have demonstrated direct patient benefit as predicted by surrogate
endpoints. One important endpoint for determining drug effects in
solid tumors is the assessment of tumor burden by measuring
response to treatment (Therasse et al. Journal of the National
Cancer Institute 92(3):205-216 2000). The clinical criteria (RECIST
criteria) for such evaluation have been promulgated by Response
Evaluation Criteria in Solid Tumors Working Group, a group of
international experts in cancer. Drugs with a demonstrated effect
on tumor burden, as shown by objective responses according to
RECIST criteria, in comparison to the appropriate control group
tend to, ultimately, produce direct patient benefit. In the
pre-clinical setting tumor burden is generally more straightforward
to assess and document. In that pre-clinical studies can be
translated to the clinical setting, drugs that produce prolonged
survival in pre-clinical models have the greatest anticipated
clinical utility. Analogous to producing positive responses to
clinical treatment, drugs that reduce tumor burden in the
pre-clinical setting may also have significant direct impact on the
disease. Although prolongation of survival is the most sought after
clinical outcome from cancer drug treatment, there are other
benefits that have clinical utility and it is clear that tumor
burden reduction, which may correlate to a delay in disease
progression, extended survival or both, can also lead to direct
benefits and have clinical impact (Eckhardt et al. Developmental
Therapeutics: Successes and Failures of Clinical Trial Designs of
Targeted Compounds; ASCO Educational Book, 39.sup.th Annual
Meeting, 2003, pages 209-219).
[0046] Accordingly, it is an objective of the invention to identify
Laminin Receptor 1 Precursor Protein (designated 37LRP) by way of
binding of a particular epitope of 37LRP with an isolated
monoclonal antibody, designated 5LAC-23, or a fragment thereof
defined as an antigenic fragment which binds to said particular
epitope, which isolated monoclonal antibody was produced by a
method for producing cancerous disease modifying antibodies from
cells derived from a particular individual which are cytotoxic with
respect to cancer cells while simultaneously being relatively
non-toxic to non-cancerous cells.
[0047] It is a further objective of this invention to provide a
conjugated moiety capable of binding with a particular epitope of
37LRP recognized by 5LAC-23, and herein referred to as a
drug-antibody conjugate, wherein the antibody is 5LAC-23 or a
fragment thereof which binds to said particular epitope, and the
conjugate can be a radionuclide, or an active antitumor drug in the
form of a biological or chemical toxin, or a compound having
equivalent antitumor activity including but not limited to
chemotherapeutic drugs.
[0048] It is another objective of this invention to teach a method
for delivering the active antitumor drug, enzyme or radionuclide
effective as an antitumor drug or as an aid in methods of
diagnostic imaging to the site of tumor cells in a mammal
comprising administering to the mammal the conjugated moiety in
accordance with the instant invention, whereby selective binding of
the conjugated moiety and antigenic epitope occurs.
[0049] It is still an additional objective of the invention to
teach a method for determining the presence of cancerous cells by
any means for evidencing selective binding of the 5LAC-23 antibody
with the 37LRP precursor protein at a level sufficient to indicate
the presence of malignancy.
[0050] A still further objective of the instant invention is to
teach a method for diagnosis, prognosis, therapy, imaging and
monitoring of cancerous or precancerous cells utilizing a method
which relies upon the binding of 5LAC-23, an antigenic binding
fragment (as hereinbefore defined) or a conjugate moiety (as
hereinbefore defined) with a particular antigenic moiety of
37LRP.
[0051] Other objects and advantages of this invention will become
apparent from the following description wherein, by way of
illustration and example, certain embodiments of this invention are
set forth.
BRIEF DESCRIPTION OF THE FIGURES
[0052] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0053] FIGS. 1A and 1B. Western blot of total cell lysates (T) and
cytoplasmic fraction (C) made from OVCAR-3 Cells stained with
either an isotype control (Panel A) or 5LAC-23 (Panel B). A
distinct band is detected with 5LAC-23 and is indicated by a black
arrow.
[0054] FIGS. 2A and 2B. Western blots of OVCAR-3 cytoplasmic
proteins separated by 2-D electrophoresis. Panel A shows a blot
probed with the IgM isotype control while panel B shows the blot
probed with 5LAC-23. The arrows correspond to the spots recognised
by 5LAC-23.
[0055] FIGS. 3A, 3B and 3C. 2-Dimensional SDS-PAGE and Western blot
of OVCAR-3 cytoplasmic proteins. Panel A shows a Silver stained gel
of the cytoplasmic fractions. Panel C demonstrates the position of
the protein recognised by 5LAC-23 while Panel B demonstrates a
similar blot probed with an isotype control antibody. The arrows
correspond to the spot recognised by 5LAC-23
[0056] FIGS. 4A, 4B, 4C and 4D. Western blots of OVCAR-3
cytoplasmic fractions probed with the anti-37LRP antibody H-150
(A), the anti-37LRP antibody F-18 (B), an IgM isotype control (C)
and 5LAC-23 (B).
[0057] FIGS. 5A and 5B. 2-Dimensional Western blots of cytoplasmic
proteins from OVCAR-3 cells. The arrow on Blot A indicates the
major spot that 5LAC-23 binds to. The line in Blot B indicates the
spots that are specific for the anti-37LRP(H-150) antibody. Other
spots revealed on blots are due to interactions with the isotype of
the antibody or with the secondary antibodies used.
[0058] FIGS. 6A, 6B, 6C and 6D. Immunostains of CHO cells
(10.times. magnification) that were transfected with a plasmid
expressing a human cDNA clone encoding for 37LRP (pCMV-XL537LRP)
with 5LAC-23 (bottom row) and an IgM isotype control (top row).
Cells were transfected with Fugene reagent alone (Column A), or
increasing amounts of pCMV-XL537LRP (1 micrograms: Column B; 2
micrograms: Column C; 4 micrograms: Column D). Increasing numbers
of positive cells (brown) can be seen with increasing amounts of
plasmid when cells were stained with 5LAC-23.
[0059] FIGS. 7A, 7B and 7C. Immunostains of CHO cells (40.times.)
transfected with a plasmid expressing a human cDNA clone encoding
for 37LRP (pCMV-XL537LRP; 2 micrograms). Cells were stained with
5LAC-23 (A), the anti-37LRP antibody H-150 (B) and the anti-67LR
antibody MLuC5 (C) and positive cells can be seen in all
immunostains.
[0060] FIGS. 8A, 8B and 8C. Expression of human 37LRP in E. coli
detected by various antibodies by Western analysis. Plasmids that
were included in the bacterial reaction mixtures included Control
Vector GFP with a C-terminal His.sub.6-tag (Lane 1), pIVEX2.3dLRP
(37LRP; Lane 2) and pIVEX2.4dLRP.sub.NHis6 (37LRP with a N-terminal
His.sub.6-tag; Lane 3). Blots were probed with an IgM isotype
control (Panel A), 5LAC-23 (Panel B) and the anti-37LRP antibody
H-150 (Panel C).
[0061] FIGS. 9A, 9B and 9C. Staining of paraffin-embedded tissues
with 5LAC-23 (Bottom row) and an IgM isotype control (Top row).
Sections were taken from normal stomach (Column A), normal liver
(Column B) and a hepatocellular carcinoma (Column C).
[0062] FIGS. 10A, 10B, and 10C. Binding of various antibodies to
cryo-preserved liver tissue from matched normal (Top row) and
matched tumor tissue (Bottom row). Antibodies used were an IgM
isotype control (Column A), 5LAC-23 (Column B) and an
anti-cytokeratin 8 (Column C).
[0063] FIGS. 11A and 11B. Immunohistochemical staining of the
normal lung epithelial cell line Beas-2B with 5LAC-23 ( ) and an
IgM isotype control ( ). The cells were grown with (Column B) or
without (Column A) Vitrogen.
[0064] FIGS. 12A and 12B. Expression of the epitopes of the
anti-37LRP antibody H-150 (A) and 5LAC-23 (B) in a number of tumor
and transformed cell lines as detected by Western blotting under
non-reducing conditions. Cell lines included wild type CHO cells
(Lane 1) and the human cell lines: HB4aR4.a (Lane 2), HMT 3522
(Lane 3), MCF-7 (Lane 4), MDA-MB-231 (Lane 5); MDA-MB-361 (Lane 6),
OVCAR-3 (Lane 7), Chang's Liver (Lane 8), HepG2 (Lane 9); A375
(Lane 10), DLD-1 (Lane 11), LS174T (Lane 12) and SW620 (Lane
13).
DETAILED DESCRIPTION OF THE INVENTION
[0065] The hybridoma cell lines 5LAC-23 were deposited, in
accordance with the Budapest Treaty, with the American Type Culture
Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209
on Dec. 9, 2003, under Accession Number PTA-5690. In accordance
with 37 CFR 1.808, the depositors assure that all restrictions
imposed on the availability to the public of the deposited
materials will be irrevocably removed upon the granting of a
patent.
Example 1
Identification of Binding Proteins by Western Blotting
[0066] To identify the antigen(s) recognised by the antibody
5LAC-23, cell lysates and cytoplasmic fractions expressing the
antigen were subjected to gel electrophoresis, and transferred to
membranes. Western blotting was used to determine proteins detected
by this antibody.
[0067] 1. 1-Dimensional SDS-PAGE
[0068] Previous work demonstrated weak binding of 5LAC-23 to the
ovarian cancer cell line OVCAR-3 by FACS analysis and that 5LAC-23
was shown to have cytotoxic effects against this cell line (U.S.
patent application Ser. No. 10/810,163, the contents of which are
herein incorporated by reference). Total cell lysates were prepared
in RIPA buffer [50 mM Tris-HCl, pH 7.2; 150 mM NaCl; 0.1% (w/v)
SDS; 1% (w/v) sodium deoxycholate; 1% (w/v) Triton X-100] while
cell fractions were made using the Mem-PER Eukaryotic Membrane
Protein extraction Kit (Cat. No. 89826; Pierce; Tattenhall,
Cheshire, UK). The hydrophilic fraction generated was essentially
enriched by removal of membrane components and was considered to be
the cytoplasmic fraction. Protease inhibitors (SIGMA P8340) were
included in all lysis steps. Aliquots of cell preparations were
loaded onto 12% gels and ran at 60V for 30 minutes and then 150V
until the dye front reached the bottom of the gels. Gels were
prepared for transfer of proteins onto PVDF membranes at 30V for 2
hours, using a NOVEX XCell II Blot Module (Invitrogen, Paisley,
UK). Following transfer, membranes were blocked with 5% skimmed
milk powder in Tris-buffered saline containing 0.5% Tween (TBST)
overnight at 4.degree. C. Membranes were incubated with primary
antibody for 4 hours at room temperature. Primary antibodies
included 5LAC-23 (5 .mu.g/mL), and isotype control (mouse
anti-trinitrophenol, IgM, .kappa.; Clone G155-228; Cat. No. 553472;
BD PharMingen; Oxford, Oxon, UK; 5 .mu.g/ml). After membranes were
washed three times with TBST, membranes were incubated with a
horseradish peroxidase (HRP) conjugated goat anti-mouse IgM, .mu. a
chain specific antibody (1/10,0000; Cat. No. 115-035-075; Jackson
Immunologicals West Grove, Pa., USA) for 1 h. After washing the
membranes five times, HRP was detected using ECL Western Blotting
Detection Reagents (Amersham).
[0069] Binding of 5LAC-23 to cytoplasmic fractions of OVCAR-3 cells
yielded a band with approximate MW of 40 kDa (FIG. 1), as indicated
by the black arrow. This band was very weak in total cell lysates
made from OVCAR-3 cells, implicating that the antigen could be
enriched by generating cytoplasmic fractions. The band was not
detected by the isotype control indicating that the interaction
with 5LAC-23 was specific. Although a dominant band was also
observed at approx. 70 kD, this band was also detected by the
isotype control with less intensity. From this experiment, 5LAC-23
appears to bind specifically to a protein of approximately 40
kD.
[0070] 2. 2-Dimensional SDS-PAGE
[0071] Total cytoplasmic proteins prepared as described above, were
precipitated using the Plus One 2-D Clean-Up Kit (Cat. No.
80-6484-51; Amersham, Little Chalfont, Bucks, UK) and then
resuspended in rehydration buffer containing ampholytes in the pH
range 3-10. First dimension isoelectric focusing (IEF) was
performed on an IPGphor (Amersham) with 7 cm immobilised pH 3-10
gradient (IPG)-based strips (Amersham, Little Chalfont, Bucks, UK)
in the presence of rehydration solution (8M urea, 2% CHAPS;
Amersham). Voltage limits were 30V for 14 hours to allow
rehydration to take place, then 200V for 1 hour, 500V for 1 hour,
1000V for 30 minutes and 8000V until 8000 Vh was reached. Following
IEF separation, strips were equilibrated in an SDS-PAGE
equilibration buffer without DTT with 2.5% IAA for 15 minutes. The
strips were placed on top of a 10% gel and sealed with 0.5%
agarose. SDS-PAGE ran at 60V for 30 minutes and then 150V until the
dye front reached the bottom of the gels. Gels were prepared for
transfer of proteins onto PVDF membranes, using Hoefer TE 77
Semi-Dry Transfer Unit (Amersham).
[0072] Following transfer, membranes were blocked with 5% skimmed
milk powder in Tris-buffered saline containing 0.5% Tween (TBST)
overnight at 4.degree. C. Membranes were incubated with primary
antibody for 4 hours at room temperature. Primary antibodies
included 5LAC-23 (5 .mu.g/mL), and isotype control (mouse
anti-trinitrophenol, IgM, .kappa.; Clone G155-228; Cat. No. 553472;
BD PharMingen; Oxford, Oxon, UK; 5 .mu.g/mL). After membranes were
washed three times with TBST, membranes were incubated with a
horseradish peroxidase (HRP) conjugated goat anti-mouse IgM, .mu.
chain specific antibody (1/5000-10,0000; Cat. No. 115-035-075;
Jackson Immunologicals West Grove, Pa., USA) for 1 h. After washing
the membranes five times, HRP was detected using ECL Western
Blotting Detection Reagents (Amersham).
[0073] FIG. 2 demonstrates the Western blot obtained from OVCAR-3
cytoplasmic fractions incubated with 5LAC-23. A single distinct
spot can be seen in the blot probed with 5LAC-23 (FIG. 2b),
compared to the blot incubated with the isotype control (FIG. 2a).
The unique spot is indicated with an arrow and has an acidic pI
with a molecular weight similar to the 36 kD protein marker.
[0074] Two dimensional electrophoresis was repeated using larger
strips (18 cm; Amersham) in order to confirm that 5LAC-23 bound to
an acidic protein, to improve separation of protein spots and to be
able to obtain enough protein for subsequent mass spectrometry
analysis. Rehydration and IEF were carried out according to the
programmed settings: 30V 14 h; 200V 30 min; 500V 30 min; 1000V 1 h
on gradient; 6500V 3 h on gradient; 8000V. The total Vh was
54,000-60,000. Following IEF separation, strips were equilibrated
in an SDS-PAGE equilibration buffer with 2.5% IAA for 15 min. The
strips were placed on top of a 12% gel and sealed with 0.5% agarose
and SDS-PAGE was performed overnight at 60V. One of the gels was
stained for protein using the PlusOne Sliver Staining kit (Cat. No.
17-1150-01; Amersham) following the manufacturer's instructions to
be compatible with MS analysis. Other gels were prepared for
transfer of proteins onto PVDF membranes as described above.
Membranes were probed with 5LAC-23 and an isotype control as
described above. Following transfer, gels were also stained for
protein as described above to assist with alignment of protein
spots.
[0075] The protein spot from OVCAR-3 cytoplasmic fractions that
5LAC-23 binds to can be seen clearly in FIG. 3. FIG. 3a shows a
silver stained gel of OVCAR-3 cytoplasmic fractions. FIG. 3b
reveals that the isotype control did not bind to any protein spots
while a single distinct spot was apparent in the blot probed with
5LAC-23 (FIG. 3c) with molecular weight similar to the 36 kD
protein marker. Binding of 5LAC-23 was specific as the spot was not
detected with the isotype control (FIG. 3b). This experiment
confirmed that the antigenic moiety bound by 5LAC-23 was
approximately 36 KD, and had an acidic pI.
Example 2
Identification of Binding Proteins by Mass Spectroscopy
[0076] The region of the gel corresponding to the 37-40 kD protein
spot recognised by 5LAC-23 was excised using a sterile pipette tip.
Gel plugs were then used for identification of proteins by mass
spectroscopy.
[0077] The samples were subjected to in-gel digestion with trypsin
using a MWG Roboseq 4204 robot (MWG Biotech). Peptides were
released from the gel plug with 1% formic acid and 2% acetonitrile.
A portion of the resulting digest supernatant was analysed on a
MicroMass Q-TOF Global using a 75 mm C18 column for peptide
separation. The data were searched using MASCOT.
[0078] The proteins identified by MS analysis in the region of the
gel that was recognised by 5LAC-23 are presented in Table 2. The
antigen for 5LAC-23 identified by mass spectroscopy was Laminin
receptor 1.
TABLE-US-00002 TABLE 2 Proteins identified that 5LAC-23 recognised
from cytoplasmic fractions of the human ovarian cancer cell line
OVCAR-3 NCBI Observed MW Protein ID Score Accession # 37-40 kD
Laminin receptor 1 136 250127 37-40 kD Ribosomal protein 136
59859883 RS.40K, cytosolic
Example 3
Confirmation of Antigen for 5LAC-23
[0079] 1. Confirmation by Co-Localisation Studies
[0080] Confirmation of the putative antigen was assisted by
determining whether known anti-37LRP antibodies could co-localise
with 5LAC-23. Proteins in cytoplasmic fractions from OVCAR-3 cells
were separated by SDS-PAGE and blotted onto nitrocellulose
membranes. Western blotting was performed as described above for
1-Dimensional SDS-PAGE. Primary antibodies included 5LAC-23 (5
.mu.g/mL), an IgM isotype control (as described above; 5 .mu./mL),
the anti-37LRP antibody H-150 (0.2 .mu.g/mL; Cat. No. sc-20979;
Santa Cruz Biotechnology; Santa Cruz, Calif., USA; This antibody
was raised against a recombinant protein corresponding to AA
110-250 of human 37LRP), the anti-37LRP antibody F-18 (0.4
.mu.g/mL; Cat. No. sc-21534; Santa Cruz) and normal rabbit IgG (0.2
.mu.g/mL; Cat. No. AB-105-C; R&D Systems; Abingdon, Oxon, UK).
Secondary antibodies included HRP-conjugated goat anti-mouse IgM
described above (1/5000-10,000; Jackson Immunologicals) for
detection of IgMs, HRP-conjugated anti-rabbit immunoglobulins
(1/2000; Cat. no. P0448; DAKO, Carpentaria, Calif., USA) and
HRP-conjugated anti-goat immunoglobulins (1/2000; Cat. No.
P0449).
[0081] The two anti-37LRP antibodies, H-150 and F-18, bind to a
band with approximate MW 40 kD (FIGS. 4A and B). 5LAC-23 also binds
to a band of a similar size (FIG. 4D), while the IgM isotype
control does not, confirming that the interactions of 5LAC-23 with
this protein are specific.
[0082] Further 2-D Westerns of cytoplasmic proteins from OVCAR-3
cells were prepared as described in the 2-Dimensional SDS-PAGE
section except that IEF was performed using pharmylates with pI
3.5-5 to restrict the pH range. The primary and secondary
antibodies used for blotting are described above.
[0083] The binding of 5LAC-23 and H-150 to the Western blots is
shown in FIG. 5 and has been compared to blots probed with the
appropriate isotype controls (data not shown). The protein spot
recognised by 5LAC-23 is shown in FIG. 5A, indicated by the black
arrow. The anti-37LRP antibody, H-150, bound to a broader smear of
protein highlighted by the black line (FIG. 5B) that coincides with
the vicinity of the gel that 5LAC-23 also bound to. It is likely
that the smear recognised by H-150 contains at least 3 protein
spots, and may indicate that different isoforms of the protein are
present in the cytoplasmic fraction from OVCAR-3 cells. Other
proteins detected on the blots are due to interactions due to the
isotypes of the antibodies or the secondary antibody and are not
specific to either 5LAC-23, nor H-150. Both the anti-37LRP antibody
and 5LAC-23 bound to proteins of approximately 40 kD providing
further evidence that 37LRP is indeed the antigen for 5LAC-23.
However, although the molecular weights were similar, 5LAC-23
demonstrated a unique binding pattern compared to the other
anti-37LRP antibody. This suggests that the epitopes recognized by
the 2 antibodies are different, with 5LAC-23 demonstrating more
restricted binding.
[0084] 2. Confirmation by Transfection Studies
[0085] Confirmation of the putative antigen was carried out by
determining whether 5LAC-23 could bind to cells that were
transfected with a cDNA clone of 37LRP. A clone of the cDNA
encoding for 37LRP was obtained in the plasmid pCMV6-XL5 (referred
to as pCMV-XL537LRP; Item no. TC107938; Accession Number:
NM-002295; ORIGENE Technologies; Rockville, Mass., USA). Chinese
Hamster Ovary (CHO) cells were grown to be 60-70% confluent in
6-well plates (F12 Ham Nutrient Mixture; 10% FBS; 2 mM Glutamine).
Cells were transfected with pCMV-XL537LRP using Fugene Transfection
Reagent according to the manufacturer's protocol (Cat. No. 1988
387; Roche Diagnostics; Lewes, East Sussex, UK). Cells were grown
for at least 48 hours before immunostaining. Cells were washed
twice with PBS and then fixed with ice cold acetone:methanol (1:1)
for 3 minutes. The acetone:methanol was removed and the cells were
air dried. They were washed three times with PBS and then blocked
with 2% FBS in PBS for 30 minutes. Primary antibody was added and
cells incubated for 1 hour at room temperature. Primary antibodies
included 5LAC-23 (5 .mu.g/ml), an IgM isotype control (as described
above; 5 .mu.g/mL), the anti-37LRP antibody (H-150; 0.2 .mu.g/mL;
Santa Cruz Biotechnology), the anti-67LR antibody (MLuC5; Cat. no.
ab3099; 4 .mu.g/mL; Abcam limited, Cambridge, Cambs, UK) and normal
rabbit IgG (0.2 .mu.g/mL; Cat. No. AB-105-C; R&D Systems;
Abingdon, Oxon, UK). After the cells were washed three times with
PBS, secondary antibody, (HRP-conjugated goat anti-mouse IgM,
1/1000; Jackson Immunologicals) was added and incubated for 1 hour.
Secondary antibodies included HRP-conjugated goat anti-mouse IgM
described above (1/1000; Jackson Immunologicals) for detection of
IgMs and HRP-conjugated anti-rabbit immunoglobulins (1/200; DAKO).
The cells were washed three times before HRP was detected using a
DAB-Substrate Kit (Cat. No. SK-4100; Vector laboratories;
Peterborough, Cambs., UK) according to the manufacturer's
instructions.
[0086] The immunostains reveal that the number of positive (brown)
cells stained with 5LAC-23 increases as the amount of DNA
(pCMV-XL537LRP) is increased (FIG. 6). The isotype control stained
the CHO cells with some background staining but this is similar
regardless of the quantity of DNA that has been included in the
transfection procedure (FIG. 6, Top row), indicating that the
binding of 5LAC-23 to the transiently transfected cells is
specific. These results confirm that the binding protein for
5LAC-23 is the 37kd LRP.
[0087] In addition to transfected cells staining with 5LAC-23, some
cells also stained with two other antibodies directed against the
37LRP and the 67LR (FIG. 7). The anti-37LRP antibody H-150
recognised some transfected cells localising to the cytoplasm (FIG.
7B). The second antibody MLuC5, which recognises the 67LR, also
bound to some cells (FIG. 7C) although in a different pattern to
that of 5LAC-23 and H-150 (FIGS. 7A and 7C). Other investigators
have found that MLuC5 has failed to bind to transfected CHO cells.
This may be due to experimental differences, such as the use of a
different promoter, secondary antibody or clone, or variations in
staining technique. Note that the staining of MLuC5 is restricted
to compartments that may be lysosomal membranes, enabling release
of the receptor for attachment upon contact with laminin. These
results confirm that the transfected protein, 37LRP, is
successfully expressed in CHO cells. These results also provide
evidence that the 67LR is related to the 37LRP polypeptide and that
it can be synthesised in CHO cells. SDS-PAGE results suggest that
5LAC-23 binds to the precursor molecule rather than the 67 kD
laminin receptor protein, as 5LAC-23 binds to a protein which is
approximately 37-45 kD (Example 1). Results from this experiment
revealing the immunostaining pattern of transfected cells shows
that the location of the 5LAC-23 is predominantly cytoplasmic, more
similar to H-150 binding than to MluC5 binding. In toto, the sum of
this evidence suggests that the antigen for 5LAC-23 is the 37LRP
precursor molecule rather than the 67LR.
[0088] 2. Confirmation by Bacterial Expression of 37LRP
[0089] In order to further evaluate the putative antigen of
5LAC-23, the 37LRP cDNA was cloned into an expression vector for
biosynthesis of the protein in a cell free in vitro translation
system. The plasmid pCMV-XL537LRP (described above; ORIGENE) was
used as the template for amplification of the 37LRP cDNA with
primers 5'-GGGAAATTTTCCATATGTCCGGAGC-3' (SEQ ID NO: 5) (includes a
synthetic Nde I site) and 5'-CCTATGCAAGCCCGGGTTAAGACCAG-3' (SEQ ID
NO: 6) (includes a stop codon and synthetic Sma I site). PCR
amplifications were performed using Turbo DNA polymerase
(Stratagene). The DNA template was denatured for 5 min at
94.degree. C., followed by 30 cycles (45 minutes at 94.degree. C.,
45 minutes at 60.degree. C., 1 minute at 72.degree. C.) and
extended for 10 minutes at 72.degree. C. The 37LRP PCR product was
cloned into either pIVEX2.3d and pIVEX2.4d (Cat. no. 03 269 019
001; Roche) using the Nde I and Sma I sites. The plasmids that were
generated include pIVEX2.3dLRP (37LRP without His.sub.6-tag) and
pIVEX2.4dLRP.sub.NHis6 (37LRP with a His.sub.6-tag at N-terminal
end). Expression of 37LRP proteins was performed using cell free in
vitro translation system in bacteria (RTS 100 E. coli HY Kit; Cat
no. 3186 148; Roche Diagnostics, Lewes, UK) following the
manufacturer's instructions. An aliquot of the reaction mixture was
loaded onto 10% gels and transferred to nitrocellulose. Membranes
were blocked with 5% skimmed milk in TBST overnight at 4.degree. C.
Primary antibody was added and blots were incubated at room
temperature for 3 hours. After five washes with TBST, secondary
antibody was added for 1 hour at room temperature. Primary
antibodies included 5LAC-23 (5 .mu.g/mL), an IgM isotype control
(as described above; 5 .mu.g/mL), the anti-37LRP antibody (H-150;
0.2 .mu.g/mL; Santa Cruz Biotechnology) and normal rabbit IgG (0.2
.mu.g/mL; R&D Systems). Secondary antibodies included
HRP-conjugated goat anti-mouse IgM described above (1/5000-10,000;
Jackson Immunologicals) for detection of IgMs and HRP-conjugated
anti-rabbit immunoglobulins (1/2000; DAKO). After washing five
times, HRP-conjugated antibodies were detected with ECL Western
Blotting Detection Reagents (Amersham).
[0090] Western blot analysis revealed that both the anti-37LRP
antibody, H-150, and 5LAC-23 bind to proteins synthesised in the
bacterial reaction mixes only when a template for 37LRP
(pIVEX2.3dLRP (37LRP and pIVEX2.4dLRP.sub.NHis6) was included in
the reaction mixture (FIG. 8). 5LAC-23 and H-150 did not bind to
the reaction mixture when a control plasmid expressing GFP was
included in the reaction mixture. Antibodies against a C-terminal
His.sub.6-tag confirmed that the GFP protein was synthesised (data
not shown). Reaction mixtures appeared to generate at least 2
protein products from the pIVEXLRP constructs, probably due to two
start codons in these constructs (one immediately after the
Ribosomal binding site and one included in the Nde I site used for
cloning), or to post-translational modifications. Although 5LAC-23
and H-150 recognise two similar protein bands, the pattern of
binding is quite distinct. 5LAC-23 preferentially binds to the top
band which appears as more of a smear suggesting possible
post-translational modifications. H-150 binds to distinct bands
(note each band is a doublet; not visible in photographs). There
appears to be some binding to the top 37LRP band by the IgM isotype
control (FIG. 8; Panel A) which may be due to non-specific binding
due to an overload of protein. These results further confirm the
binding of 5LAC-23 to the 37 kD LRP, and provide additional
evidence that the binding of this antibody is to a unique epitope,
distinct from the binding of the known anti-37LRP antibody H-150.
Using the RTS system from Roche, a number of post-translational
modifications can be excluded from biosynthesis of the 37LRP
product. These include N- and O-linked glycosylation,
phosphorylation and disulphide bond formation. It is possible that
other molecules may be added to the polypeptide (such as lipids and
sulphated groups) and may be part of the epitope that 5LAC-23 binds
to. ScanProsite (Expasy) predicts that the 37LRP sequence has a
number of phosphorylation sites, 2 N-myristolyation sites and a
tyrosine sulphation site. Potential myristyl groups can be excluded
from the epitope of 5LAC-23 since E. coli cannot accommodate this
modification. The absence of phosphorylation may be interesting in
that if 5LAC-23 binds to a peptide sequence, this sequence may be
phosphorylated in normal human cells, and de-phosphorylated in
tumor cells, thus exposing the antigen of 5LAC-23.
Example 4
IHC Studies of Antigen Distribution in Humans in Cryo-Preserved
Normal Tissues
[0091] IHC studies were conducted on cryo-preserved tissues to
characterize 5LAC-23 antigen distribution in normal human tissues.
Cryo-preserved slides of normal human tissues were made available
from Covance (UK). They were fixed in acetone for 10 minutes then
washed in wash buffer (PBS with 0.02% Tween-20) twice. Endogenous
peroxidase activity was blocked by incubation in 0.6% hydrogen
peroxide in methanol for 15 minutes. Slides were washed in buffer
prior to blocking in 1% horse serum in wash buffer for 20 minutes
at RT. They were blocked in 2% BSA (SP-5050; Vector Laboratories
Ltd) for a further 20 minutes. Endogenous biotin sites were blocked
using an avidin/biotin blocking kit (SP-2001; Vector Laboratories
Ltd) according to the manufacturer's instructions. 5LAC-23,
anti-cytokeratin-8 (M0631; Dako Cytomation) and IgM anti-KLH
isotype control (550340; BD PharMingen) were incubated with the
slides at 5 .mu.g/mL in 2% BSA/wash buffer for 60 minutes at RT.
Slides were washed three times prior to incubation in goat
anti-mouse IgM biotinylated secondary (B9265; Sigma-Aldrich Company
Ltd) diluted 1:100 in 1% horse serum/wash buffer for 30 minutes at
RT. Slides were washed three times and incubated in avidin-HRP,
made up according to the mouse IgG Vectastain ABC kit (PK-6102;
Vector Laboratories Ltd) for 30 minutes at RT. After washing three
times, the slides were colour developed using DAB according to the
manufacturer's instructions (SK-4100; Vector Laboratories Ltd).
Following a water wash the slides were counterstained with Harris's
haematoxylin then washed in copious amounts of water before
dehydration and mounted in DPX mounting medium (M/D110/08; Fischer
Scientific Ltd).
[0092] On cryo-preserved normal tissues, 5LAC-23 binds weakly to
normal brain and kidney tubules and none of the other tissues
tested (Table 3).
TABLE-US-00003 TABLE 3 Staining of frozen normal tissue arrays 20
.mu.g/mL isotype 5 .mu.g/mL Cytokeratin-8 5 .mu.g/mL 5LAC-23 Skin -
++g epi mc - Brain - - +c Colon - ++epi mc - Breast - ++epi mc -
Lung - ++epi c - Muscle - - - Heart - - - Kidney - ++tubules
+tubules Spleen - +bv - Liver - ++mc heps - g = granular mc =
membraneous/cytoplasmic bv = blood vessels epi = epithelial tissue
c = cytoplasmic heps = hepatocytes
[0093] These results suggested that the antigen for 5LAC-23 was not
widely expressed on normal tissues, and that the antibody would
bind only to a limited number of tissues in humans.
Example 5
Human Normal and Tumor IHC
[0094] The human tissue binding results were then extended by
examining the binding in a wider panel of formalin-fixed human
tissues. Formalin-fixed paraffin-embedded normal organ and tumor
array slides (BA3; AMS Biotechnology Ltd) were de-waxed through
alcohol. Slides were briefly dipped in wash buffer (PBS with 0.02%
Tween-20). Antigen retrieval was performed by micro-waving at full
power for 20 minutes in low pH target retrieval solution (S1699;
Dako Cytomation). Endogenous peroxide activity was blocked by
incubation in 0.6% hydrogen peroxide in methanol for 15 minutes.
Slides were washed in buffer prior to blocking in 1% horse serum in
wash buffer for 20 minutes at RT. Endogenous biotin sites were
blocked using an avidin/biotin blocking kit (SP-2001; Vector
Laboratories Ltd) according to the manufacturer's instructions.
5LAC-23 and IgM anti-KLH isotype control (550340; BD PharMingen)
were incubated with the slides at 0.75 .mu.g/mL in 1% horse serum
wash buffer for 90 minutes at RT. Slides were washed twice prior to
incubation in goat anti-mouse IgM biotinylated secondary (B9265;
Sigma-Aldrich Company Ltd) diluted 1:100 in 1% horse serum/wash
buffer for 30 minutes at RT. After washing twice, slide were
incubated in avidin-HRP, made up according to the mouse IgG
Vectastain ABC kit (PK-6102; Vector Laboratories Ltd) for 30 mins
at RT. Slides were washed twice then colour developed using DAB
according to the kit (SK-4100; Vector Laboratories Ltd). Following
a water wash, the slides were counterstained with Harris's
haematoxylin then washed in copious amounts of water before
dehydration and mounting in DPX mounting medium (M/D110/08; Fischer
Scientific Ltd).
[0095] Table 4 demonstrates that 5LAC-23 binds weakly to skeletal
muscle, normal liver and normal stomach when the tissues are
paraffin-embedded. As in the previous example, 5LAC-23 binding is
restricted in normal tissues. 5LAC-23 binds most strongly to a
hepatocellular carcinoma (HCC) and weakly to a stomach
adenocarcinoma. It does not bind to any of the other normal or
tumor tissues tested. These IHC studies revealed that there is a
clear differential of binding to the HCC compared to normal liver
and most other normal tissues.
TABLE-US-00004 TABLE 4 Staining of human normal organ and tumour
array slides with 5LAC-23 5LAC23 Isotype staining Staining Normal
organ/Tumor (0.75 .mu.g/mL) (0.75 .mu.g/mL) Skin - - - - Breast - -
- - Spleen - - - - Skeletal Muscle - + - - Lung - - - - Liver +c -
- - Gastric Body +g +g - - Colon - - - - Kidney - - - - Prostate -
- - - Placenta - - - - Brain - - - - Infiltrating breast duct - - -
- carcinoma Lung, SCC - - - - Liver, HCC ++c +c - - Oesophagus, SCC
- - - - Stomach, - +c - - adenocarcinoma Small intestine, malignant
- - - - stromal tumor Rectum, adenocarcinoma - - - - Kidney, RCC -
- - - Bladder, TCC - - - - Uterus, endometrial - - - - carcinoma
Ovary, mucinous - - - - cystadenocarcinoma Metastatic malignant N/a
N/a N/a N/a melanoma sc = scattered cells g = granular mc =
membraneous/cytoplasmic bv = blood vessels ctis = connective tissue
epi = epithelial tissue c = cytoplasmic heps = hepatocytes
[0096] An example of staining with 5LAC-23 of normal stomach
(Column A), normal liver (Column B) and liver tumor (Column C) can
be seen in FIG. 9. There is no binding to any of the tissues with
the isotype control indicating that the binding of 5LAC-23 is
specific. 5LAC-23 bound predominantly to the cytoplasm of cells,
although some membranous localisation was observed. 5LAC-23 does
bind very weakly to normal liver and normal stomach, but binds
strongly to the malignant liver.
Example 6
Binding of 5LAC-23 to Human Liver Tumor Sections
[0097] An IHC study was undertaken to determine the cancer
association of the 5LAC-23 antigen with human liver cancers.
Formalin-fixed paraffin-embedded liver array slides (CS1; AMS
Biotechnology Ltd) were de-waxed through alcohol. Slides were
briefly dipped in wash buffer (PBS with 0.02% Tween-20). Antigen
retrieval was performed by micro-waving at full power for 20
minutes in low pH target retrieval solution (S1699; Dako
Cytomation). Endogenous peroxide activity was blocked by incubation
in 0.6% hydrogen peroxide in methanol for 15 minutes. Slides were
washed in buffer prior to blocking in 1% horse serum in wash buffer
for 20 minutes at RT. Endogenous biotin sites were blocked using an
avidin/biotin blocking kit (SP-2001; Vector Laboratories Ltd)
according to the manufacturer's instructions. 5LAC-23 and IgM
anti-KLH isotype control (550340; BD PharMingen) were incubated
with the slides at 0.75 .mu.g/mL in 1% horse serum wash buffer for
90 minutes at RT. Slides were washed twice prior to incubation in
goat anti-mouse IgM biotinylated secondary (B9265; Sigma-Aldrich
Company Ltd) diluted 1:100 in 1% horse serum/wash buffer for 30
minutes at RT. After washing twice, slide were incubated in
avidin-HRP, made up according to the mouse IgG Vectastain ABC kit
(PK-6102; Vector Laboratories Ltd) for 30 minutes at RT. Slides
were washed twice then colour developed using DAB according to the
kit (SK-4100; Vector Laboratories Ltd). Following a water wash, the
slides were counterstained with Harris's haematoxylin then washed
in copious amounts of water before dehydration and mounting in DPX
mounting medium (M/D110/08; Fischer Scientific Ltd).
[0098] 5LAC-23 bound to 73% of HCC sections, on a HCC tissue array
slide (See Table 5), although in one or two samples only a few
cells were stained. Staining was predominantly cytoplasmic. These
results indicate that the 5-LAC-23 antigen is not only highly
expressed in liver cancers compared to other tissue types, but that
it is expressed in the majority of human liver cancers from
different patients.
TABLE-US-00005 TABLE 5 IHC of a Human liver array slide with
5LAC-23 and an IgM isotype control. Sec. Isotype No. Age Sex Organ
Diagnosis Stage 5LAC-23 control 1 53 F skin metastatic HCC IVB - --
2 57 M liver HCC IIA - -- 3 41 M liver HCC IIIA +Cytoplasmic -- 4
45 M skin metastatic HCC IVB - -- 5 54 F chest wall metastatic HCC
IVB +Cytoplasmic -- 6 40 M lumbar vertebra metastatic HCC IVB
+Cytoplasmic -- 7 52 M liver HCC IIIA +Cytoplasmic -- 8 43 M bile
duct HCC IVA - -- 9 51 M colon metastatic HCC IVB - -- 10 49 M bile
duct HCC IVA +Blood -- 11 56 M liver HCC IIIA +Cytoplasmic -- 12 56
F lung metastatic HCC IVB - -- 13 40 M lymph node metastatic HCC
IIIB +Cytoplasmic -- 14 37 M liver HCC IVB +Cytoplasmic -- 15 75 M
liver HCC I +Cytoplasmic -- 16 37 M neck metastatic HCC IVB
+Scattered cells -- 17 32 M liver HCC IIIA - -- 18 50 M femur
metastatic HCC IVB +Scattered cells; granular -- 19 57 M liver HCC
IIIA +Cytoplasmic -- 20 62 M lung metastatic HCC IVA +Scattered
cells -- 21 61 M lung metastatic HCC IVB - -- 22 65 M liver
moderately differentiated HCC II +Cytoplasmic; Membraneous -- 23 52
M liver moderately differentiated HCC I +Cytoplasmic -- 24 65 M
liver poorly differentiated HCC II +Cytoplasmic -- 25 72 M liver
well & poorly differentiated HCC II - -- 26 58 F liver well
differentiated HCC II +Cytoplasmic -- 27 52 M liver well
differentiated HCC II +Cytoplasmic -- 28 40 M liver moderately
differentiated HCC II +Cytoplasmic -- 29 53 F liver moderately
differentiated HCC IIIA +Cytoplasmic -- 30 67 M liver moderately
differentiated HCC II +Cytoplasmic -- 31 58 F liver moderately
differentiated HCC II +Cytoplasmic -- 32 20 M liver well
differentiated HCC IVA +Scattered cells 33 48 M liver moderately
differentiated HCC II ++Cytoplasmic -- 34 47 M liver HCC I
+Scattered cells -- 35 35 M omentum metastatic HCC IVB +one cell;
Cytoplasmic -- 36 69 F liver moderately differentiated HCC II
+Ctyoplasmic -- 37 65 M liver well differentiated HCC II
+Cytoplasmic -- 38 63 F liver moderately differentiated HCC II
+Scattered cells -- 39 60 F liver poorly differentiated HCC II
+Scattered cells 40 65 F liver moderately differentiated HCC IIIA -
-- 41 69 F liver poorly differentiated HCC IIIA +Scattered cells --
42 66 F liver well differentiated HCC II +Cytolpasmic -- 43 65 F
liver moderately differentiated HCC I +Cytoplasmic -- 44 64 M liver
poorly differentiated HCC IIIA +Cytoplasmic -- 45 59 M liver
moderately differentiated HCC IIIA +Cytoplasmic --
Example 7
Matched Normal and Liver Tumor Staining
[0099] Cryo-preserved slides of matched adjacent normal and primary
human hepatocellular carcinoma liver tissue (T6235149; AMS
Biotechnology Ltd) were defrosted and air dried. They were fixed in
acetone for 10 mins then washed in wash buffer (PBS with 0.02%
Tween-20) twice. Endogenous peroxide activity was blocked by
incubation in 0.6% hydrogen peroxide in methanol for 15 minutes.
Slides were washed in buffer prior to blocking in 1% horse serum in
wash buffer for 20 minutes at RT. They were blocked in 2% BSA
(SP-5050; Vector Laboratories Ltd) for a further 20 minutes.
Endogenous biotin sites were blocked using an avidin/biotin
blocking kit (SP-2001; Vector Laboratories Ltd) according to the
manufacturer's instructions. 5LAC-23, anti-cytokeratin-8 (M0631;
Dako Cytomation) and IgM anti-KLH isotype control (550340; BD
PharMingen) were incubated with the slides at 5 .mu.g/mL in 2%
BSA/wash buffer for 60 minutes at RT. Slides were washed three
times prior to incubation in goat anti-mouse IgM biotinylated
secondary (B9265; Sigma-Aldrich Company Ltd) diluted 1:100 in 1%
horse serum/wash buffer for 30 minutes at RT. Slides were washed
three times and incubated in avidin-HRP, made up according to the
mouse IgG Vectastain ABC kit (PK-6102; Vector Laboratories Ltd) for
30 minutes at RT. After washing three times, the slides were colour
developed using DAB according to the manufacturer's instructions
(SK-4100; Vector Laboratories Ltd). Following a water wash the
slides were counterstained with Harris's haematoxylin then washed
in copious amounts of water before dehydration and mounted in DPX
mounting medium (M/D110/08; Fischer Scientific Ltd).
[0100] FIG. 10 shows cryo-preserved liver sections from matched
normal and tumor tissue from the same individual. Note that
cytokeratin 8 (Panel C) localises to normal hepatocytes and binding
is restricted to the normal tissue, indicating that some of the
sections are a mixture of normal and cancerous tissue. 5LAC-23 only
binds to the tumor sample and is specific to the central region
that represents the malignant tissue in this section. The sections
are negative when stained with the isotype control, indicating that
the binding of 5LAC-23 is specific. The staining pattern, from
5LAC-23, showed that in patient samples, the antibody was highly
specific for malignant cells thereby making it an attractive
druggable target.
Example 8
Induction of Binding in Normal Cells by Altered Growth
Conditions
[0101] Experiments were carried out to investigate whether 5LAC-23
expression could be induced in normal cells under selected
conditions. Beas-2B `normal` lung epithelial cells were grown on
flasks uncoated and coated with vitrogen (0.03 mg/ml vitrogel;
Cohesion Technologies Inc.). Cells were grown in bronchial
epithelial growth medium (CC-3170; Clonetics) in a humidified
atmosphere of 95% air/5% CO.sub.2 at 37.degree. C. When
approximately 80% confluent, cells were removed with cell
dissociation solution (SIGMA Cat. No. C5914), washed twice in PBS
and fixed in 10% formalin for 30 minutes. Cell pellets were
dehydrated in alcohol before being suspended in paraffin wax and
incubated at 45.degree. C. for 60 minutes. The paraffin wax was
refreshed three times with a 60 minute incubation at 45.degree. C.
After cooling and setting, 3 .mu.m sections were cut on a Leica RM
2135 microtome and baked onto glass slides. Sections were stained
with 5 .mu.g/mL 5LAC-23 or IgM isotype control as for the matched
liver samples described above.
[0102] Binding of 5LAC-23 to the normal lung epithelial cell line
Beas-2B can be seen only when the cells are grown with Vitrogen
(FIG. 11). No binding of the IgM isotype control can be seen
indicating that the binding of 5LAC-23 was specific. These results
may imply that under certain conditions, such as in the presence of
growth factors, adhesion molecules and extracellular matrix
molecules, the expression of antigen of 5LAC-23 can be induced,
even in normal cells. Abnormalities in these growth conditions
often occur in the malignant and pre-malignant states, and may
contribute to altered expression of 37LRP, such as that observed in
the hepatocellular carcinomas.
Example 9
Distribution of 5LAC-23 in Various Human Cell Lines by Western
Analyses
[0103] A survey of human cell lines was performed by Western
analyses to assess the distribution of 37LRP and the epitope of
5LAC-23. Cell lysates were prepared from a number of human tumor or
transformed cell lines in RIPA buffer with protease inhibitors and
Western blots were prepared as described in the 1-Dimensional
SDS-PAGE section. Lysates were made from breast cell lines HB4aR4.a
(normal breast cells transformed with Ras), HMT 3522 (normal
cells), MCF-7 (tumor cells), MDA-MB-231 (tumor cells); MDA-MB-361
(brain metastasis from breast tumor); an ovarian tumor cell line
OVCAR-3; liver cell lines Chang's Liver and HepG2; a melanoma cell
line A375; and the colon tumor cell lines DLD-1, LS174T and SW620.
Wild type CHO cells were also included since a 37LRP protein has
previously been described from the Chinese hamster (Cricetulus
griseus; NCBI Accession number: 298088). Blots were probed with
either H-150 or 5LAC-23 as described in the Co-localisation
section.
[0104] The anti-37LRP antibody H-150 recognises a protein band in
all of the cell lines tested, including wild type CHO cells (FIG.
12A). Note that there are at least two protein bands in the colon
cell line, DLD-1, detected with H-150 indicating the existence of
different human isoforms. Although both H-150 and 5LAC-23 bound to
a similar sized band in the Westerns, 5LAC-23 bound to the cell
lysates in a different pattern to that of H-150 (FIG. 12B). H-150
detects the 37LRP in all of the cell lines, the expression of the
epitope of 5LAC-23 varies across the different cell lysates and in
some is not present. This difference between antibodies is not due
to lower affinity of 5LAC-23 for its antigen compared to H-150 for
its' antigen as the binding of both antibodies to the CHO cell
lysates is similar. 5LAC-23 detects a unique smear of approx. 110
kD in the LS 174T lysates under non-reducing conditions (FIG. 12B,
Lane 12). This smear disappears under reducing conditions but there
is no increase in the band corresponding to 37LRP precursor (data
not shown). These results present further evidence that the epitope
recognized by 5LAC-23 on 37LRP is unique, compared to the known
anti-LRP antibody H-150.
[0105] 5LAC-23 did not bind to wild type CHO cells by an
immunohistochemistry (Transfection section, Example 3), yet does
bind to wt CHO lysates by Western under both reducing and
non-reducing conditions (FIG. 12). This suggests that the epitope
of 5LAC-23 may be conformationally dependent and/or that the
epitope is not exposed or accessible to the antibody under native
conditions.
[0106] In toto, this data demonstrates that the 5LAC-23 antigen is
a cancer associated antigen and is expressed in humans, and is a
pathologically relevant cancer target. Further, this data also
demonstrates the binding of the 5LAC-23 antibody to human cancer
tissues, and can be used appropriately for assays that can be
diagnostic, predictive of therapy, or prognostic. In addition, the
cell localization of this antigen is indicative of the cancer
status of the cell due to the lack of expression of the antigen in
most non-malignant cells, and this observation permits the use of
this antigen, its gene or derivatives, its protein or its variants
to be used for assays that can be diagnostic, predictive of
therapy, or prognostic.
[0107] In all, this invention teaches the use of the 5LAC-23
antigen as a target for diagnostics, theranostics, prognostics or
therapeutics. Furthermore, this invention also teaches the use of
detecting the 5LAC-23 antigen in cancerous cells that can be useful
for the diagnosis, prediction of therapy, and prognosis of mammals
bearing tumors that express this antigen.
[0108] All patents and publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0109] It is to be understood that while a certain form of the
invention is illustrated, it is not to be limited to the specific
form or arrangement of parts herein described and shown. It will be
apparent to those skilled in the art that various changes may be
made without departing from the scope of the invention and the
invention is not to be considered limited to what is shown and
described in the specification.
[0110] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. Any oligonucleotides, peptides, polypeptides, biologically
related compounds, methods, procedures and techniques described
herein are presently representative of the preferred embodiments,
are intended to be exemplary and are not intended as limitations on
the scope. Changes therein and other uses will occur to those
skilled in the art which are encompassed within the spirit of the
invention and are defined by the scope of the appended claims.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention which are obvious to those skilled
in the art are intended to be within the scope of the following
claims.
Sequence CWU 1
1
616PRTArtificialpeptide G, a synthetic peptide derived from the
sequence of 37LRP 1Leu Met Trp Trp Met Leu1 525PRTHomo sapiens 2Tyr
Ile Gly Ser Arg1 535PRTHomo sapiens 3Ile Lys Val Ala Val1
546PRTHomo sapiens 4Leu Gly Thr Ile Pro Gly1
5525DNAArtificialprimer sequence for PCR amplification 5gggaaatttt
ccatatgtcc ggagc 25626DNAArtificialprimer sequence for PCR
amplification 6cctatgcaag cccgggttaa gaccag 26
* * * * *