U.S. patent application number 11/957899 was filed with the patent office on 2008-07-24 for single-chain antibodies against human insulin-like growth factor i receptor: expression, purification, and effect on tumor growth.
This patent application is currently assigned to CITY OF HOPE. Invention is credited to Yoko Fujita-Yamaguchi.
Application Number | 20080177047 11/957899 |
Document ID | / |
Family ID | 34222249 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080177047 |
Kind Code |
A1 |
Fujita-Yamaguchi; Yoko |
July 24, 2008 |
Single-chain Antibodies Against Human Insulin-Like Growth Factor I
Receptor: Expression, Purification, and Effect on Tumor Growth
Abstract
A method of inhibiting the growth of hormone dependent tumor
cells in a mammal comprises administering to said mammal an
insulin-like growth factor receptor (IGF-IR) recombinant antibody,
wherein said antibody can be a single-chain recombinant antibody,
which can be humanized, capable of blocking agonist interaction
with the IGF-IR.
Inventors: |
Fujita-Yamaguchi; Yoko;
(Duarte, CA) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W., SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
CITY OF HOPE
Duarte
CA
|
Family ID: |
34222249 |
Appl. No.: |
11/957899 |
Filed: |
December 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10864818 |
Jun 10, 2004 |
7329745 |
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11957899 |
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10134519 |
Apr 30, 2002 |
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10864818 |
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09609776 |
Jul 3, 2000 |
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10134519 |
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60211187 |
Jun 13, 2000 |
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Current U.S.
Class: |
530/389.1 |
Current CPC
Class: |
C07K 16/2863 20130101;
A61K 39/39541 20130101; C07K 2317/24 20130101; A61K 39/39541
20130101; A61K 2039/505 20130101; C07K 2319/00 20130101; A61K
2300/00 20130101; C07K 2317/73 20130101; C07K 2317/622
20130101 |
Class at
Publication: |
530/389.1 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. A purified recombinant antibody comprising antigen binding
portions having the binding specificity of the antigen binding
portions of murine antibody 1H7.
2. A purified recombinant antibody comprising antigen binding
portions of a V.sub.L domain and a V.sub.H domain comprising amino
acid sequences as set forth in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID
NO:8, and in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11,
respectively.
3. The recombinant antibody of claim 2, wherein the V.sub.L and
V.sub.H domains have amino acid sequences as set forth in SEQ ID
NO:3 and SEQ ID NO:4, respectively.
Description
[0001] This application is a divisional of U.S. Ser. No.
10/864,818, filed Jun. 10, 2004, which is a continuation-in-part of
U.S. application Ser. No. 10/134,519, filed Apr. 30, 2002, which is
a continuation of U.S. application Ser. No. 09/609,776, filed Jul.
3, 2000, which claims priority from provisional application number
60/211,187, filed Jun. 13, 2000. Each of these applications are
incorporated by reference into this application in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention is in the field of methods for
treatment of hormone dependent cancers.
BACKGROUND OF THE INVENTION
[0003] All references cited herein are incorporated by reference
into this application in their entirety.
[0004] Insulin and Insulin-like Growth Factors stimulate the growth
of human breast cancer cells in vitro. The Insulin-like Growth
Factors I (IGF-I) and II (IGF-II) interact with cell surface
receptors eliciting their cellular response. The IGF-I receptor
(IGF-IR) is the cell surface receptor for IGF-I having high binding
affinity for this growth factor. However, IGF-IR is also thought to
have a high binding affinity for IGF-II. Interaction of either of
these two growth factors to the IGF-IR elicits intracellular
responses through protein tyrosine phosphorylations, which can be
blocked through the inhibition of the interaction of either IGF-I
or IGF-II to the receptor.
[0005] These intracellular responses of IGF-IR signaling are
implicated in the inducement of cell growth, proliferation and
anti-apoptosis. It has been shown that the IGF-IR can not only
induce normal cell growth but also induces tumor cell growth in
both breast cancer and prostate cancer. In addition, the
anti-apoptotic activity of IGF-IR protects cancerous tumor cells
from chemotherapeutic treatments in breast cancers.
[0006] Therefore, a need exists for a method of inhibiting IGF-IR
in order to inhibit tumor cell growth and increase sensitivity to
chemotherapeutic agents. The activity of the IGF-IR can be
inhibited by various methods. One of these methods comprises
inhibiting the activation of the IGF-IR by preventing binding of
agonist such as IGF-I or IGF-II. This can be achieved by blocking
the IGF-IR binding site with antagonists.
[0007] Antibodies can be effective antagonists in inhibiting the
interaction of the IGF-IR with IGF-I or IGF-II. .alpha.IR-3
(Arteaga, C. L. and Osborne, C. K.; Cancer Research 49, 6237-6241,
1989) is an antibody with high affinity for the IGF-IR and has been
found to inhibit the interaction of IGF-I with the IGF-IR. In in
vitro experimentation this murine antibody has been found to
inhibit the growth of various tumor cells from breast cancer cell
lines. In various tumor cells (MCF-7, MDA-231, ZR75-1, and HS578T)
this .alpha.IR-3 could inhibit the IGF-I mediated DNA synthesis in
vitro. However, in estrogen dependent tumor cells, such as MCF-7,
ZR75-1 and T47D, the inhibition with .alpha.IR-3 of the IGF-IR in
vivo failed to block estrogen stimulated DNA synthesis or
proliferation. In contrast, in T61 tumor cells the .alpha.IR-3
antibody could inhibit tumor cell growth in vivo when used in
combination with down-regulation of IGF-II synthesis by
simultaneous treatment with estradiol and tamoxifen. It appears
that .alpha.IR-3 is a better antagonist for IGF-I blockage compared
to its ability to inhibit interaction of IGF-II with IGF-IR.
[0008] Another murine antibody against the .alpha.-subunit of
IGF-IR, 1H7 (Li S. et al. Biochemical and Biophysical Research
Communications, 196, 92-98, 1993), has shown good results in
inhibiting the activation of IGF-IR. In in vitro experimentation
with NIH3T3 cells over-expressing human IGF-IR the 1H7 antibody
inhibits basal, IGF-I or IGF-II stimulated DNA synthesis. A second
antibody raised against the IGF-IR .alpha.-subunit, 2C8, however,
is unable to block IGF-IR activation by either IGF-I or IGF-II
while having binding affinities for the receptor.
[0009] While these two murine antibodies, .alpha.IR-3 and 1H7, have
shown results in inactivation of the IGF-IR in vitro, their ability
to inhibit estrogen dependent tumor cell growth in vivo is limited.
Furthermore, the monoclonal murine antibodies have their obvious
disadvantages in their use for human treatment or other mammals. In
addition, their relative complexity limits the ability to
manipulate the antibodies to optimize their use in the treatment of
mammalian hormone dependent cancers. Accordingly, improvements are
sought.
SUMMARY OF THE INVENTION
[0010] In accordance with the present invention, a method of
inhibiting the growth of hormone dependent tumor cells in a mammal
comprises administering to said mammal an anti insulin-like growth
factor I receptor (IGF-IR) recombinant antibody. In a preferred
embodiment, the method comprises administering a single chain
antibody (scFv). In a further preferred embodiment the method
comprises administering a chimeric single chain antibody in which a
constant domain has been linked to the single chain antibody.
[0011] There also is provided a novel IGF-IR antagonist comprising
a recombinant antibody which blocks agonist interaction with the
IGF-IR. The antibody comprises antigen binding portions that have
the specificity of the antigen binding sites of the murine 1H7
antibody. The recombinant antibody can be a single chain or double
chain antibody. In one embodiment of the invention, the antibody is
in the form of a novel chimeric single-chain antibody against
IGF-IR.
[0012] In a preferred embodiment of the invention, the antibody is
in the form of the single chain recombinant antibody of SEQ ID
NO:1.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1. This figure is a schematic representation of soluble
forms of anti (human insulin-like growth factor I receptor)
single-chain antibodies (.alpha.IGF-IR scFvs). A is a single-chain
antibody (.alpha.IGF-IR scFv). B is a chimeric .alpha.IGF-IR
scFv-Fc [C-terminal tag: SEQ ID NO:22].
[0014] FIG. 2. This figure illustrates the effects of .alpha.IGF-IR
scFv-Fc and mAb on .sup.125I-IGF-II (A) and .sup.125I-IGF-I (B)
binding to purified human IGF-1 receptor. The binding activity is
calculated as the percentage IGF-binding in the absence of
antibodies, and expressed as average .+-.SD of four independent
experiments for A or seven independent experiments for B, except
that two control experiments with 2C8 mAb were performed for B.
Antibodies used are .alpha.IGF-IR scFv-Fc (.tangle-solidup.), 1H7
(.box-solid.), and control 2C8 (.tangle-solidup.).
[0015] FIG. 3. This figure illustrates the effects of .alpha.IGF-IR
scFv-Fc and 1H7 on cell growth. NIH3T3 cells over-expressing IGF-IR
were cultured in the absence (.box-solid.), or presence of 10 nM
(.tangle-solidup.), 100 nM () or 1000 nM (.diamond-solid.) 1H7 (A)
or .alpha.IGF-IR scFv-Fc (B).
[0016] FIG. 4. This figure illustrates the effects of MCF-7 tumor
cell growth in nude mice in the absence (.box-solid.) or presence
(.tangle-solidup.) of .alpha.IGF-IR scFv-Fc.
[0017] FIG. 5. This figure illustrates the effects of .alpha.IGF-IR
scFv-Fc on MCF-7 tumor cell growth in vivo in the presence or
absence of the anti-neoplastic agent Doxorubicin. On day 0 MCF-7
cells were implanted in the mouse followed by a treatment of PBS
(control) (.box-solid.), .alpha.IGF-IR scFv-Fc (.tangle-solidup.),
Doxorubicin () or .alpha.IGF-IR scFv-Fc+Doxorubicin
(.diamond-solid.) beginning at day 4.
[0018] FIG. 6. This figure illustrates the effects of .alpha.IGF-IR
scFv-Fc on T61 tumor cell growth in vivo in the presence or absence
of the estrogen antagonist Tamoxifen (TAM). On day 36 following
implantation of T61 tumor cells the mice were treated with PBS
(control) ( ), .alpha.IGF-IR scFv-Fc (.DELTA.), tamoxifen
(.box-solid.) or .alpha.IGF-IR scFv-Fc+tamoxifen (*).
[0019] FIG. 7. This figure shows the amino acid sequence of SP-3b1,
a soluble single chain recombinant antibody, the amino acid
sequence of which is comparable to the amino acid sequence of 1H7,
(SEQ ID No:1), including the CDRs in both the V.sub.L and V.sub.H
domains of the single chain recombinant antibody.
[0020] FIG. 8. This figure shows the nucleic acid sequence of
SP-3b1, a soluble single chain recombinant antibody, the amino acid
sequence of which is comparable to the amino acid sequence of 1H7,
(SEQ ID NO:2), including the regions coding for the CDRs of both
the V.sub.L and V.sub.m domains.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention provides a method of inhibiting
hormone dependent tumor growth by blocking the activation of the
Insulin-like Growth Factor I receptor (IGF-IR). This blockage can
be accomplished by exposing hormone dependent tumor cells to an
antagonist of IGF-IR. Inhibition of IGF-IR can lead to a decrease
in cell growth and can also render the hormone dependent tumor
cells more susceptible to therapeutic agents. Alternatively, the
antagonist interaction with IGF-IR, inhibiting the activation of
the receptor, can lead to apoptosis of the hormone dependent tumor
cells.
[0022] Therefore, the invention provides a method of treatment of
mammals suffering from hormone dependent tumor cell growth by
administering to the mammal an anti-IGF-IR recombinant antibody.
Preferably the invention provides for a treatment of mammals
suffering from estrogen dependent cancer, such as breast cancer. In
addition, the treatment comprises administering to said mammal an
anti-IGF-IR recombinant antibody in combination with one or more
therapeutic agents, such as tamoxifen, which are effective in
reducing the growth of hormone-dependent tumors.
[0023] In a preferred embodiment of the invention the anti-IGF-IR
antibody is a recombinant antibody wherein the antigen binding
portions of the antibody are comparable to the antigen binding
portions of murine antibody 1H7. Comparable antigen binding
portions are ones in which the amino acid sequences have the
binding specificity of the amino acid sequence of the antigen
binding portions of the murine antibody 1H7. The CDRs within the
binding portion have at least 90% identity to the corresponding CDR
of 1H7, preferably 95% identity and most preferably full identity.
Particularly preferred is a single chain recombinant antibody, such
as the .alpha.IGF-IR scFv or .alpha.IGF-IR scFv-Fc antibody
comprising antigen binding portions comparable to the antigen
binding portions of murine antibody 1H7, or even more preferred is
the single chain recombinant antibody of SEQ ID NO:1. The single
chain antibodies are advantageous because of the relative ease in
their expression, purification and manipulation. The expression of
such antibodies in expression systems makes them more susceptible
to large scale production and purification. In addition,
manipulation of such single chain antibodies may consist of
altering such antibodies to covalently attach other therapeutic
agents. Such agents can, for example, include toxins, enzymes, or
radionucleotides. The recombinant single chain antibody conjugated
with such agents can block IGF-IR induced tumor cell growth and
target such agents to said tumor cells which have been made more
susceptible to apoptosis by the inhibition of IGF-IR.
[0024] The single chain antibody comprises at least an Fv domain
capable of blocking IGF-IR interaction with IGF-I or IGF-II. The
IGF-IR scFv comprises both the antigen binding region of a light
chain variable domain, V.sub.L, and the antigen binding region of a
heavy chain variable domain, V.sub.H, coupled by a short linker
peptide. In a preferred embodiment, the V.sub.L domain and the
V.sub.H domain are derived from the 1H7 antibody against the
.alpha.-subunit of IGF-IR. The IGF-IR scFv can be tagged with a
short peptide such as the FLAG epitope to facilitate purification
of the soluble IGF-IR scFv from the medium of the expression
system. The DNA coding for the V.sub.L and V.sub.H domains are
obtainable by sequencing said domains from a parental antibody, in
a preferred embodiment said parental antibody being 1H7. A
recombinant DNA then can be constructed comprising, in order,
coding sequences for the N-terminal signal peptide, the antigen
binding region of the V.sub.L domain, a linker peptide, the antigen
binding region of the V.sub.H domain and a C-terminal tag peptide
for purification and identification. Said genetically engineered
antibody can be expressed in myeloma or bacterial cell expression
systems. The monovalent recombinant single chain antibody IGF-IR
scFv can be purified from the medium of said expression system by
conventional protein purification methods, such as, for example,
affinity chromatography.
[0025] The linker peptide is chosen based upon known structural and
conformational information of peptide segments and is selected so
that it will not interfere with the tertiary structure of the
single chain antibody and its uses. Typically, a linker of between
about 6 and 50 amino acids is preferred for ease and economics of
preparation.
[0026] One such single chain recombinant antibody comprising
antigen binding portions comparable to the antigen binding portions
of murine antibody 1H7 is the peptide with the binding specificity
of the sequence shown in SEQ ID NO:1 shown in FIG. 1. FIG. 2 shows
the nucleic acid sequence encoding this single chain recombinant
antibody. The single chain recombinant antibody of SEQ ID NO:1
comprises a V.sub.L domain (SEQ ID NO:3) and a V.sub.H domain (SEQ
ID NO:4) which are linked by the linker peptide GGGGSGGGSGGGGSGGGS
(SEQ ID NO:5). Each of these domains (V.sub.L and V.sub.H) contain
three complimentarity determining regions (CDRs)responsible for
antigen recognition. The three CDRs of the V.sub.L domain
KASQDVNTA(SEQ ID NO:6), WASTRMMT (SEQ ID NO:7), and HQHYTTPYT(SEQ
ID NO:8), are designated CDR1.sub.L, CDR2.sub.L and CDR3.sub.L
respectively. The three CDRs of the V.sub.H domain, IYAMS (SEQ ID
NO:9), SISNGGTTYYPDSVKG (SEQ ID NO:10), and TFYYSFPRAMDY (SEQ ID
NO:11) are designated CDR1.sub.H, CDR2.sub.H, and CDR3.sub.H
respectively.
[0027] In one embodiment of the invention the soluble IGF-IR scFv
is a chimeric antibody which further comprises an Fc domain. In
this embodiment, the recombinant DNA will comprise the coding
sequence of IGF-IR scFv minus the C-terminal tag peptide, coupled
to a coding sequence for an Fc domain. Desirably, the Fc domain
comprises the C.sub.H2 and C.sub.H3 regions of an antibody heavy
chain constant domain. The recombinant DNA can be expressed in a
myeloma or bacterial expression system in accordance with
conventional techniques and said single-chain antibody IGF-IR
scFv-Fc can be purified using conventional protein purification
methods. The IGF-IR scFv-Fc exists preferably in its divalent form.
The IGF-IR scFv-Fc can comprise a humanized form of the IGF-IR
scFv, such as, for example, by using a coding sequence of a human
Fc domain when constructing the recombinant DNA. Said single chain
antibodies (IGF-IR scFv or IGF-IR scFv-Fc) subsequently can be
modified, if desired, and attached to other therapeutic agents.
[0028] To treat mammals suffering from hormone dependent cancer,
preferably from estrogen dependent breast cancer, the recombinant
single-chain antibodies (IGF-IR scFv or IGF-IR scFv-Fc) can be
administered in a pharmaceutically acceptable composition as the
sole therapeutic or in combination with one or more other
therapeutic agents, such as tamoxifen, which are effective in
reducing hormone-dependent tumor cell growth. The tamoxifen or
other therapeutic agent can be administered in accordance with
conventional therapeutic methods, such as parenteral or
subcutaneous administration. Administration of said recombinant
single chain antibodies can be used as a method of inhibiting tumor
cell growth in vivo or to induce susceptibility of said tumor cells
to therapeutic agents.
[0029] In light of the preceding description, one skilled in the
art can use the present invention to its fullest extent. The
following examples, therefore, are to be construed as illustrative
only and not limiting in relation to the remainder of the
disclosure.
EXAMPLE 1
[0030] Cloning of 1H7 variable Domains by RT-PCR.
[0031] Heavy and light chains of mouse monoclonal antibody 1H7 (Li,
S. et al. Biochemical and Biophysical Research Communications, 196,
92-98, 1993) were separated by sodium dodecyl
sulfate/polyacrylamide gel electrophoresis (SDS-PAGE; 12.5%
polyacrylamide gel), under reducing conditions, blotted onto a
polyvinylidene difluoride membrane, and subjected to N-terminal
amino acid sequence determination by Edman degradation. Degenerate
oligonucleotides, used as upstream primers, were synthesized on the
N-terminal sequences of the heavy and light chains of 1H7 while the
constant region oligonucleotides for the downstream primers were
designed and synthesized according to the published nucleotide
sequences. Primers (Table 1) containing the EcoRI site were used to
amplify the heavy- and light-chain variable regions (V.sub.H and
V.sub.L, respectively) from 1H7 poly(A) rich mRNA by reverse
transcriptase polymerase chain reaction (RT-PCR). PCR products were
ligated into the EcoRI site of pBleuscriptII SK. Escheria coli
XL1-Blue was transformed with the vectors encoding PCR-generated
V.sub.H and V.sub.L sequences.
[0032] The N-terminal amino acid sequences of the heavy- and
light-chains of 1H7 were determined to be EVKVVESGGGLVKPG (SEQ ID:
NO 12) and DIVMTQSHKFMSTSV (SEQ ID: NO 13) respectively.
TABLE-US-00001 TABLE 1 Primers for PCR amplification of variable
regions of heavy and light chains of 1H7. Light-chain primers Amino
Acid 1 2 3 4 5 6 Asp Ile Val Met Thr Gln [SEQ ID NO: 14 5' end
primer gggaattc G A C A T T G T G A T G A C C C A A 3' [SEQ ID NO:
15] T C C A G T C-region amino acid Ser Ile Phe Pro Pro Ser [SEQ ID
NO: 16] C-region primer 5'T C C A T C T T C C C A C C A T C C
gaattccg3' [SEQ ID NO: 17] Heavy-chain primers Amino Acid 1 2 3 4 5
6 [SEQ ID NO: 18] Glu Val Lys Val Val Glu 5' End primer gggaattc G
A A G T A A A A G T A G T A G A A 3' [SEQ ID NO: 19] G C G C C G G
G G C-region amino acid Val Tyr Pro Leu Ala Pro [SEQ ID NO: 20]
C-region primer 5'G T C T A T C C A C T G G C C C C T gaattccg3'
[SEQ ID NO: 21]
EXAMPLE 2
Design of .alpha.IGF-IR Antibodies.
[0033] Two soluble forms of 1H7-based .alpha.IGF-IR antibodies,
scFv and scFv-Fc, were designed as schematically presented in FIG.
3. ScFv is a monovalent antibody and has an expected M.sub.r of 27
kDa. ScFv-Fc is a divalent antibody that contains the human IgG1 Fc
domain and has an expected M.sub.r of 120 kDa.
[0034] The gene encoding the .alpha.IGF-IR scFv was constructed
using the N-terminal signal peptide derived from the mT84.66 light
chain, V.sub.L DNA, an oligonucleotide encoding the linker peptide
(GGGGSGGGS).sub.2(SEQ ID NO:5), V.sub.H DNA, and a C-terminal tag
(including DYKD; [SEQ ID NO: 22]), and assembled using
splice-overlap extension PCR. The resulting DNA encoding
.alpha.IGF-IR scFv is shown in FIG. 2 (SEQ ID NO:2). The
.alpha.IGF-IR scFv construct was cloned into pcDNA3 (Invitrogen,
San Diego, Calif.), containing the cytomegalovirus promoter and
neo.sup.r selection marker (pcDNA/.alpha.IGF-IR scFv).
[0035] To construct the gene encoding .alpha.IGF-IR scFv-Fc, a SalI
fragment containing the human IgG1 Fc (cDNA clone from Dr. J.
Schlom, Laboratory of Tumor Immunology and Biology, division of
Cancer Biology and Diagnosis, NCI, Bethesda, Md.) was inserted into
the unique XhoI site of pcDNA/.alpha.IGF-IR scFv. The HindIII
fragment encoding .alpha.IGF-IR scFv-Fc, isolated from the
pcDNA/.alpha.IGF-IR scFv-Fc plasmid, was inserted into the HindIII
site of pEE12-1. This plasmid encodes a glutamine synthase gene
that provides a selection system for myeloma NS0 cells in
L-glutamine-deficient selection medium.
EXAMPLE 3
[0036] Cell Culture, Transfection and Purification of .alpha.IGF-IR
scFv or .alpha.IGF-IR scFv-Fc.
[0037] Murine myeloma Sp2/0 cells were transfected with
pcDNA/.alpha.IGF-IR scFv by electroporation, and incubated at
37.degree. C. for 3 days in a humidified 5% CO.sub.2 atmosphere. On
day 4, cells were collected, counted and placed in 24-well plates
(10.sup.5 cells/well) in regular medium containing 400 .mu.g/ml
G418. Murine myeloma NS0 cells were grown in selective medium
consisting of L-glutamine-free Celltech DME (JRH Biosciences,
Lenexa, Kans.), dialyzed fetal calf serum (Gibco/BRL, Gaithersburg,
Md.), and glutaminase synthase supplement (JRH Biosciences, Lenexa,
Kans.). Murine myeloma NS0 cells were stably transfected with
pEE12-1/.alpha.IGF-IR scFv-Fc by electroporation and transferred to
non-selective culture medium in a 96-well plate (50 .mu.l/well),
and incubated overnight. The next day 150 .mu.l of selection medium
was added to each well, and the cells were incubated for three
weeks until discrete surviving colonies appeared.
[0038] To purify .alpha.IGF-IR scFv by affinity chromatography, 150
ml of conditioned medium (CM), collected from Sp2/0 cells, were
applied to 6 ml .alpha.FLAG M2 mAb (Eastman Kodak Co., Rochester,
N.Y.) conjugated to Sepharose 4B (0.2 mg/ml gel), and .alpha.IGF-IR
scFv-Fc was eluted from the column with FLAG peptide. Eluates were
concentrated and dialyzed, using an Ultrafree-4 spin column
(Millipore, Bedford, Mass.). Based on the recovery of approximately
4 .mu.g of .alpha.IGF-IR scFv protein from purifying 150 ml CM, the
level of .alpha.IGF-IR scFv expression was estimated to be
approximately 20 ng/ml CM.
[0039] To purify .alpha.IGF-IR scFv-Fc approximately 40 ml cell
culture supernatants collected from .alpha.IGF-IR scFv-Fc
expressing NS0 transfectants were adjusted to pH 8.0 by adding 1/20
volume 1.0 M TRIS (pH 8.0), and passed through a
protein-A-Sepharose CL 4B column. .alpha.IGF-IR scFv-Fc was eluted
from the column with 100 mM glycine buffer pH 3.0, collected in 1.5
ml conical tubes containing 1/10 volume 1M TRIS (pH 8.0). The
estimated expression level of .alpha.IGF-IR scFv-Fc in this
expression system ranged between 45 .mu.g/ml and 85 .mu.g/ml.
EXAMPLE 4
[0040] Inhibition of Agonist Binding to Purified IGF-IR by 1H7 and
.alpha.IGF-IR scFv-Fc.
[0041] The affinity constants of 1H7 (10.sup.9 M.sup.-1) and
.alpha.IGF-IR scFv-Fc (10.sup.8 M.sup.-1) for IGF-IR were
determined using a BIAcore instrument (BIAcore Inc., Piscataway,
N.J.). Analytes, at various concentrations, were passed over
IGF-IR-immobilized chips (0.3 .mu.g/chip) at a flow rate of 5
.mu.l/min.
[0042] The in vitro potency of inhibition of purified IGF-IR by
.alpha.IGF-IR scFv-Fc for both IGF-I and IGF-II binding is seen in
FIG. 4.
EXAMPLE 5
[0043] Effect of .alpha.IGF-IR scFv-Fc on Cell Growth.
[0044] Using the MTT method the effect of extracellular addition of
.alpha.IGF-IR scFv-Fc or 1H7 on cell growth was determined on
NIH3T3 cells over expressing IGF-IR. Cell growth was significantly
inhibited after four days of treatment with 10 nM or 100 nM 1H7
mAb, see FIG. 5. Also, after 4 days of treatment with .alpha.IGF-IR
scFv-Fc cell growth appeared to be inhibited in a dose dependent
manner as is shown in FIG. 5.
EXAMPLE 6
[0045] Effect of .alpha.IGF-IR scFv-Fc on Tumor Growth in vivo.
[0046] The human breast MCF-7 cell line was obtained from American
Type Culture Collection (Rockville, Md.). MCF-7 cells were cultured
in Dulbecco's modified Eagle's medium supplemented with 5% fetal
calf serum. Female athymic mice (BALB/C nude, Charles River
Facility for NCI, Frederick, Md.), 4 weeks old, that had received
0.25 mg 17.beta.-estradiol pellet one week previously were
inoculated in the flank with 10.sup.7 MCF-7 cells (day 0). On day
3, intraperitoneal or subcutaneous injections near the tumor sites
of .alpha.IGF-IR scFv-Fc into each of three mice (500 .mu.g/0.1 ml
phosphate buffered saline, PBS/mouse, twice a week) was started,
and continued for two weeks.
[0047] The recombinant single chain antibody .alpha.IGF-IR scFv-Fc
inhibits MCF-7 tumor cell growth in athymic mice. As shown in FIG.
6, inhibition of tumor cell growth is significant, although, the
results for individual mice varied. In several mice MCF-7 tumor
cell growth was completely suppressed from day 3 to day 17, and in
one mouse the tumor disappeared.
EXAMPLE 7
[0048] Effect of .alpha.IGF-IR scFv-Fc in combination with
anti-neoplastic Agent Doxorubicin (DOX) on Tumor Growth invivo.
[0049] In combination with Doxorubicin (DOX), .alpha.IGF-IR scFv-Fc
inhibits tumor cell growth, as shown in FIG. 7. Female athymic mice
were treated similarly as described above in example 6, with the
exception that the pellet with which the mice were inoculated
contained 0.72 mg 17.beta.-estradiol. The treatment of the mice was
started on day 4 by either intraponeal injections of .alpha.IGF-IR
scFv-Fc as in example 6 three times per week, by intraponeal
injections of DOX (2 mg/kg bodyweight) once a week, or by a
combination of both.
EXAMPLE 8
[0050] Effect of .alpha.IGF-IR scFv-Fc in Combination with
Anti-estrogen Agent Tamoxifen (TAM) on Tumor Growth in vivo.
[0051] In combination with the anti-estrogen drug Tamoxifen (TAM),
.alpha.IGF-IR scFv-Fc inhibits T61 tumor cell growth in vivo as is
shown in FIG. 8. The observed inhibition of a combination treatment
in these T61 tumor cells shows a synergistic effect. Female athymic
mice were inoculated with T61 tumor cells. The treatment of the
mice was started on day 36 by either intraponeal injections of
.alpha.IGF-IR scFv-Fc as in example 6 three times per week, by
subcutaneous implantation of 5 mg of a TAM pellet, or a combination
of both.
Sequence CWU 1
1
221245PRTMus musculus 1Asp Ile Val Met Thr Gln Ser His Lys Phe Met
Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Asn Ile Thr Cys Lys Ala Ser
Gln Asp Val Asn Thr Ala20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ser Pro Lys Leu Leu Ile35 40 45Tyr Trp Ala Ser Thr Arg His Thr
Gly Val Pro Asp Arg Phe Thr Gly50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Leu Thr Leu
Tyr Tyr Cys His Gln His Tyr Thr Thr Pro Tyr85 90 95Thr Phe Gly Gly
Gly Thr Asn Leu Glu Ile Lys Gly Gly Gly Gly Ser100 105 110Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Lys115 120
125Val Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu
Lys130 135 140Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
Ala Met Ser145 150 155 160Trp Val Arg Gln Thr Pro Glu Lys Lys Leu
Glu Trp Val Ala Ser Ile165 170 175Ser Asn Gly Gly Thr Thr Tyr Tyr
Pro Asp Ser Val Lys Gly Arg Phe180 185 190Thr Ile Ser Arg Asp Asn
Ala Arg Asn Ile Leu Tyr Leu Gln Met Asn195 200 205Ser Leu Arg Ser
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Thr Phe210 215 220Tyr Tyr
Ser Phe Pro Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser225 230 235
240Val Thr Val Ser Ser2452735DNAMus musculus 2gacattgtga tgacccagtc
tcacaaattc atgtccacat cggtaggaga cagggtcaac 60atcacctgca aggccagtca
ggatgtgaat actgctgtgg cctggtatca acaaaaacca 120gggcaatctc
ctaaactcct gatttactgg gcatccaccc ggcacactgg agtccctgat
180cgcttcacag gcagtggatc tgggacagat tttactctca ccatcagcag
tgtgcaggct 240gaagacctga cactttatta ctgtcatcaa cattatacca
ctccgtacac gttcggaggg 300gggaccaatc tggaaataaa aggcggaggc
ggtagcggcg gtggttcagg aggtggcggc 360agtggtggag gatctgaagt
aaaagtggtg gaatctgggg gaggcttagt gaagcctgga 420gggtccctga
aactctcctg tgcagcctct ggattcactt tcagtatcta tgccatgtca
480tgggttcgcc agactccaga gaagaaactg gagtgggtcg catccattag
taatggtggt 540accacctact atccagacag tgtgaagggc cgattcacca
tctccagaga taatgccagg 600aacatcctgt acctgcaaat gaacagtctg
aggtctgagg acacggccat gtattactgt 660gcaaggtacc ttctactata
gttttccccg agctaggact actggggtca aggaacctcg 720gtcaccgtct cctca
7353107PRTMus musculus 3Asp Ile Val Met Thr Gln Ser His Lys Phe Met
Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Asn Ile Thr Cys Lys Ala Ser
Gln Asp Val Asn Thr Ala20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ser Pro Lys Leu Leu Ile35 40 45Tyr Trp Ala Ser Thr Arg His Thr
Gly Val Pro Asp Arg Phe Thr Gly50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Leu Thr Leu
Tyr Tyr Cys His Gln His Tyr Thr Thr Pro Tyr85 90 95Thr Phe Gly Gly
Gly Thr Asn Leu Glu Ile Lys100 1054120PRTMus musculus 4Glu Val Lys
Val Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr20 25 30Ala
Met Ser Trp Val Arg Gln Thr Pro Glu Lys Lys Leu Glu Trp Val35 40
45Ala Ser Ile Ser Asn Gly Gly Thr Thr Tyr Tyr Pro Asp Ser Val Lys50
55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr
Tyr Cys Ala85 90 95Arg Thr Phe Tyr Tyr Ser Phe Pro Arg Ala Met Asp
Tyr Trp Gly Gln100 105 110Gly Thr Ser Val Thr Val Ser Ser115
120518PRTMus musculus 5Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly1 5 10 15Gly Ser69PRTMus musculus 6Lys Ala Ser
Gln Asp Val Asn Thr Ala1 578PRTMus musculus 7Trp Ala Ser Thr Arg
Met Met Thr1 589PRTMus musculus 8His Gln His Tyr Thr Thr Pro Tyr
Thr1 595PRTMus musculus 9Ile Tyr Ala Met Ser1 51016PRTMus musculus
10Ser Ile Ser Asn Gly Gly Thr Thr Tyr Tyr Pro Asp Ser Val Lys Gly1
5 10 151112PRTMus musculus 11Thr Phe Tyr Tyr Ser Phe Pro Arg Ala
Met Asp Tyr1 5 101215PRTMus musculus 12Glu Val Lys Val Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly1 5 10 151315PRTMus musculus 13Asp
Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val1 5 10
15146PRTMus musculus 14Asp Ile Val Met Thr Gln1 51526DNAMus
musculus 15gggaattcga yatygtsatg achcar 26166PRTMus musculus 16Ser
Ile Phe Pro Pro Ser1 51726DNAMus musculus 17tccatcttcc caccatccga
attccg 26186PRTMus musculus 18Glu Val Lys Val Val Glu1 51926DNAMus
musculus 19gggaattcga rgtvaargtv gtvgar 26206PRTMus musculus 20Val
Tyr Pro Leu Ala Pro1 52126DNAMus musculus 21gtctatccac tggcccctga
attccg 26224PRTMus musculus 22Asp Tyr Lys Asp1
* * * * *