U.S. patent application number 14/266391 was filed with the patent office on 2014-08-21 for j591 minibodies and cys-diabodies for targeting human prostate specific membrane antigen (psma) and methods for their use.
This patent application is currently assigned to ImaginAb, Inc.. The applicant listed for this patent is ImaginAb, Inc.. Invention is credited to David T. Ho, Arye Lipman, Tove Olafsen.
Application Number | 20140234215 14/266391 |
Document ID | / |
Family ID | 44115507 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234215 |
Kind Code |
A1 |
Ho; David T. ; et
al. |
August 21, 2014 |
J591 MINIBODIES AND CYS-DIABODIES FOR TARGETING HUMAN PROSTATE
SPECIFIC MEMBRANE ANTIGEN (PSMA) AND METHODS FOR THEIR USE
Abstract
In one embodiment, a minibody monomer that binds PSMA is
provided. The minibody monomer is encoded by a nucleotide sequence
comprising, from N-terminus to C-terminus, an scFv sequence that
can bind PSMA, an artificial hinge sequence, and a human IgG CH3
sequence. In another embodiment, a CysDB monomer that binds PSMA is
provided. The CysDB monomer may be encoded by a nucleotide sequence
comprising, from N-terminus to C-terminus, an scFv sequence that
can bind PSMA and a cysteine tail. In other embodiments, methods
for diagnosing or treating a cancer associated with PSMA expression
in a subject are provided.
Inventors: |
Ho; David T.; (Long Beach,
CA) ; Olafsen; Tove; (Reseda, CA) ; Lipman;
Arye; (El Segundo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ImaginAb, Inc. |
Inglewood |
CA |
US |
|
|
Assignee: |
ImaginAb, Inc.
Inglewood
CA
|
Family ID: |
44115507 |
Appl. No.: |
14/266391 |
Filed: |
April 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12959340 |
Dec 2, 2010 |
8772459 |
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14266391 |
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61266134 |
Dec 2, 2009 |
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Current U.S.
Class: |
424/1.49 ;
424/135.1; 424/178.1; 530/387.3 |
Current CPC
Class: |
C07K 2317/622 20130101;
C07K 2317/24 20130101; C07K 2317/526 20130101; C07K 16/3069
20130101; A61K 51/1072 20130101; A61P 35/00 20180101; C07K 2317/77
20130101; C07K 2317/90 20130101; C07K 2317/64 20130101; C07K
2317/56 20130101 |
Class at
Publication: |
424/1.49 ;
530/387.3; 424/135.1; 424/178.1 |
International
Class: |
C07K 16/30 20060101
C07K016/30 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
Contract No. HHSN261200900051C, awarded by the National Cancer
Institute (NCI). The government has certain rights in the
invention.
Claims
1.-14. (canceled)
15. A method for diagnosing a cancer associated with PSMA
expression in a subject, the method comprising: administering an
anti-PSMA minibody or an anti-PSMA cys-diabody that is conjugated
to a diagnostic agent and can bind PSMA to a subject having or
suspected of having a cancer associated with PSMA expression;
exposing the subject to an imaging method to visualize the labeled
minibody or cys-diabody in vivo; and determining that the subject
has a cancer associated with PSMA expression when the labeled
minibody or cys-diabody localizes to a tumor site.
16. The method of claim 15, wherein the minibody comprises SEQ ID
NO:10 or SEQ ID NO:11.
17. The method of claim 15, wherein cys-diabody, comprises SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.
18. The method of claim 15, wherein the anti-PSMA minibody or
anti-PSMA cys-diabody targets neovasculature of a solid tumor.
19. The method of claim 15, wherein the cancer associated with PSMA
expression in a subject is prostate cancer, lung cancer, colorectal
cancer, breast cancer, renal cancer, liver cancer, bladder cancer,
pancreatic cancer or melanoma.
20. A method for treating a cancer associated with PSMA expression
in a subject, the method comprising: administering a
therapeutically effective amount of a pharmaceutical composition to
the subject, the composition comprising an anti-PSMA minibody or an
anti-PSMA cys-diabody.
21. The method of claim 20, wherein the minibody comprises SEQ ID
NO:10 or SEQ ID NO:11.
22. The method of claim 20, wherein the cys-diabody, comprises SEQ
ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.
23. The method of claim 20, wherein the anti-PSMA minibody or
anti-PSMA cys-diabody is conjugated to a therapeutic agent.
24. The method of claim 23, wherein the therapeutic agent is
selected from the group consisting of chemotherapeutic agents,
therapeutic antibodies and antibody fragments, toxins,
radioisotopes, enzymes, nucleases, hormones, immunomodulators,
antisense oligonucleotides, chelators, boron compounds, photoactive
agents and dyes.
25. The method of claim 20, wherein the anti-PSMA minibody or
anti-PSMA cys-diabody targets neovasculature of a solid tumor.
26. The method of claim 19, wherein the cancer associated with PSMA
expression in a subject is prostate cancer, lung cancer, colorectal
cancer, breast cancer, renal cancer, liver cancer, bladder cancer,
pancreatic cancer or melanoma.
27. The method of claim 15, wherein: (a) the minibody comprises: a
heavy chain CDR1 that is a CDR1 in SEQ ID NO: 10; a heavy chain
CDR2 that is a CDR2 in SEQ ID NO: 10; a heavy chain CDR3 that is a
CDR3 in SEQ ID NO: 10; a light chain CDR1 that is a CDR1 in SEQ ID
NO: 10; a light chain CDR2 that is a CDR2 in SEQ ID NO: 10; and a
light chain CDR3 that is a CDR3 in SEQ ID NO: 10; (b) the minibody
comprises: a heavy chain CDR1 that is a CDR1 in SEQ ID NO: 11; a
heavy chain CDR2 that is a CDR2 in SEQ ID NO: 11; a heavy chain
CDR3 that is a CDR3 in SEQ ID NO: 11; a light chain CDR1 that is a
CDR1 in SEQ ID NO: 11; a light chain CDR2 that is a CDR2 in SEQ ID
NO: 11; and a light chain CDR3 that is a CDR3 in SEQ ID NO: 11; (c)
the cys-diabody comprises: a heavy chain CDR1 that is a CDR1 in SEQ
ID NO: 12; a heavy chain CDR2 that is a CDR2 in SEQ ID NO: 12; a
heavy chain CDR3 that is a CDR3 in SEQ ID NO: 12; a light chain
CDR1 that is a CDR1 in SEQ ID NO: 12; a light chain CDR2 that is a
CDR2 in SEQ ID NO: 12; and a light chain CDR3 that is a CDR3 in SEQ
ID NO: 12; (d) the cys-diabody comprises: a heavy chain CDR1 that
is a CDR1 in SEQ ID NO: 13; a heavy chain CDR2 that is a CDR2 in
SEQ ID NO: 13; a heavy chain CDR3 that is a CDR3 in SEQ ID NO: 13;
a light chain CDR1 that is a CDR1 in SEQ ID NO: 13; a light chain
CDR2 that is a CDR2 in SEQ ID NO: 13; and a light chain CDR3 that
is a CDR3 in SEQ ID NO: 13; (e) the cys-diabody comprises: a heavy
chain CDR1 that is a CDR1 in SEQ ID NO: 14; a heavy chain CDR2 that
is a CDR2 in SEQ ID NO: 14; a heavy chain CDR3 that is a CDR3 in
SEQ ID NO: 14; a light chain CDR1 that is a CDR1 in SEQ ID NO: 14;
a light chain CDR2 that is a CDR2 in SEQ ID NO: 14; and a light
chain CDR3 that is a CDR3 in SEQ ID NO: 14; or (f) the cys-diabody
comprises: a heavy chain CDR1 that is a CDR1 in SEQ ID NO: 15; a
heavy chain CDR2 that is a CDR2 in SEQ ID NO: 15; a heavy chain
CDR3 that is a CDR3 in SEQ ID NO: 15; a light chain CDR1 that is a
CDR1 in SEQ ID NO: 15; a light chain CDR2 that is a CDR2 in SEQ ID
NO: 15; and a light chain CDR3 that is a CDR3 in SEQ ID NO: 15.
28. The method of claim 20, wherein: (a) the minibody comprises: a
heavy chain CDR1 that is a CDR1 in SEQ ID NO: 10; a heavy chain
CDR2 that is a CDR2 in SEQ ID NO: 10; a heavy chain CDR3 that is a
CDR3 in SEQ ID NO: 10; a light chain CDR1 that is a CDR1 in SEQ ID
NO: 10; a light chain CDR2 that is a CDR2 in SEQ ID NO: 10; and a
light chain CDR3 that is a CDR3 in SEQ ID NO: 10; (b) the minibody
comprises: a heavy chain CDR1 that is a CDR1 in SEQ ID NO: 11; a
heavy chain CDR2 that is a CDR2 in SEQ ID NO: 11; a heavy chain
CDR3 that is a CDR3 in SEQ ID NO: 11; a light chain CDR1 that is a
CDR1 in SEQ ID NO: 11; a light chain CDR2 that is a CDR2 in SEQ ID
NO: 11; and a light chain CDR3 that is a CDR3 in SEQ ID NO: 11; (c)
the cys-diabody comprises: a heavy chain CDR1 that is a CDR1 in SEQ
ID NO: 12; a heavy chain CDR2 that is a CDR2 in SEQ ID NO: 12; a
heavy chain CDR3 that is a CDR3 in SEQ ID NO: 12; a light chain
CDR1 that is a CDR1 in SEQ ID NO: 12; a light chain CDR2 that is a
CDR2 in SEQ ID NO: 12; and a light chain CDR3 that is a CDR3 in SEQ
ID NO: 12; (d) the cys-diabody comprises: a heavy chain CDR1 that
is a CDR1 in SEQ ID NO: 13; a heavy chain CDR2 that is a CDR2 in
SEQ ID NO: 13; a heavy chain CDR3 that is a CDR3 in SEQ ID NO: 13;
a light chain CDR1 that is a CDR1 in SEQ ID NO: 13; a light chain
CDR2 that is a CDR2 in SEQ ID NO: 13; and a light chain CDR3 that
is a CDR3 in SEQ ID NO: 13; (e) the cys-diabody comprises: a heavy
chain CDR1 that is a CDR1 in SEQ ID NO: 14; a heavy chain CDR2 that
is a CDR2 in SEQ ID NO: 14; a heavy chain CDR3 that is a CDR3 in
SEQ ID NO: 14; a light chain CDR1 that is a CDR1 in SEQ ID NO: 14;
a light chain CDR2 that is a CDR2 in SEQ ID NO: 14; and a light
chain CDR3 that is a CDR3 in SEQ ID NO: 14; or (f) the cys-diabody
comprises: a heavy chain CDR1 that is a CDR1 in SEQ ID NO: 15; a
heavy chain CDR2 that is a CDR2 in SEQ ID NO: 15; a heavy chain
CDR3 that is a CDR3 in SEQ ID NO: 15; a light chain CDR1 that is a
CDR1 in SEQ ID NO: 15; a light chain CDR2 that is a CDR2 in SEQ ID
NO: 15; and a light chain CDR3 that is a CDR3 in SEQ ID NO: 15.
29. The method of claim 15, wherein the minibody comprises: the
heavy chain CDR1 comprises amino acid residues 51 to 55 of SEQ ID
NO: 10, the heavy chain CDR2 comprises amino acid residues 70 to 86
of SEQ ID NO: 10, and the heavy chain CDR3 comprises amino acid
residues 119 to 124 of SEQ ID NO: 10.
30. The method of claim 15, wherein the minibody comprises: the
light chain CDR1 comprises amino acid residues 177 to 187 of SEQ ID
NO: 10, the light chain CDR2 comprises amino acid residues 203 to
209 of SEQ ID NO: 10, and the light chain CDR3 comprises amino acid
residues 242 to 250 of SEQ ID NO: 10.
31. The method of claim 15, wherein the minibody comprises: the
heavy chain CDR1 comprises amino acid residues 51 to 55 of SEQ ID
NO: 10), the heavy chain CDR2 comprises amino acid residues 70 to
86 of SEQ ID NO: 10, the heavy chain CDR3 comprises amino acid
residues 119 to 124 of SEQ ID NO: 10, the light chain CDR1
comprises amino acid residues 177 to 187 of SEQ ID NO: 10, the
light chain CDR2 comprises amino acid residues 203 to 209 of SEQ ID
NO: 10, and the light chain CDR3 comprises amino acid residues 242
to 250 of SEQ ID NO: 10.
32. The method of claim 15, wherein the minibody comprises: the
heavy chain CDR1 comprises amino acid residues 46 to 52 of SEQ ID
NO: 10, the heavy chain CDR2 comprises amino acid residues 72 to 77
of SEQ ID NO: 10, and the heavy chain CDR3 comprises amino acid
residues 119 to 224 of SEQ ID NO: 10.
33. The method of claim 15, wherein the minibody comprises: the
light chain CDR1 comprises amino acid residues 177 to 187 of SEQ ID
NO: 10, the light chain CDR2 comprises amino acid residues 203 to
209 of SEQ ID NO: 10, and the light chain CDR3 comprises amino acid
residues 242 to 250 of SEQ ID NO: 10.
34. The method of claim 15, wherein the minibody comprises: the
heavy chain CDR1 comprises amino acid residues 46 to 52 of SEQ ID
NO: 10, the heavy chain CDR2 comprises amino acid residues 72 to 77
of SEQ ID NO: 10, the heavy chain CDR3 comprises amino acid
residues 119 to 224 of SEQ ID NO: 10, the light chain CDR1
comprises amino acid residues 177 to 187 of SEQ ID NO: 10, the
light chain CDR2 comprises amino acid residues 203 to 209 of SEQ ID
NO: 10, and the light chain CDR3 comprises amino acid residues 242
to 250 of SEQ ID NO: 10.
Description
PRIORITY CLAIM
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/266,134, filed Dec. 2, 2009, the subject matter
of which is hereby incorporated by reference as if fully set forth
herein.
BACKGROUND
[0003] Advances in antibody engineering have enabled the
development of various antibody fragments featuring different
pharmacokinetic and binding properties (Wu et al 2005, Wu et al
2008, Wu et al 2009). A minibody is an antibody format which
features a smaller molecular weight (.about.80 kD) than the
full-length antibody while maintaining the bivalent binding
property against an antigen (Hu et al 1996). Because of its smaller
size, the minibody features faster clearance from the system and
enhanced penetration when targeting tumor tissue. With the ability
for strong targeting combined with rapid clearance, the minibody is
an optimized antibody format that may be used for diagnostic
imaging (Wu et al 2005). Since the discovery of the first minibody
against the tumor-associated target CEA, many minibodies have been
developed against different cancer targets for preclinical
diagnostic imaging including human epidermal growth factor
receptor-2 (HER2) in breast cancer, B-lymphocyte antigen CD20 in
non-Hodgkins' lymphoma, and prostate stem cell antigen (PSCA) in
prostate cancer (Hu et al 1996, Leyton et al 2008, Olafsen et al
2004, Olafsen et al 2009). For example, an .sup.123I-labeled CEA
minibody has been evaluated in the clinic for imaging patients with
colorectal cancer by SPECT and similar studies have been performed
with an .sup.111In-DOTA labeled minibody (Wong et al 2004). The
development of novel imaging agents is particularly critical for
the diagnosis, management, and treatment of specific cancers which
are poorly imaged with current technology such as prostate
cancer.
[0004] The development of imaging agents for all types of cancer is
needed to enable the targeting, staging, and monitoring of the
disease. Current methods for diagnostic imaging of prostate cancer
remain relatively inaccurate. With an estimated 234,460 new cases
and 27,350 deaths in 2006, an imaging agent capable of accurately
diagnosing, staging, and monitoring prostate cancer is needed
(Olson et al 2007).
[0005] Prostate Specific Membrane Antigen (PSMA), a cell-surface
biomarker that is associated with prostate cancer (Slovin 2005), is
a single-pass Type II transmembrane protein possessing glutamate
carboxypeptidase activity, although the functional role of PSMA is
not well understood (Olson et al 2007). Expression of PSMA is
relatively limited in normal tissues outside of the prostate
including the brain, small intestines, liver, proximal kidney
tubules, and salivary gland (Olson et al 2007).
[0006] PSMA expression in prostate cancer increases with tumor
aggressiveness and is the highest in high-grade tumors, metastatic
lesions, and androgen-independent disease (Olson et al 2007).
Therefore, PSMA is a cancer biomarker that is a good candidate for
targeting by an imaging agent. PSMA expression is also upregulated
in the neovasculature of many non-prostatic solid tumors including
lung, colon, breast, renal, liver and pancreatic carcinomas as well
as sarcomas and melanoma (Olson et al 2007).
[0007] Full-length antibodies that target PSMA have been developed,
some of which are in various stages of preclinical and clinical
development (Olson et al 2007). PSMA was originally defined by a
murine antibody (mAb), 7E11, which recognized an intracellular
epitope of PSMA (Olson et al 2007). The 7E11 mAb was later
developed into a FDA-approved SPECT imaging agent called
Prostascint for the detection and imaging of prostate cancer in
soft tissue (Olson et al 2007). However, since 7E11 recognizes an
intracellular epitope, Prostascint is a relatively poor imaging
agent which is limited to detecting necrotic tumor tissue (Olson et
al 2007). Having the pharmacokinetic properties of a full-length
antibody, Prostascint also requires a long period of time between
injection and imaging (Olson et al 2007). Furthermore, Prostascint
is a murine antibody which elicits strong immune responses that
prevent multiple dosing (Olson et al 2007).
[0008] Another full-length antibody that targets PSMA, J591, was
discovered and subsequently deimmunized, the deimmunized version
known as huJ591 (Liu et al 1997, Bander et al 2003). The
deimmunized huJ591 is an anti-human PSMA antibody that recognizes
and binds an extracellular epitope on PSMA (Bander et al 2003). The
huJ591 antibody is being developed as a potential
radioimmunotherapy agent against prostate cancer. In Phase I
trials, DOTA-conjugated huJ591 antibody labeled with gamma emitting
isotopes Indium 111 and Lutetium 177 demonstrated excellent
targeting to metastatic sites, no immunogenicity, and multiple
doses were well tolerated (Bander et al 2003, Milowsky et al 2004,
Bander et al 2005, Olson et al 2007). Beyond prostate cancer, Phase
I studies with .sup.111In-DOTA huJ591 demonstrated specific
targeting of tumor neovasculature of advanced solid tumors
(Milowsky et al 2007).
SUMMARY
[0009] In one embodiment, a minibody that binds PSMA is provided.
According to this embodiment, the minibody is encoded by a
nucleotide sequence comprising, from N-terminus to C-terminus, an
scFv sequence that can bind prostate specific membrane antigen
(PSMA), an artificial hinge sequence, and a human IgG1 CH3
sequence. The minibody monomer may also include an N-terminus
signal sequence to enable secretion of the minibody when expressed
in a cell.
[0010] The minibody scFv as described herein comprises a variable
heavy domain (VH) linked to a variable light domain (VL) by a
linker sequence. In one aspect, the scFv is in a VHVL orientation
such that the VH is upstream of the VL. A minibody monomer having
such an scFv may have a nucleotide sequence comprising SEQ ID NO:1
or SEQ ID NO:2. In another aspect, the scFv is in a VLVH
orientation such that the VL is upstream of the VH.
[0011] The minibody monomer may be expressed by a cell. In such
embodiments, a CysDB monomer expressed by a cell may include the
amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11.
[0012] In another embodiment, a cys-diabody (CysDB) that binds PSMA
is provided. According to this embodiment, the CysDB monomer is
encoded by a nucleotide sequence comprising, from N-terminus to
C-terminus, an scFv sequence that can bind PSMA and a cysteine
tail. The CysDB may also include an N-terminus signal sequence to
enable secretion of the minibody when expressed in a cell.
[0013] The CysDB scFv as described herein comprises a variable
heavy domain (VH) linked to a variable light domain (VL) by a
linker sequence. In one aspect, the scFv is in a VHVL orientation
such that the VH is upstream of the VL. A CysDB monomer having such
an scFv may have a nucleotide sequence comprising SEQ ID NO:6 or
SEQ ID NO:7. In another aspect, the scFv is in a VLVH orientation
such that the VL is upstream of the VH. A CysDB monomer having such
an scFv may have a nucleotide sequence comprising SEQ ID NO:8 or
SEQ ID NO:9.
[0014] The CysDB may be expressed by a cell. In some embodiments, a
CysDB expressed by a cell may include the amino acid sequence SEQ
ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.
[0015] In another embodiment, a method for diagnosing a cancer
associated with PSMA expression in a subject is provided. Such a
method includes administering an anti-PSMA minibody or a
cys-diabody conjugated to a diagnostic agent to a subject having or
suspected of having a cancer associated with PSMA expression;
exposing the subject to an imaging method to visualize the labeled
minibody or cys-diabody in vivo; and determining that the subject
has a cancer associated with PSMA expression when the labeled
minibody or cys-diabody localizes to a tumor site.
[0016] In another embodiment, a method for treating a cancer
associated with PSMA expression in a subject is provided. Such a
method includes administering a therapeutically effective amount of
a pharmaceutical composition to the subject, the composition
comprising an anti-PSMA minibody or an anti-PSMA cys-diabody. In
one aspect, the anti-PSMA minibody or anti-PSMA cys-diabody is
conjugated to a therapeutic agent.
[0017] The cancer associated with PSMA expression in a subject may
be lung cancer, colorectal cancer, breast cancer, renal cancer,
liver cancer, bladder cancer, pancreatic cancer or melanoma.
[0018] A minibody that may be used in the methods as described
above may be any suitable minibody as described herein, or may
comprise SEQ ID NO:10 or SEQ ID NO:11. A cys-diabody that may be
used in methods as described above may be any suitable minibody as
described herein, or may comprise SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14 or SEQ ID NO:15.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A is a schematic diagram of the J591 Minibody. This
diagram depicts the minibody in the VHVL orientation binding the
target PSMA.
[0020] FIG. 1B is a schematic diagram of an expression construct
for the J591 minibody in VHVL orientation. SP=signal peptide,
VH--variable heavy domain, VL--variable light domain, L--18 amino
acid linker, H./E--artificial hinge/extension, CH3 from human
IgG1.
[0021] FIG. 2 is a comparison between the amino acid sequences of
deimmunized (Line 3; SEQ ID NO:5; SEQ ID NO:19), Murine (Line 2;
SEQ ID NO:4; SEQ ID NO:18), and Human Composite (Line 1; SEQ ID
NO:3; SEQ ID NO:17) J591 V-regions. Highlighted residues along the
HC line (Line 1) designate differences between the HC and murine
V-regions. Highlighted residues along the deimmunized line (Line 3)
designate differences between the deimmunized and the murine
V-regions as a result of the original deimmunization process. The
two stars designate the two Prolines introduced by the
deimmunization.
[0022] FIG. 3 is the J591 Human Composite VHVL Minibody nucleotide
sequence (SEQ ID NO:1) and corresponding translated amino acid
sequence (SEQ ID NO:10).
[0023] FIG. 4 is the J591 2P VHVL Minibody nucleotide sequence (SEQ
ID NO:2) and corresponding translated amino acid sequence (SEQ ID
NO:11).
[0024] FIG. 5 is a schematic diagram of the cys-diabody (CysDB)
(A), a schematic diagram of an expression construct for a CysDB in
VLVH orientation (B), and a schematic diagram of an expression
construct for a CysDB in VHVL orientation (C). SS=signal sequence,
VH=variable heavy domain, VL=variable light domain, L linker (may
be 5 or 8 amino acids), GGS=cysteine tail (Gly-Gly-Cys).
[0025] FIG. 6 is the J591 cys-diabody (CysDB) VH-5-VL nucleotide
sequence (SEQ ID NO:6) and corresponding translated amino acid
sequence (SEQ ID NO:12).
[0026] FIG. 7 is the J591 cys-diabody (CysDB) VH-8-VL nucleotide
sequence (SEQ ID NO:7) and corresponding translated amino acid
sequence (SEQ ID NO:13).
[0027] FIG. 8 is the J591 cys-diabody (CysDB) VL-5-VH nucleotide
sequence (SEQ ID NO:8) and corresponding translated amino acid
sequence (SEQ ID NO:14).
[0028] FIG. 9 is the J591 cys-diabody (CysDB) VL-8-VH nucleotide
sequence (SEQ ID NO:9) and corresponding translated amino acid
sequence (SEQ ID NO:15).
[0029] FIG. 10 is a Vector Map for pcDNA 3.1/myc-His (-) Versions
A, B, C. This expression vector from Invitrogen Corp. features the
CMV promoter for mammalian expression and Neomycin resistance for
selection.
[0030] FIG. 11 is a representative Western blot analysis confirming
the expression of the J591 minibodies by CHO-K1 cells. Lane 1
corresponds to a Molecular weight marker sample, Lane 2 corresponds
to an Empty Vector sample, Lane 3 corresponds to a positive control
minibody sample, Lane 4 corresponds to the J591 HC VLVH sample,
Lane 5 corresponds to the J591 HC VHVL sample, Lane 6 corresponds
to the J591 2P VLVH sample, and Lane 7 corresponds to the J591 2P
VHVL sample.
[0031] FIG. 12A-D are graphs that represent flow cytometry analysis
of the J591 minibodies. Histograms plot cell count versus PE signal
(FL2-H). FIG. 12A shows a graph representing the flow cytometry
analysis for the J591 HC VLVH minibody, FIG. 12B shows a graph
representing the flow cytometry analysis for the J591 HC VHVL
minibody, FIG. 12C shows a graph representing the flow cytometry
analysis for the J591 2P VLVH minibody, and FIG. 12D shows a graph
representing the flow cytometry analysis for the J591 2P VHVL
minibody.
[0032] FIG. 13 is an SDS-PAGE analysis of the purified J591
minibody. The purified J591 minibody protein was loaded onto the
SDS-PAGE gel under non-reducing conditions (lane 1) and reducing
conditions (lane 2). The gel was stained with GelCode Blue (Pierce,
Thermo Scientific). The minibody was diluted 1/5 for loading on the
gel.
[0033] FIG. 14 is a size exclusion chromatography (SEC) analysis of
purified J591 minibody. The graph plots the 220 nm UV absorbance
(mAU) vs. time (min). 4 .mu.g of the J591 minibody was loaded onto
a TSK-GEL Super SW3000 column. A protein molecular weight standard
was also run separately on the column to provide reference. The
percentage of the aggregate versus the minibody protein (labeled
here as monomer) was determined by calculating the area under the
curve.
[0034] FIG. 15 illustrates that the J591 minibody protein binds
PSMA by ELISA. 96-well ELISA plates were coated with purified
recombinant PSMA protein at 1 .mu.g/ml. Purified J591 minibody
protein (1, ) was introduced at a starting concentration of 2
.mu.g/ml and serially diluted ten times by third dilutions.
Identical dilutions were performed for the negative control
minibody (2, .box-solid.). Samples were performed in triplicate at
each dilution, and the error bars represent standard deviation.
Following the primary incubation, bound minibodies were detected
using a goat anti-human IgG (Fc specific) antibody conjugated to
alkaline phosphatase and developed with a pNPP solution. Absorbance
was detected at 405 nm.
[0035] FIGS. 16A-D are graphs that represent flow cytometry
analysis, illustrating that the J591 minibody binds PSMA+ cell
lines. All histograms plot cell count vs. PE signal (FL2-H). The
J591 minibody protein and the negative control minibody (1), both
at 20 .mu.g/ml, were tested for binding to the PSMA+ cell line
LNCaP (A and B) and CWR22rv1 (C and D). Cells were subsequently
stained with a secondary anti-human IgG (Fc specific)-PE conjugated
antibody. 1.times.10.sup.5 cells/point and analysis was performed
with 5,000 events/point. (A) J591 minibody (2) binding LNCaP cells
(B) J591-DOTA minibody (2) binding LNCaP cells (C) J591 minibody
(2) binding CWR cells (D) J591-DOTA minibody (2) binding CWR
cells.
[0036] FIG. 17 are representative images that show the
internalization of J591 minibody in LNCaP cells. LNCaP cells were
plated on poly-d-lysine-coated coverslips in 12-well plates.
Following 2 days of growth, the cells were pre-chilled for 30
minutes at 4 C before incubation with the primary antibody or
minibody for 30 minutes at 4 C. At the indicated time points after
primary incubation, the cells were fixed, permeabilized, and
stained with secondary anti-human IgG-Alexa 488. The coverslips
were simultaneously mounted on to slides and counterstained with
DAPI within the mounting media. Slides were viewed using a
63.times. oil-immersion lens on a Leica SP2-1P-FCS confocal
microscope.
[0037] FIG. 18 are representative images that show the
internalization of J591 minibody in CWR22rv1 cells. CWR22rv1 cells
were plated on poly-d-lysine-coated coverslips in 12-well plates.
Following 2 days of growth, the cells were pre-chilled for 30
minutes at 4 C before incubation with the primary antibody or
minibody for 30 minutes at 4 C. At the indicated time points
post-primary incubation, the cells were fixed, permeabilized, and
stained with secondary anti-human IgG-Alexa 488. The coverslips
were simultaneously mounted on to slides and counterstained with
DAPI within the mounting media. Slides were viewed using a
63.times. oil-immersion lens on a Leica SP2-1P-FCS confocal
microscope.
[0038] FIG. 19 is a graph illustrating uptake and retention of
cell-associated radioactivity of .sup.131I-labelled and
.sup.111In-DOTA labelled J591 minibody. The uptake and retention of
cell-associated radioactivity over time upon binding to CWR22rv1
cells. The radioactivity from the cell membrane, cell lysate
(internalized), and total (membrane+internalized) fractions are
expressed as counts per minute (cpm). CWR22rv1 cells were seeded
into 24-well plates at 5.times.10.sup.5 cells/well the day before
the experiment. Cells were pre-chilled at 4 C before incubation
with an excess of (A) .sup.131I-labelled or (B) .sup.111In-DOTA
labelled J591 minibody. At each time point, the supernatant
containing the radiolabeled minibody was removed, the cells were
stripped with an acidic glycine buffer to obtain the membrane
fraction, and the cells were lysed. Each time point was performed
in triplicate. The Y-bars represent standard deviation.
[0039] FIG. 20 is a graph comparing cell-associated radioactivity
of .sup.131I labelled versus .sup.111In-DOTA labelled J591
minibody. The total cell-associated radioactivity
(membrane+internalized) expressed as a percentage of the initial
cell-associated radioactivity over time upon binding to CWR22rv1
cells. This plot shows both the .sup.131I-labelled (bottom line)
and the .sup.111In-DOTA labelled J591 minibody (top line).
[0040] FIG. 21 illustrates representative serial microPET/CT images
of a mouse bearing CWR22rv1 and PC3 xenografts injected with
.sup.64Cu-DOTA-J591 minibody. A representative mouse was serially
scanned at multiple times postinjection. The CWR22rv1 tumor is
depicted as the (+) tumor and the PC3 tumor as the (-) tumor. (A)
CT scan at 4 hours postinjection. Coronal and tranverse planes are
shown. (B) PET/CT overlay image at 4 hours postinjection. Coronal
and transverse planes are shown. (C) Coronal PET/CT overlay 3D
projection of the representative mouse at 4 hours postinjection (D)
Coronal PET/CT overlay 3D projection of the representative mouse at
43 hours postinjection.
[0041] FIG. 22 is a bar graph illustrating the biodistribution of
.sup.64Cu-DOTA-J591 minibody at 19 hours and 43 hours
post-injection. Graph plotting the biodistribution of the
.sup.64Cu-DOTA-J591 minibody in the xenograft tumors and selected
normal tissues of interest. Biodistribution is plotted as % of the
injected dose divided by weight in grams (% D/g). Each data point
represents the average % ID/g for the group of mice at 19 hrs (n=8)
and 43 hrs postinjection (n=4). The error bars represent the
standard deviation.
[0042] FIG. 23 is illustrates representative serial microPET images
of a mouse bearing CWR22rv1 and PC3 xenografts injected with
.sup.1241-J591 minibody. A representative mouse was serially
scanned at multiple times postinjection. The CWR22rv1 tumor is
depicted as the (+) tumor and the PC3 tumor as the (-) tumor. (A)
CT scan at 4 hours postinjection. Coronal and tranverse planes are
shown. (B) PET/CT overlay image at 4 hours postinjection. Coronal
and transverse planes are shown. (C) Coronal PET/CT overlay images
at 4, 20, and 44 hours postinjection.
[0043] FIG. 24 is a bar graph illustrating the biodistribution of
.sup.1241-J591 minibody at 19 hours and 44 hours post-injection.
Graph plotting the biodistribution of the .sup.1241-J591 minibody
in the xenograft tumors and selected normal tissues of interest.
Biodistribution is expressed as % of the injected dose divided by
weight in grams (% ID/g). Each data point represents the average %
ID/g for the group of mice (n=4 at 19 hours, n=2 at 44 hours) at 19
hrs and 44 hrs postinjection. The error bars represent the standard
deviation.
[0044] FIG. 25 is a bar graph illustrating the expression level of
the following minibody variants in transient transfected CHO-K1
cells: (1) J591 HC VLVH minibody (J591 VLVH Mb), (2) J591 HC VHVL
minibody (J591 VHVL Mb), (3) J591 2P VLVH minibody (J591 VLVH**Mb)
and (4) J591 2P VHVL minibody (J591 VHVL**Mb). The huJ591 HC VHVL
exhibited the highest expression (6.7 .mu.g/mL) from transient
transfection.
[0045] FIG. 26 is a bar graph showing the biodistribution ratios
(i.e., positive tumor to tissue ratios) at 4 hours, 20 hours and 43
hours after injection of the .sup.64Cu-DOTA-J591 minibody. The
biodistribution ratios included ratios of positive tumor (Pos)
compared to liver (Liv), kidneys (Kid) and soft tissue (Soft).
Error bars represent mean standard errors (SEM).
[0046] FIG. 27 is a bar graph showing the biodistribution ratios
(i.e., positive tumor to tissue ratios) at 4 hours, 20 hours and 43
hours after injection of the .sup.1241-J591 minibody. The
biodistribution ratios included ratios of positive tumor (Pos)
compared to liver (Liv), kidneys (Kid) and soft tissue (Soft).
Error bars represent mean standard errors (SEM).
DETAILED DESCRIPTION
[0047] The disclosure is directed to an antibody or functional
antibody fragment that targets prostate specific membrane antigen
(PSMA). The PSMA antibody or functional antibody fragment thereof
may be conjugated to a substance such as a diagnostic agent, a
therapeutic agent or a nanoparticle to form an anti-PSMA conjugate.
Also disclosed are methods that include the use of the PSMA
antibody, the functional PSMA antibody fragment or the anti-PSMA
conjugate for diagnosing, visualizing, monitoring, or treating
cancer or other conditions associated with overexpression of
PSMA.
[0048] PSMA Antibodies and Functional Fragments Thereof.
[0049] PSMA antibodies or a functional PSMA antibody fragments are
provided herein according to the embodiments described herein. A
PSMA antibody or functional antibody fragment is a molecule that
includes one or more portions of an immunoglobulin or
immunoglobulin-related molecule that specifically binds to, or is
immunologically reactive with a PSMA. The term modified antibody
includes, but is not limited to genetically engineered or otherwise
modified forms of immunoglobulins, such as intrabodies, chimeric
antibodies, fully human antibodies, humanized antibodies, and
heteroconjugate antibodies (e.g., bispecific antibodies, diabodies,
triabodies, and tetrabodies). The term functional antibody fragment
includes one or more antigen binding fragments of antibodies alone
or in combination with other molecules, including, but not limited
to Fab', F(ab').sub.2, Fab, Fv, rIgG, scFv fragments, single domain
fragments, peptibodies, minibodies and cys-diabodies. The term scFv
refers to a single chain Fv antibody in which the variable domains
of the heavy chain and of the light chain of a traditional two
chain antibody have been joined to form one chain.
[0050] In one embodiment, the modified antibody or functional
antibody fragment is an anti-PSMA minibody. In one embodiment, the
anti-PSMA antibody is a J591 minibody. The anti-PSMA minibody has
an anti-PSMA antibody fragment with optimized pharmacodynamic
properties for in vivo imaging and biodistribution as described
below. A "minibody" is a homodimer, wherein each monomer is a
single-chain variable fragment (scFv) linked to a human IgG1 CH3
domain by a linker, such as ana hinge sequence. In one embodiment,
the hinge sequence is a human IgG1 hinge sequence
(EPKSCDKTHTCPPCPAPELLGGP; SEQ ID NO:16). In another embodiment, the
hinge sequence is an artificial hinge sequence. The artificial
hinge sequence may include a portion of a human IgG1 hinge and a
GlySer linker sequence. In one embodiment, the artificial hinge
sequence includes approximately the first 14 or 15 residues of the
human IgG1 hinge followed by a GlySer linker sequence that is 8, 9
or 10 amino acids in length. In another embodiment, the artificial
hinge sequence includes approximately the first 15 residues of the
IgG1 hinge followed by a GlySer linker sequence that is 10 amino
acids in length.
[0051] The scFv may have a VHVL or a VLVH orientation, wherein a
VHVL orientation means that the variable heavy domain (VH) of the
scFv is upstream from the variable light region (VL) and a VLVH
orientation means that the VL of the scFv is upstream from the VH.
As used herein, "upstream" means toward the N-terminus of an amino
acid or toward the 5' end of a nucleotide sequence. The VH and VL
are linked to each other by an amino acid linker sequence. The
amino acid linker may be any suitable length. In one embodiment,
the linker is Gly-Ser-rich and approximately 15-20 amino acids in
length. In another embodiment, the linker is Cly-Ser rich and is 18
amino acids in length.
[0052] According to the embodiments described herein, each monomer
of the anti-PSMA minibody may be encoded by a nucleotide sequence
that includes the following elements, from N-terminus to
C-terminus: (a) an scFv sequence that can bind PSMA, (b) an
artificial hinge sequence, and (c) a human IgG CH3 sequence. The
minibodies may be expressed by a cell, a cell line or other
suitable expression system as described herein. Thus, a signal
sequence may be fused to the N-terminus of the scFv to enable
secretion of the minibody when expressed in the cell or cell line.
In some embodiments, the nucleotide sequence is SEQ ID NO:1 or SEQ
ID NO:2. When expressed by a cell or cell line, the nucleotide is
transcribed and translated into an amino acid sequence. In some
embodiments, the expressed amino acid sequence is SEQ ID NO:10 or
SEQ ID NO:11.
[0053] In another embodiment, the modified antibody or functional
antibody fragment is an anti-PSMA cys-diabody (CysDB) is provided.
A "diabody" comprises a first polypeptide chain which comprises a
heavy (VH) chain variable domain connected to a light chain
variable domain (VL) on the first polypeptide chain (VH-VL)
connected by a peptide linker that is too short to allow pairing
between the two domains on the first polypeptide chain and a second
polypeptide chain comprising a light chain variable domain (VL)
linked to a heavy chain variable domain VH on the second
polypeptide chain (VL-VH) connected by a peptide linker that is too
short to allow pairing between the two domains on the second
polypeptide chain. The short linkages force chain pairing between
the complementary domains of the first and the second polypeptide
chains and promotes the assembly of a dimeric molecule with two
functional antigen binding sites. Therefore, a peptide linker may
be any suitable length that promotes such assembly, for example,
between 5 and 10 amino acids in length. As described further below,
some cys-diabodies may include a peptide linker that is 5 or 8
amino acids in length. The anti-PSMA CysDB is a homodimer antibody
format formed with two identical monomers that include single chain
Fv (scFv) fragments with an approximate molecular weight of 55 kDa.
In one embodiment, the anti-PSMA is a J591 CysDB. Like the
anti-PSMA minibodies described above, the anti-PSMA CysDBs
described herein have an anti-PSMA antibody fragment with optimized
pharmacodynamic properties that may be used for in vivo imaging and
biodistribution.
[0054] According to the embodiments described herein, each monomer
of a CysDB may be encoded by a nucleotide sequence that includes
the following elements, from N-terminus to C-terminus: (a) an scFv
sequence that can bind PSMA and (b) a cysteine tail. The CysDBs may
be expressed by a cell or a cell line as described herein. Thus, a
signal sequence may be fused to the N-terminus of the scFv to
enable secretion of the minibody when expressed in the cell or cell
line. In some embodiments, the nucleotide sequence is SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. When expressed by a cell
or cell line, the nucleotide is transcribed and translated into an
amino acid sequence. In some embodiments, the expressed amino acid
sequence is SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID
NO:15.
[0055] According to some embodiments, the CysDB scFv sequence is
similar to the minibody scFv sequences described above scFv. Thus,
the scFv may have a VHVL or a VLVH orientation, wherein a VHVL
orientation means that the variable heavy domain (VH) of the scFv
is upstream from the variable light region (VL) and a VLVH
orientation means that the VL of the scFv is upstream from the VH.
The antibody variable regions are linked together by a GlySer
linker as described above. A Cysteine tail (Gly-Gly-Cys), is added
at the C-terminus. This Cysteine tail allows the diabody complex to
form covalent Cysteine bonds and provides the option for available
sulfur residues for site-specific conjugation of functional
moieties such as radiolabels.
[0056] Multiple CysDBs have been successfully engineered from
various parental antibodies against different targets including
CEA, Her2 (trastuzumab/Herceptin.RTM.), PSCA, and CD20
(rituximab/Rituxan.RTM.). Different variations of the CysDB format
have been evaluated with four particular versions demonstrating the
most promise with respect to binding and expression level. For each
individual antibody, the heavy and light chain variable domains
associate in different ways. For this reason, the use of different
linker lengths allows for conformational flexibility and
range-of-motion to ensure formation of the disulfide bonds. In some
embodiments, the two linker length variants have either a 5 amino
acid linker or an 8 amino acid linker. Each linker length variant
may be developed using both orientations (VL-linker-VH-Cys tail and
VH-linker-VL-Cys tail) to ensure the proper folding and stability
is achieved. According to some embodiments, four CysDB variants
that may be used in methods described herein have been constructed:
VH5VL, VH8VL, VL5VH, and VL8VH (see FIGS. 6-9). Although each of
the CysDB variants has been successfully expressed, results may
vary depending on the parental antibody used. Producing and testing
the expression and binding of all four variants ensures
identification of an optimal format for protein production for each
new CysDB. Evaluating the set of variants is important to ensure
that a high-quality, stable protein is produced where the disulfide
bridge is available. Therefore, engineering a CysDB actually
involves using two distinct linker lengths, not one--as in the
minibody, as well as both orientations of the variable regions,
VH/VL and VL/VH.
[0057] In some embodiments, a mammalian cell line (e.g., CHO-K1
cell line) may used as an expression system to produce the
minibodies, cys-diabodies or other antibody fragments described
herein. However, because the minibodies, cys-diabodies and other
antibody fragments described herein are non-glycosylated, the cell
line or expression, a mammailan expression system is not required,
as such post-translational modifications are not needed. As such, a
wide variety of mammalian and non-mammalian expression systems may
be used to produce the PSMA antibody fragments (e.g., anti-PSMA
minibodies and cys-diabodies) according to the embodiments of the
disclosure including, but not limited to mammalian expression
systems (e.g., CHO-K1 cells), bacterial expression systems (e.g.,
E. Coli, B. subtilis) yeast expression systems (e.g., Pichia, S.
cerevisiae) or any other known expression system.
[0058] As described in detail in the Examples below, four minibody
variants that differ in the svFv region were made and expressed in
CHO-K1 cells. Specific binding to PSMA was demonstrated by ELISA
and flow cytometry. One of the variants with high expression and
PSMA binding (J591 HC VHVL) was selected for protein production,
purification and further evaluation. Protein production of the J591
HC VHVL minibody was successfully scaled-up to produce sufficient
amounts for the internalization and microPET imaging experiments
described below.
[0059] Confocal microscopy studies of the J591 minibody showed
increased intracellular staining in CWR22rv1 and LNCaP cells over
time, similar to that of the intact huJ591 mAb, suggesting that the
J591 minibodies undergo rapid internalization. To further evaluate
internalization of the J591 minibody, two radiolabeling strategies
were employed: radioiodination with I-131 and DOTA conjugation for
radiometal labeling with In-111. The .sup.111In-DOTA J591 minibody
showed a 260% increase in cell-associated radioactivity over a 3
hour time period In contrast, initial cell binding of
.sup.131I-J591 minibody was followed by a significant loss to 80%
of the initial activity.
[0060] The J591 minibody is rapidly internalized upon binding to
PSMA+ cell lines CWR22rv1 and LNCaP. For .sup.131I-labeled J591
minibody, the total cell-associated radioactivity decreased over
time suggesting loss of label likely attributed to dehalogenation
and/or rapid metabolism and release from the cells of the
.sup.131I-J591 minibody. In contrast, the total cell-associated
radioactivity of the .sup.111In-DOTA-J591 minibody increased
significantly over time (.about.2.5 fold) which is consistent with
the residualizing label being trapped in the lysosomes. Based on
the persistence of total cell-associated radioactivity over time,
the residualizing .sup.111In-DOTA radiolabeling strategy appeared
to be the appropriate approach for in vivo imaging of the
internalizing PSMA antigen.
[0061] Anti-PSMA Derivatives and Conjugates
[0062] In some embodiments, the PSMA antibodies or functional
antibody fragments may include antibody derivatives that are
modified. For example, the antibody derivatives include, but are
not limited to, antibodies that have been modified by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, and linkage to a cellular ligand or other protein. Any of
numerous chemical modifications may be carried out by known
techniques, including, but not limited to, specific chemical
cleavage, acetylation, gormylation and metabolic synthesis of
tunicamycin. Additionally, the derivative may contain one or more
non-natural amino acids.
[0063] In other embodiments, the PSMA antibody or functional
antibody fragment may be conjugated to another substance to form an
anti-PSMA conjugate. The anti-PSMA conjugates described herein can
be prepared by known methods of linking antibodies with lipids,
carbohydrates, protein or other atoms and molecules. In one aspect,
the anti-PSMA conjugate is formed by site-specific conjugation
using a suitable linkage or bond. Site-specific conjugation is more
likely to preserve the binding activity of an antibody or
functional antibody fragment. The substance may be conjugated or
attached at the hinge region of a reduced antibody component or
antibody fragment via disulfide bond formation. For example,
introduction of cysteine residues at the C-terminus of an scFv
fragment, such as those introduce in the cys-diabodies described
above, allows site-specific thiol-reactive coupling at a site away
from the antigen binding site to a wide variety of agents.
Alternatively, other linkages or bonds used to form the anti-PSMA
conjugate may include, but is not limited to, a covalent bond, a
non-covalent bond, a sulfide linkage, a hydrazone linkage, a
hydrazine linkage, an ester linkage, an amido linkage, and amino
linkage, an imino linkage, a thiosemicabazone linkage, a
semicarbazone linkage, an oxime linkage and a carbon-carbon
linkage.
[0064] In one embodiment, the anti-PSMA conjugate may include a
PSMA antibody or functional PSMA antibody fragment conjugated to a
diagnostic agent. A "diagnostic agent" is an atom, molecule, or
compound that is useful in diagnosing, detecting or visualizing a
disease. According to the embodiments described herein, diagnostic
agents may include, but are not limited to, radioactive substances
(e.g., radioisotopes, radionuclides, radiolabels or radiotracers),
dyes, contrast agents, fluorescent compounds or molecules,
bioluminescent compounds or molecules, enzymes and enhancing agents
(e.g., paramagnetic ions). In addition, it should be noted that
some nanoparticles, for example quantum dots and metal
nanoparticles (described below) may also be suitable for use as a
detection agent.
[0065] Radioactive substances that may be used as diagnostic agents
in accordance with the embodiments of the disclosure include, but
are not limited to, .sup.18F, .sup.32P, .sup.33P, .sup.45Ti,
.sup.47Sc, .sup.52Fe, .sup.59Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu,
.sup.67Ga, .sup.68Ga, .sup.75Sc, .sup.77As, .sup.86Y, .sup.90Y.
.sup.89Sr, .sup.89Zr, .sup.94Tc, .sup.94Tc, .sup.99mTc, .sup.99Mo,
.sup.105Pd, .sup.105Rh, .sup.111Ag, .sup.111In, .sup.123I,
.sup.124I, .sup.125I, .sup.131I, .sup.142Pr, .sup.143Pr,
.sup.149Pm, .sup.153Sm, .sup.154-1581Gd, .sup.161Tb, .sup.166Dy,
.sup.166Ho, .sup.169Er, .sup.175Lu, .sup.177Lu, .sup.186Re,
.sup.188Re, .sup.189Re, .sup.194Ir, .sup.198Au, .sup.199Au,
.sup.211At, .sup.211Pb, .sup.212Bi, .sup.212Pb, .sup.213Ra,
.sup.223Ra and .sup.225Ac. Paramagnetic ions that may be used as
diagnostic agents in accordance with the embodiments of the
disclosure include, but are not limited to, ions of transition and
lanthanide metals (e.g. metals having atomic numbers of 6 to 9,
21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V,
Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er,
Tm, Yb and Lu.
[0066] When the diagnostic agent is a radioactive metal or
paramagnetic ion, the agent may be reacted with a reagent having a
long tail with one or more chelating groups attached to the long
tail for binding these ions. The long tail may be a polymer such as
a polylysine, polysaccharide, or other derivatized or derivatizable
chain having pendant groups to which may be bound to a chelating
group for binding the ions. Examples of chelating groups that may
be used according to the disclosure include, but are not limited
to, ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NETA,
porphyrins, polyamines, crown ethers, bis-thiosemicarbazones,
polyoximes, and like groups. The chelate is normally linked to the
PSMA antibody or functional antibody fragment by a group which
enables formation of a bond to the molecule with minimal loss of
immunoreactivity and minimal aggregation and/or internal
cross-linking. The same chelates, when complexed with
non-radioactive metals, such as manganese, iron and gadolinium are
useful for MRI, when used along with the antibodies and carriers
described herein. Macrocyclic chelates such as NOTA, DOTA, and TETA
are of use with a variety of metals and radiometals including, but
not limited to, radionuclides of gallium, yttrium and copper,
respectively. Other ring-type chelates such as macrocyclic
polyethers, which are of interest for stably binding nuclides, such
as .sup.223Ra for RAIT may be used. In certain embodiments,
chelating moieties may be used to attach a PET imaging agent, such
as an Al-.sup.18F complex, to a targeting molecule for use in PET
analysis.
[0067] Contrast agents that may be used as diagnostic agents in
accordance with the embodiments of the disclosure include, but are
not limited to, barium, diatrizoate, ethiodized oil, gallium
citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide,
iodoxamic acid, iogulamide, iohexyl, iopamidol, iopanoic acid,
ioprocemic acid, iosefamic acid, ioseric acid, iosulamide
meglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid,
iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine,
metrizamide, metrizoate, propyliodone, thallous chloride, or
combinations thereof.
[0068] Bioluminescent and fluorescent compounds or molecules and
dyes that may be used as diagnostic agents in accordance with the
embodiments of the disclosure include, but are not limited to,
fluorescein, fluorescein isothiocyanate (FITc), Oregon Green.TM.,
rhodamine, Texas red, tetrarhodimine isothiocynate (TRITc), Cy3,
Cy5, etc.), fluorescent markers (e.g., green fluorescent protein
(GFP), phycoerythrin, etc.), autoquenched fluorescent compounds
that are activated by tumor-associated proteases, enzymes (e.g.,
luciferase, horseradish peroxidase, alkaline phosphatase, etc.),
nanoparticles, biotin, digoxigenin or combination thereof.
[0069] Enzymes that may be used as diagnostic agents in accordance
with the embodiments of the disclosure include, but are not limited
to, horseradish peroxidase, alkaline phosphatase, acid phoshatase,
glucose oxidase, .beta.-galactosidase, .beta.-glucoronidase or
.beta.-lactamase. Such enaymes may be used in combination with a
chromogen, a fluorogenic compound or a luminogenic compound to
generate a detectable signal.
[0070] In another embodiment, the anti-PSMA conjugate may include a
PSMA antibody or functional PSMA antibody fragment conjugated to a
therapeutic agent. A "therapeutic agent" as used herein is an atom,
molecule, or compound that is useful in the treatment of cancer or
other conditions associated with PSMA. Examples of therapeutic
agents include, but are not limited to, drugs, chemotherapeutic
agents, therapeutic antibodies and antibody fragments, toxins,
radioisotopes, enzymes (e.g., enzymes to cleave prodrugs to a
cytotoxic agent at the site of the tumor), nucleases, hormones,
immunomodulators, antisense oligonucleotides, chelators, boron
compounds, photoactive agents and dyes.
[0071] Chemotherapeutic agents are often cytotoxic or cytostatic in
nature and may include alkylating agents, antimetabolites,
anti-tumor antibiotics, topoisomerase inhibitors, mitotic
inhibitors hormone therapy, targeted therapeutics and
immunotherapeutics. In some embodiments the chemotherapeutic agents
that may be used as diagnostic agents in accordance with the
embodiments of the disclosure include, but are not limited to,
13-cis-Retinoic Acid, 2-Chlorodeoxyadenosine, 5-Azacitidine,
5-Fluorouracil, 6-Mercaptopurine, 6-Thioguanine, actinomycin-D,
adriamycin, aldesleukin, alemtuzumab, alitretinoin,
all-transretinoic acid, alpha interferon, altretamine,
amethopterin, amifostine, anagrelide, anastrozole,
arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin,
aminoglutethimide, asparaginase, azacytidine, bacillus
calmette-guerin (BCG), bendamustine, bevacizumab, bexarotene,
bicalutamide, bortezomib, bleomycin, busulfan, calcium leucovorin,
citrovorum factor, capecitabine, canertinib, carboplatin,
carmustine, cetuximab, chlorambucil, cisplatin, cladribine,
cortisone, cyclophosphamide, cytarabine, darbepoetin alfa,
dasatinib, daunomycin, decitabine, denileukin diftitox,
dexamethasone, dexasone, dexrazoxane, dactinomycin, daunorubicin,
decarbazine, docetaxel, doxorubicin, doxifluridine, eniluracil,
epirubicin, epoetin alfa, erlotinib, everolimus, exemestane,
estramustine, etoposide, filgrastim, fluoxymesterone, fulvestrant,
flavopiridol, floxuridine, fludarabine, fluorouracil, flutamide,
gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin,
granulocyte--colony stimulating factor, granulocyte
macrophage-colony stimulating factor, hexamethylmelamine,
hydrocortisone hydroxyurea, ibritumomab, interferon alpha,
interleukin-2, interleukin-11, isotretinoin, ixabepilone,
idarubicin, imatinib mesylate, ifosfamide, irinotecan, lapatinib,
lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C,
lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine,
mesna, methotrexate, methylprednisolone, mitomycin C, mitotane,
mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin,
oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitumumab, PEG
Interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase,
pentostatin, plicamycin, prednisolone, prednisone, procarbazine,
raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine,
sargramostim, satraplatin, sorafenib, sunitinib, semustine,
streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus,
temozolamide, teniposide, thalidomide, thioguanine, thiotepa,
topotecan, toremifene, tositumomab, trastuzumab, tretinoin,
trimitrexate, alrubicin, vincristine, vinblastine, vindestine,
vinorelbine, vorinostat, or zoledronic acid.
[0072] Therapeutic antibodies and functional fragments thereof,
that may be used as diagnostic agents in accordance with the
embodiments of the disclosure include, but are not limited to,
alemtuzumab, bevacizumab, cetuximab, edrecolomab, gemtuzumab,
ibritumomab tiuxetan, panitumumab, rituximab, tositumomab, and
trastuzumab
[0073] Toxins that may be used as diagnostic agents in accordance
with the embodiments of the disclosure include, but are not limited
to, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
[0074] Radioisotopes that may be used as diagnostic agents in
accordance with the embodiments of the disclosure include, but are
not limited to, .sup.32P, .sup.89Sr, .sup.93Y. .sup.99mTc,
.sup.99Mo, .sup.131I, .sup.153Sm, .sup.177Lu, .sup.186Re,
.sup.213Bi, .sup.223Ra and .sup.225Ac.
[0075] In another embodiment, the anti-PSMA conjugate may include a
PSMA antibody or functional PSMA antibody fragment conjugated to a
nanoparticle. The term "nanoparticle" refers to a microscopic
particle whose size is measured in nanometers, e.g., a particle
with at least one dimension less than about 100 nm. Nanoparticles
are particularly useful as detectable substances because they are
small enough to scatter visible light rather than absorb it. For
example, gold nanoparticles possess significant visible light
extinction properties and appear deep red to black in solution. As
a result, compositions comprising PSCA-specific antibody or
fragments conjugated to nanoparticles can be used for the in vivo
imaging of tumors or cancerous cells in a subject. At the small end
of the size range, nanoparticles are often referred to as clusters.
Metal, dielectric, and semiconductor nanoparticles have been
formed, as well as hybrid structures (e.g. core-shell
nanoparticles). Nanospheres, nanorods, and nanocups are just a few
of the shapes that have been grown. Semiconductor quantum dots and
nanocrystals are examples of additional types of nanoparticles.
Such nanoscale particles, when conjugated to a PSMA antibody or
functional antibody fragment, can be used as imaging agents for the
in vivo detection of tumor cells as described above. Alternatively,
nanoparticles can be used in therapeutic applications as drug
carriers that, when conjugated to a PSCA-specific antibody or
fragment of the present invention, deliver chemotherapeutic agents,
hormonal therapaeutic agents, radiotherapeutic agents, toxins, or
any other cytotoxic or anti-cancer agent known in the art to
cancerous cells that overexpress PSCA on the cell surface.
[0076] Any of the anti-PSMA conjugates described above may be
further conjugated with one or more additional therapeutic agents,
diagnostic agents, nanoparticles, carriers or a combination
thereof. For example, a PSMA antibody or functional PSMA antibody
fragment may be radiolabeled with .sup.131I and conjugated to a
lipid carrier, such that the anti-PSMA-lipid conjugate forms a
micelle. The micelle may incorporate one or more therapeutic or
diagnostic agents. Alternatively, in addition to the carrier, the
PSMA antibody or functional PSMA antibody fragment may be
conjugated to .sup.131I (e.g., at a tyrosine residue) and a drug
(e.g., at the epsilon amino group of a lysine residue), and the
carrier may incorporate an additional therapeutic or diagnostic
agent.
[0077] Methods for Diagnosing, Staging and Monitoring Cancer
[0078] The PSMA antibody, functional PSMA antibody fragment or
anti-PSMA conjugate may be used to target a PSMA positive cell,
such as cancer cells that overexpress PSMA. Therefore, methods for
diagnosing, detecting, visualizing, monitoring or treating a cancer
or other condition associated with PSMA expression may include
administering the PSMA antibody, functional PSMA antibody fragment
or anti-PSMA conjugate to a subject having or suspected of having a
cancer or other condition associated with PSMA expression. As used
herein, the term "subject" refers to any animal (e.g., a mammal),
including but not limited to humans, non-human primates, rodents,
dogs, pigs, and the like.
[0079] Cancers that are associated with PSMA expression may include
those having cancer tumor tissue that overexpresses PSMA (e.g.,
prostate cancer) or those having solid tumor neovasculature that
overexpresses PSMA (e.g., prostate cancer, lung cancer, colon (or
colorectal) cancer, breast cancer, renal cancer, liver cancer,
bladder cancer and pancreatic cancer as well as sarcomas and
melanoma). Most solid tumor neovasculature expresses PSMA, making
PSMA a neovasculature biomarker. Thus, in addition to cancer cells
that expresses PSMA, a cancer that is associated with PSMA
expression may include any cancer tissue with neovasculature
including, but not limited to, carcinomas such as prostate cancer,
lung cancer, colon (or colorectal) cancer, breast cancer, renal
cancer, liver cancer, bladder cancer and pancreatic cancer as well
as sarcomas and melanoma.
[0080] In one embodiment, a method for diagnosing, detecting,
visualizing or monitoring a cancer associated with PSMA expression
includes administering a diagnostic anti-PSMA conjugate to a
subject having or suspected of having a cancer. The diagnostic
anti-PSMA conjugate includes a PSMA antibody or a functional PSMA
antibody fragment conjugated to one or more diagnostic agents as
described above. In one embodiment, the PSMA antibody, or a
functional PSMA antibody fragment is a minibody or a CysDB, derived
from a J591 antibody such as those J591 minibodies and J591 CysDBs
described herein. The diagnostic anti-PSMA conjugate may be
conjugated to or associated with one or more additional substances
described herein, such as a therapeutic anti-PSMA conjugate (as
described below), unconjugated therapeutic agents, contrast
solutions, carrier lipids or nanoparticles.
[0081] The diagnostic anti-PSMA conjugate used in the method
described above is suitable for in vivo or in vitro detection or
visualization methods. In one embodiment, an in vitro diagnostic or
prognostic assay will be performed to determine the expression
level of PSMA in a tissue sample taken from a subject having or
suspected of having a cancer associated with PSMA as compared to a
normal (i.e., non cancerous) or control tissue sample (i.e., known
cancerous or benign tissue sample). Various assays for determining
such express ion levels are contemplated and include
immunohistochemistry, fluorescent in situ hybridization (FISH) and
shed antigen assays, southern blotting, or PCR techniques.
[0082] In another embodiment, the diagnostic anti-PSMA conjugate
may be used with an in vivo imaging modality to visualize the
target cells within the topography of the subject's body. According
to the method described herein, determining that the subject has a
cancer associated with PSMA expression is accomplished by
visualizing the lableled minibody or CysDB, wherein the visualized
labeled minibody or CysDB localizes to a tumor site. In addition to
diagnosing a cancer associated with PSMA expression, the PSMA
minibody may also be used to stage, and monitor cancer progression
according to method that are similar to those described above.
[0083] Suitable methods of in vivo imaging that may be used in
accordance with the methods described herein include, but are not
limited to, magnetic resonance imaging (MRI), positron emission
tomography (PET) or microPET, computed tomography (CT), PET/CT
combination imager, cooled charged coupled device (CCD), camera
optical imaging, optical imaging (e.g., bioluminescent optical
imaging, fluorescent optical imaging, or absorption of reflectance)
and single photon emission computed tomography (SPECT),
[0084] As described in the examples below, a minibody or CysDB as
described herein that is labeled with an appropriate radioisotope
(e.g., residualizing .sup.124I, .sup.64Cu-DOTA or .sup.89Zr-DOTA),
may be used as a clinical imaging agent to target PSMA in vivo
according to the methods described herein. These J591 minibodies
and CysDBs may also be developed as a potential single photon
emission computed tomography (SPECT) imaging agent according to
embodiments described herein. The J591 minibody may be used as a
SPECT imaging agent by changing the radiolabel, for example,
.sup.111In-DOTA-J591.
[0085] The J591 minibodies described herein were evaluated for
tumor targeting by small-animal PET (microPET) and biodistribution
experiments following radiolabeling with the positron emitters
I-124 (t.sub.1/2=4.2 d) and Cu-64 (t.sub.1/2=12.7 h) to compare
retention of cell-associated radioactivity in vivo.
[0086] Both .sup.124I and .sup.64Cu-DOTA labelled J591 minibodies
rapidly targeted the CWR22rv1 tumor with high uptake and
specificity. Serial imaging of mice carrying PSMA positive CWR22rv1
and negative PC-3 xenografts resulted in high contrast images and
excellent tumor uptakes with both labels. At 19 hours p.i.,
8.2(.+-.1.2) % ID/g and 8.8(.+-.2.0) % ID/g were achieved with
.sup.64Cu-DOTA- and .sup.124I-J591 minibodies, respectively. At 43
hours post injection (p.i.), tumor uptake increased to
13.3(.+-.8.3) % ID/g with the .sup.64Cu-DOTA-J591 minibodies, which
declined to 3.25(.+-.0.9) % ID/g with the .sup.124I-J591
minibodies. Positive to negative tumor ratios were 3.1 and 4.9 at
19 hours and 5.4 and 7.3 at 43 hours for .sup.64Cu-DOTA- and
.sup.1241-J591 minibodies, respectively. Persistent high liver
uptake [21.4(.+-.3.1) % ID/g at 19 hr and 14.4(.+-.2.1) % ID/g at
43 hr] was seen with .sup.64Cu-DOTA-J591 minibodies, whereas the
.sup.1241-J591 minibodies exhibited rapid background clearance
resulting in higher contrast images. The similar tumor uptakes of
both radiolabeled minibodies at 19 hours were unexpected, and
suggestive of slower in vivo internalization. Thus, the J591
minibodies radiolabeled with I-124 is an efficient tracer for
detecting PSMA positive cells.
[0087] Methods for Treating Cancer
[0088] In some embodiments, methods for treating cancer or other
condition associated with overexpression of PSMA are provided. Such
methods include administering to a subject a therapeutically
effective amount of a pharmaceutical composition that includes a
PSMA antibody, or a functional PSMA antibody fragment as described
above. In one embodiment, the PSMA antibody, or a functional PSMA
antibody fragment is a minibody or a CysDB, derived from a J591
antibody such as those J591 minibodies and J591 CysDBs described
herein.
[0089] "Treating" or "treatment" of a condition may refer to
preventing the condition, slowing the onset or rate of development
of the condition, reducing the risk of developing the condition,
preventing or delaying the development of symptoms associated with
the condition, reducing or ending symptoms associated with the
condition, generating a complete or partial regression of the
condition, or some combination thereof.
[0090] A "therapeutically effective amount" or a: "therapeutically
effective dose is an amount of a compound that produces a desired
therapeutic effect in a subject, such as preventing or treating a
target condition or alleviating symptoms associated with the
condition. The precise therapeutically effective amount is an
amount of the composition that will yield the most effective
results in terms of efficacy of treatment in a given subject. This
amount will vary depending upon a variety of factors, including but
not limited to the characteristics of the therapeutic compound
(including activity, pharmacokinetics, pharmacodynamics, and
bioavailability), the physiological condition of the subject
(including age, sex, disease type and stage, general physical
condition, responsiveness to a given dosage, and type of
medication), the nature of the pharmaceutically acceptable carrier
or carriers in the formulation, and the route of administration.
One skilled in the clinical and pharmacological arts will be able
to determine a therapeutically effective amount through routine
experimentation, namely by monitoring a subject's response to
administration of a compound and adjusting the dosage accordingly.
For additional guidance, see Remington: The Science and Practice of
Pharmacy 21.sup.st Edition, Univ. of Sciences in Philadelphia
(USIP), Lippincott Williams & Wilkins, Philadelphia, Pa.,
2005.
[0091] In one embodiment, the pharmaceutical composition may
include a therapeutic anti-PSMA conjugate, wherein the conjugate
includes a PSMA antibody or a functional PSMA antibody fragment
conjugated to one or more therapeutic agent as described above. In
one embodiment, the PSMA antibody, or a functional PSMA antibody
fragment is a minibody or a CysDB, derived from a J591 antibody
such as those J591 minibodies and J591 CysDBs described herein. For
example, the J591 minibodies or cys-diabodies described herein may
be used in a radioimmunotherapy approach, wherein one or more of
the 3B J591 minibodies is radiolabeled with an appropriate
beta-emitting radiolabel such as Yttrium-90. The radiolabeled 3B
J591 minibody or minibodies may be used to deliver cell damage and
death to local cancerous tissue that expresses PSMA. Further, the
use of radiolabeled J591 minibodies and cys-diabodies would likely
exhibit improved tumor penetration as compared to radiolabeled
full-length parental huJ591 antibody.
[0092] The therapeutic anti-PSMA conjugate may be conjugated to or
associated with one or more additional substances described herein,
such as diagnostic anti-PSMA conjugates (described above),
unconjugated diagnostic agents, contrast solutions, carrier lipids
or nanoparticles.
[0093] In some embodiments, the pharmaceutical composition may also
include a pharmaceutically acceptable carrier. A pharmaceutically
acceptable carrier may be a pharmaceutically acceptable material,
composition, or vehicle that is involved in carrying or
transporting a compound of interest from one tissue, organ, or
portion of the body to another tissue, organ, or portion of the
body. For example, the carrier may be a liquid or solid filler,
diluent, excipient, solvent, or encapsulating material, or some
combination thereof. Each component of the carrier must be
"pharmaceutically acceptable" in that it must be compatible with
the other ingredients of the formulation. It also must be suitable
for contact with any tissue, organ, or portion of the body that it
may encounter, meaning that it must not carry a risk of toxicity,
irritation, allergic response, immunogenicity, or any other
complication that excessively outweighs its therapeutic
benefits.
[0094] The pharmaceutical compositions described herein may be
administered by any suitable route of administration. A route of
administration may refer to any administration pathway known in the
art, including but not limited to aerosol, enteral, nasal,
ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical
cream or ointment, patch), or vaginal. "Transdermal" administration
may be accomplished using a topical cream or ointment or by means
of a transdermal patch. "Parenteral" refers to a route of
administration that is generally associated with injection,
including infraorbital, infusion, intraarterial, intracapsular,
intracardiac, intradermal, intramuscular, intraperitoneal,
intrapulmonary, intraspinal, intrasternal, intrathecal,
intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,
transmucosal, or transtracheal.
[0095] The following examples are intended to illustrate various
embodiments of the invention. As such, the specific embodiments
discussed are not to be construed as limitations on the scope of
the invention. It will be apparent to one skilled in the art that
various equivalents, changes, and modifications may be made without
departing from the scope of invention, and it is understood that
such equivalent embodiments are to be included herein. Further, all
references cited in the disclosure are hereby incorporated by
reference in their entirety, as if fully set forth herein.
Example 1
Generation of the J591 Minibodies
[0096] J591 Minibody Construct.
[0097] Third generation J591 minibodies are engineered antibody
fragments that incorporate modified variable regions of the
full-length parental huJ591 antibody. The minibody format is a
homodimer wherein each monomer is a single-chain variable fragment
(scFv) linked to a human IgG1 CH3 domain (FIG. 1A). The scFv can
have a VHVL or a VLVH orientation. As shown in FIG. 1B, the J591
minibody expression construct for a an scFv having a VHVL
orientation has a variable heavy (VH) domain that is linked to a
variable light (VL) region by an 18 amino acid linker (L) sequence.
In a VLVH orientation, the VL and VH would be switched in FIG. 1B
such that the VL region is upstream of the VH domain.
[0098] Four J591 minibody sequences were synthesized for use in the
expression studies described below: The minibody sequences that
were constructed as follows: [0099] 1) J591 Human Composite (HC)
with a VHVL orientation (J591 HC VHVL; SEQ ID NO:1); [0100] 2) J591
Human Composite (HC) with a VLVH orientation (J591 HC VLVH); [0101]
3) J591 with the 2 Proline substitutions (2P) with a VHLV
orientation (J591 2P VHVL; SEQ ID NO:2); and [0102] 4) J591 with
the 2 Proline substitutions (2P) with a VLVH orientation (J591 2P
VLVH).
[0103] FIG. 3 shows the sequence of the J591 HC VHVL minibody (SEQ
ID NO:1) and FIG. 4 shows the sequence of the J591 2P VHVL minibody
(SEQ ID NO:2). The 18 amino acid linker (L) has a specific sequence
of GlySer-rich residues (see FIG. 1, for sequence see FIGS. 3 and
4). The scFv is tethered to the human IgG1 CH3 domain by an
artificial hinge sequence wherein the first 15 residues are that of
the human IgG1 hinge followed by a 10 amino acid GlySer linker
sequence (see FIG. 1, for sequence see FIGS. 3 and 4). This
specific hinge sequence has also been successfully incorporated
into previous minibodies. The minibody (either VH-VL-CH3 or
VL-VH-CH3 orientation) exists as a stable dimer due to the
association between the CH3 domains as well as the formation of
disulfide bonds within the hinge regions. To enable secretion of
the minibody, a kappa light chain signal sequence leads the
expression construct and fused at the N-terminus of the variable
heavy domain (see FIG. 1B, for sequence see FIGS. 3 and 4).
[0104] A set of J591 minibodies were engineered by making amino
acid substitutions in the parental huJ591 variable heavy and light
domains. Sequence analysis of the full length parental huJ591
variable regions identified an unusually high number of
conformationally restrictive Proline residues, which are recognized
to decrease the flexibility of protein structure. A comparison of
sequence alignment between the deimmunized J591 (SEQ ID NO:5; SEQ
ID NO:19) and the original murine J591 (SEQ ID NO:4; SEQ ID NO:18)
revealed that the deimmunization process introduced additional
Proline residues (see FIG. 2). After sequence and protein modeling
analysis, two changes to the protein were made to improve the
flexibility and folding ability. First, two Proline residues were
changed in the variable light domain (P42Q and P100A) to the
residues found in the murine sequence (see FIG. 2, here forth
referred to as 2P). Second, substitutions were calculated for both
variable regions using Human Composite (HC) Antibody technology
(Antitope) that deimmunizes the sequence by avoiding potential
epitopes instead of destroying epitopes (see FIG. 2, here forth
referred to as HC).
[0105] Expression of the J591 Minibodies.
[0106] Expression vectors for each of the four minibody sequences
above were generated. Each of the four minibody sequences was
cloned into the pcDNA3.1/myc-His (-) Version A vector for mammalian
expression (Invitrogen, Inc.) at the corresponding XbaI/HindIII
sites. The pcDNA3.1 expression vector features a CMV promoter for
mammalian expression and both mammalian (Neomycin) and bacterial
(Ampicillin) selection markers (see FIG. 10).
[0107] The four J591 minibody expression vectors were transiently
transfected into CHO-K1 cells to validate expression of the J591
minibodies. The transfections were performed using the
Lipofectamine reagent in a 6-well plate format. Following a 72 hour
transfection, the supernatants were harvested and filtered to
remove any cells.
[0108] To confirm the expression of the J591 minibodies by the
CHO-K1 cells, Western blot analyses were performed using sample of
supernatant taken from the transient transfections. Supernatant
from an empty vector transfection was included as a negative
control, and supernatant from the transfection of a different
minibody was used as a positive control. Transfection supernatants
were run out by SDS-PAGE and transferred to PVDF membrane. The
membrane was probed with an anti-human IgG (Fc-specific) antibody
conjugated with Alkaline Phosphatase (AP) and developed by
incubating with the AP substrate BCIP/NBT. FIG. 11 is a
representative blot of multiple experiments that confirmed the
expression of the J591 minibodies. Under non-reducing conditions,
the J591 minibodies run at the expected molecular weight of
approximately 90 kD (FIG. 11). A minor band representing the
monomeric form was also detected at approximately 40 kD.
[0109] Quantitative ELISAs were performed to analyze J591 minibody
expression from transient transfection. ELISA is a sandwich assay
which uses a goat anti-human IgG (Fc specific) as the capture
antibody and an AP-conjugated goat anti-human IgG (Fc specific) as
the detection antibody. Purified protein for a previously produced
minibody was used as a standard. J591 minibody supernatants were
serially diluted to find dilution points which fit in the linear
range of the standard curve. The program SoftMax Pro was used to
interpolate the concentration of the unknowns according to the
standard curve.
[0110] Supernatants from multiple transfections were assayed, and
the averages are displayed in FIG. 25. The J591 HC VHVL minibody
exhibited the highest expression (6.7 ug/ml) of the third
generation minibodies.
[0111] Binding Ability of the J591 Minibodies.
[0112] To confirm the ability of the J591 minibodies to bind
cellular PSMA, supernatant from the transient transfections
described above were analyzed by flow cytometry. As illustrated in
FIGS. 12A-12D, supernatants from the transient transfection for
each of the J591 minibodies were tested for binding to LNCaP cells
that are PSMA positive (PSMA+) (2) and compared to negative control
cells that are PSMA negative. All supernatants were normalized to a
concentration of 2.1 ug/ml of J591 minibody. Cells were
subsequently stained with a secondary anti-human IgG (Fc
specific)-PE conjugated antibody. Negative control cells were
stained with the secondary alone (1). 1.times.10.sup.5 cells/point
and analysis was performed with 10,000 events/point.
[0113] Each of the cell populations stained with the J591
minibodies demonstrated a significant increase in signal relative
to the negative control cells (see FIG. 12). The J591 minibody
supernatants did not significantly stain negative control PC3 cells
(PSMA negative) (data not shown). All four minibodies exhibited
comparable binding affinity to the LNCaP cells (see FIG. 12).
Example 2
Stable Cell Line Production
[0114] Based on the expression and binding data described above,
the J591 Human Composite VHVL (HC VHVL) minibody was selected as a
lead candidate to move forward into larger scale (.about.low
milligram quantity) protein production for subsequent in vivo
imaging studies described below. Although the Examples described
below are specific to the J591 HC VHVL minibody, however, it is
noted that any of the J591 minibodies or cys-diabodies described
herein may be purified and used in similar studies.
[0115] The J591 HC VHVL minibody was stably transfected into CHO-K1
cells using Neomycin as the selection marker. Following selection
for high-expressing clones, a clone expressing the J591 minibody at
approximately 36 mg/L (over a 4 day culture) was chosen for
scale-up production.
[0116] Protein Production Run.
[0117] To produce at least 10 mg of final purified protein, the
stable cell line was expanded to a 400 ml production run (in 2% FBS
media). Cells were seeded into eight T175 flasks, and the
production run lasted for 7 days.
[0118] Protein Purification.
[0119] At the end of the production run, the supernatant was
harvested, spun down to remove any cells, and filtered using 0.2 um
filter units. The J591 minibody was purified from the supernatant
using Protein L affinity chromatography. After loading, the column
was washed with PBS (pH=7.2) and the minibody was eluted from the
column using IgG Elution Buffer (Pierce, Thermo Scientific). Eluted
fractions were immediately neutralized using 1M Tris buffer (pH=8).
The final elution fractions were concentrated and buffer exchanged
into the final formulation of PBS (pH=7.2).
[0120] Purified Protein Analysis.
[0121] After purification, the final concentration of J591 minibody
protein was calculated using UV absorbance at 280 A. The absorbance
coefficient was 1.76 (absorbance Units at A280 per mg/ml). The
final concentration of the protein was 1.06 mg/ml.
[0122] To analyze the purity of the J591 minibody, the protein was
run under non-reducing and reducing conditions by SDS-PAGE. Under
non-reducing conditions, the minibody was detected at approximately
85 kDa (FIG. 13). A relatively minor smear was present under the 85
kDa band which may represent a small amount of degradation. The
minor band at approximately 40 kDa represents the minibody monomer.
Under reducing conditions, the minibody was detected as the
monomeric form at around 40 kDa (FIG. 13).
[0123] To examine the purity of the assembled minibody complex, the
protein was analyzed by size exclusion chromatography. 4 micrograms
of the purified protein was analyzed by SEC (FIG. 14). The major
peak corresponds with minibody homodimer. The two minor peaks which
eluted at earlier time points represent larger aggregate protein.
Analysis of the area under the peaks showed that 85% of the protein
product exists as the proper minibody homodimer vs 15%
aggregate.
Example 3
J591 Minibodies Binds and is Internalized by PSMA+ Cells
[0124] High-expressing stable cell pools were generated with
Catalent's proprietary GPEx technology using lentiviral
transductions of serum-free CHO-S cells. Using ion exchange
chromatography, the J591 minibody was purified from the cell
supernatant with sufficiently high purity for downstream
experiments. High purity of the product was confirmed by SDS-PAGE
and SEC analysis (>85% purity). The purified protein does not
have any significant bioburden (0 cfu/ml) and relatively low
endotoxin levels (between 8 and 16 EU/mg). The total yield from
this production run batch was 65 mg of J591 minibody protein.
[0125] Functional ELISA.
[0126] To confirm the ability of the J591 minibody protein to bind
purified PSMA, an indirect ELISA using purified recombinant PSMA
was performed. A negative control minibody was included in the
experiment. At the starting concentration of 2 .mu.g/ml, the J591
minibody bound the recombinant PSMA at saturation (see FIG. 15).
Subsequent serial dilutions of the J591 minibody showed
concentration-dependent binding (see FIG. 15). As expected, the
negative control minibody did not bind PSMA (see FIG. 15).
[0127] Flow Cytometry.
[0128] Following successful binding to recombinant PSMA in the
ELISA, the J591 minibody protein was tested for the ability to bind
PSMA+ cells by flow cytometry. Full-length hJ591 antibody was
included in the experiment as a positive control (data not shown)
and the negative control minibody was also included. The PSMA+
cells in this experiment were the LNCaP and CWR22rv1 cells and the
PSMA-cell line was the PC3. The J591 minibody clearly binds both
the LNCaP (see FIG. 16A) and the CWR22rv1 (see FIG. 16C) compared
to an equivalent concentration of the negative control minibody.
LNCaP cells are known to have a higher expression of PSMA than the
CWRs which may explain the higher PE signal of the cell population
(see FIG. 16, top row vs. bottom row). As anticipated, the J591
minibody did not significantly bind the PC3 cells (data not
shown).
[0129] Prior to this flow cytometry analysis, J591 minibody protein
was conjugated with the bifunctional chelator
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA) in preparation for downstream radiometal labeling.
Conjugation was performed using the water-soluble
N-hydroxysuccinimide method (Lewis et al 2001). Following DOTA
conjugation, the protein conjugate was dialyzed to change buffer
and remove excess DOTA.
[0130] To verify binding ability after conjugation, the J591-DOTA
minibody was tested for binding to PSMA by flow cytometry. Compared
to the unconjugated J591 minibody, the J591-DOTA minibody exhibited
a slight decrease in immunoreactivity as shown in the slight shift
in the PE signal of the cell population (see FIGS. 16B and 16D).
Excessive conjugation of bifunctional chelators to antibodies has
been known to be a cause for decrease in immunoreactivity (Kukis et
al 1995). Conjugation conditions can be optimized to prevent
excessive conjugation and the resulting loss of binding. However,
the slight shift in binding for the J591-DOTA minibody was
considered acceptable and the protein moved forward into
radiolabeling.
[0131] Internalization of Unlabeled Minibody.
[0132] Internalization of the J591 minibody into PSMA+ cells was
examined using immunofluorescence confocal microscopy. The two
PSMA+ cell lines used in this experiment, the LNCaP and CWR22rv1
cell lines, have been previously used in cell-binding studies and
also served in the subsequent radiolabeled internalization study.
PC3 cells were used as the PSMA-negative control cell line.
Full-length, parental J591 antibody was included in the experiment
as a positive control. A negative control minibody was also
included to further demonstrate the specificity of the J591
minibody uptake in PSMA+ cells.
[0133] Since previous internalization studies with the original
full-length J591 antibody on LNCaP cells showed strong
internalization by 180 minutes (Liu et al 1998), cells were stained
at t=0 and t=180 minutes after primary antibody incubation to
measure internalization. Localization of the antibody and minibody
were detected by a secondary anti-human IgG antibody conjugated
with the Alexa 488 fluorophore. Cells were counterstained with DAPI
for staining of the nucleus.
[0134] The J591 full-length antibody showed very sharp and distinct
staining of the plasma membrane at t=0 (see FIG. 17). After
incubating 180 minutes at 37 C, the J591 full-length antibody
internalized into the LNCaP cells as shown by the dispersion of the
of the Alexa 488 staining throughout the cell. The J591 minibody
also showed distinct plasma membrane staining at t=0 and strong
internalization by t=180 minutes (FIG. 17). Staining of the J591
minibody at t=0 was notably less distinct than the J591
full-length, perhaps suggesting a more rapid internalization for
the smaller-sized minibody within LNCaP cells. The negative control
minibody could not bind the LNCaP cells at t=0. The J591
full-length antibody and minibody could not bind the PSMA-PC3 cells
(data not shown).
[0135] Internalization of the full-length J591 antibody into
CWR22rv1 cells showed very similar staining pattern as seen for the
LNCaP cells. Staining was very sharp and distinct on the plasma
membrane at t=0 and became very dispersed by t=180 minutes (see
FIG. 18). The J591 minibody was also internalized in the CWR22rv1.
Staining at t=0 was distinctively plasma membrane and became much
more dispersed by t=180 minutes (FIG. 18). As expected, the
negative control minibody did not bind the CWR22rv1 cells (FIG.
18).
Example 4
Radiolabeled PSMA-Specific Minbodies
[0136] Radiolabeling J591 Minibody with Iodine-131.
[0137] Purified J591 minibody protein (50 .mu.g) was radiolabeled
with approximately 50 .mu.Ci of .sup.131I using the Iodogen method
from Pierce Thermo Scientific (as described in Olafsen et al 2006).
This reagent enables the chemical oxidation reaction to attach
.sup.131I to available Tyrosine residues of the J591 minibody.
Table 2 is a summary of the J591 minibody radiolabeling results,
including radiolabeling efficiency, percentage of bound
radioactivity after purification, and specific activity. The
radiolabeling efficiency was determined to be approximately 51%
using instant thin layer chromatography (ITLC) to measure the
percentage of radioactivity bound to the protein versus unbound.
(see Table 2 below). The specific activity was determined to be
0.46 .mu.Ci/.mu.g by measuring the total activity of the
radiolabeled protein using a dose calibrator and calculating the
specific activity based on the labeling efficiency (Table 2). To
remove excess unbound .sup.131I, the radiolabeled protein was
further purified using spin columns. The percentage of
radioactivity bound to the J591 minibody following purification was
dramatically increased to approximately 96% following purification
(Table 2).
[0138] DOTA-Conjugating and Radiometal Labeling the J591 with
Indium-111.
[0139] J591 minibody, previously conjugated with the bifunctional
metal chelator DOTA, was radioabeled with .sup.111In. 100 .mu.g of
the DOTA-J591 minibody was incubated with 200 .mu.Ci
.sup.111In-chloride in 0.1M metal-free ammonium acetate (pH 6.0) at
43 C for 50 minutes. The reaction was stopped by the addition of 10
mM DTPA to a final concentration of 1 mM. Radiolabeling efficiency
was determined to be approximately 60% and the specific activity
was 1.1 .mu.Ci/.mu.g (see Table 2). The radiolabeled protein was
further purified to remove excess unbound .sup.111In using spin
columns. Similar to the .sup.131I-J591 minibody, the percentage of
radioactivity bound to the J591 minibody following purification was
dramatically increased to approximately 94% (Table 2).
TABLE-US-00001 TABLE 2 Radiolabeling of the J591 Minibody with
.sup.131I and .sup.111In. Specific Radiolabeling % Bound
Radioactivity Activity Efficiency (%) Post-Purification (uCi/ug)
.sup.131I 51% 96.2% 0.46 .sup.111In-DOTA 60% 94.2% 1.1
[0140] Internalization and Retention of Radiolabeled J591
Minibody.
[0141] The .sup.131I-labeled and .sup.111In-DOTA labeled J591
minibody were tested for uptake and retention of cell-associated
radioactivity in the PSMA+ CWR22rv1 cells. The CWR22rv1 cells were
selected as the sole PSMA+ cell line for these in vitro experiments
since they will be used for the microPET imaging experiment.
Drawing from the literature and the experimental knowledge of
colleagues, the CWR22rv1 xenograft model has a higher tumor take
rate and faster in vitro and tumor growth rates than the LNCaP
model.
[0142] For the uptake and retention of the .sup.131I-labeled J591
minibody, the amount of radioactivity associated with the membrane
rapidly drops within the first 30 minutes whereas the internalized
radioactivity rapidly increases in this timeframe (see FIG. 19A).
Together these data suggests the internalization of the
.sup.131I-J591 minibody. Although the amount of internalized
.sup.131I J591 increases over time, the total cell-associated
radioactivity decreased substantially by 180 minutes relative to
the initial starting point of .about.2900 cpm (see FIG. 19A).
[0143] In sharp contrast, the uptake and retention of the
.sup.111In-DOTA labelled J591 minibody showed a relatively large
increase in total cell-associated radioactivity over time (see FIG.
19B). Similar to the .sup.131I labelled J591 minibody, the
membrane-associated radioactivity dramatically decreases as the
internalized radioactivity increases suggesting active
internalization (see FIG. 19B). Attributed in large part to the
increase in internalized radioactivity over time, the total
cell-associated radioactivity increased to approximately 20,000 cpm
by 180 minutes from a starting point of .about.7,500 cpm (FIG.
19B).
[0144] To compare the two radiolabeled J591 minibodies, the total
cell-associated radioactivity was normalized by expressing the data
in terms of percentage of the initial cell-associated radioactivity
for each respective radiolabel at t=0 (see FIG. 20). By t=180
minutes, the .sup.111In-DOTA labeled J591 minibody increases to
-250% of the initial cell-associated radioactivity whereas the
.sup.131I labeled J591 minibody decreases to .about.80% of the
initial (FIG. 20). As seen by other groups in the literature
(Vaidyanathan et al 2009), a non-residualizing .sup.131I labeling
strategy resulted in an overall decrease in cell-associated
radioactivity over time. These data clearly shows the retention and
accumulation of cell-associated radioactivity over time for the
residualizing .sup.111In-DOTA radiolabel.
[0145] Purified J591 HC VHVL minibody (or any of the minibodies
described above) may be used to demonstrate the ability to target
human PSMA in vivo in microPET imaging and biodistribution studies.
In one embodiment, the purified J591 HC VHVL minibody protein may
first be validated again to confirm its ability to bind PSMA in
vitro in preparation for the imaging studies. Upon confirmation of
binding, the J591 HC VHVL minibody may then be conjugated to the
bifunctional chelator DOTA and radiolabeled with an appropriate
positron-emitting radiometal for microPET such as Copper 64.
Radiolabeled minibody can be analyzed to ensure high radiolabeling
efficiency and immunoreactivity before proceeding to micoPET
imaging.
[0146] In some embodiments, the radiolabeled minibody can be
injected intravenously into xenograft mice implanted with either
PSMA positive or PSMA negative tumors. At specific time points
post-injection, each animal may be serially scanned by PET. After
the final scan, animals may be scanned by CT for anatomical
reference. The PET and CT images for each animal may then be
analyzed to evaluate tumor targeting and specificity.
Example 5
In Vivo Binding and Biodistribution of .sup.1241-J591 and
.sup.64Cu-DOTA-Conjugated J591 Minibodies
[0147] Radiolabeling J591 Minibody with Iodine-124.
[0148] Purified J591 minibody protein (total amount of 300 .mu.g)
was radiolabeled with approximately 1.3 mCi of .sup.124I using the
Iodogen method from Pierce Thermo Scientific (as described in
Olafsen et al 2006). This method involves a chemical oxidation
reaction to attach .sup.124I radioisotope to available Tyrosine
residues of the J591 minibody. Table 3, below, is a summary of the
J591 minibody radiolabeling results including radiolabeling
efficiency, percentage of bound radioactivity after purification,
specific activity, and immunoreactivity. Following the labeling
reaction, the radiolabeling efficiency was determined to be
approximately 62% (percentage of radioactivity bound to the protein
versus unbound) using instant thin layer chromatography (ITLC) (see
Table 3). Radiolabeled J591 minibody was partially purified using
Sephadex G-25 spin columns and re-evaluated by ITLC to determine
the percentage of bound radioactivity. The specific activity of the
radiolabelled protein was 2.6 .mu.Ci/.mu.g (Table 3), as determined
by measuring the total radioactivity of the protein using a dose
calibrator. To remove excess unbound .sup.124I from the reaction,
the radiolabeled protein was further purified using spin columns.
The percentage of radioactivity bound to the J591 minibody
following purification was dramatically increased to approximately
98% (Table 3). Immunoreactivity of the .sup.124I-J591 minibody was
determined to be 48% by testing binding to CWR22rv1 vs PC3 cells
(Table 3). Although this immunoreactivity was lower than
anticipated, the decision was made to move the .sup.124I J591
minibody forward into the imaging and biodistribution experiment
based on the previous binding performance of the minibody. Future
optimizations to the radiolabeling conditions (pH, time,
temperature, etc) and obtaining higher protein purity could
potentially improve the immunoreactivity.
TABLE-US-00002 TABLE 3 Radiolabeling of the J591 Minibody with
.sup.124I and .sup.64Cu. Radio- % Bound labeling Radioactivity
Specific Immuno- Labelling Efficiency Post- Activity reactivity
Conditions (%) Purification (uCi/ug) (%) .sup.124I Protein in PBS
62% 98.0% 2.6 48% .sup.64Cu- Protein in PBS 40% 85%* 1* 29%* DOTA
.sup.64Cu- AmmOAc, 92% 85%* 1* 29%* DOTA increase AmmCitr buffer
*Fractions from both labelling conditions were combined
[0149] Radiometal Labeling the DOTA-J591 Minibody with
Copper-64.
[0150] J591 minibody, previously conjugated with the bifunctional
metal chelator DOTA, was radiolabeled with .sup.64Cu. For the
initial radiolabeling condition, 400 .mu.g of the DOTA-J591
minibody in PBS was incubated with approximately 745 .mu.Ci
.sup.64CuCl.sub.2 in 25 mM metal-free ammonium citrate [pH 5.2] at
43 C for 60 minutes. The reaction was stopped by the addition of 10
mM EDTA to a final concentration of 1 mM. Using these labeling
conditions, radiolabeling efficiency was determined to be lower
than anticipated at approximately 40% (see Table 3).
[0151] In an attempt to improve labeling efficiency, the DOTA-J591
minibody was first dialyzed into 0.25 ammonium acetate buffer [pH
7.2] before starting the radiolabeling reaction. An additional 560
.mu.g of the DOTA-J591 minibody, in the ammonium acetate buffer,
was labeled with approximately 730 uCi of .sup.64CuCl.sub.2.
Another adjustment to improve the radiolabeling involved increasing
the percentage of ammonium citrate buffer used in the reaction.
With these adjustments, the radiolabeling efficiency was
dramatically increased to approximately 92% (Table 3).
[0152] All of the .sup.64Cu-DOTA J591 minibody fractions from both
labeling conditions were pooled together and further purified to
remove excess unbound .sup.64Cu using spin columns. The percentage
of radioactivity attached to the J591 minibody following
purification was approximately 85%, and the specific activity was 1
.mu.Ci/.mu.g (Table 3). Immunoreactivity of the radiolabeled
minibody was determined to be approximately 29% (Table 3) using the
cell-based method described previously for .sup.124I J591 minibody.
Although the immunoreactivity was lower than expected, the decision
was made to move forward into the microPET and biodistribution
experiment. In addition to the protein purity and the labeling
conditions, future efforts to optimize immunoreactivity could
include optimizing the DOTA conjugation reaction (i.e.
DOTA-molecule ratio, etc).
Example 6
Serial microPET Imaging and Biodistribution of Radiolabeled J591
Minibodies
[0153] .sup.64Cu-DOTA J591 Minibody.
[0154] To evaluate the tumor targeting and binding specificity of
the .sup.64Cu-DOTA-J591 minibody, microPET imaging and
biodistribution analysis was performed using mice implanted with
both CWR22rv1 (PSMA+) and PC3 (PSMA-) xenografts. Both xenograft
tumors were grown to a size between 39-223 mg before starting the
imaging experiment. The CT and PET/CT images, at 4 hours
post-injection, showed rapid tumor localization at the CWR22rv1
tumor compared to the PC3 tumor (FIGS. 21A and 21B show a
representative mouse). As expected for a radiometal labeled
minibody, prominent activity was detected in thorax and
particularly localized to the liver. Localization of the
radiometals such as .sup.64Cu has been well-studied in the
literature (Yazaki et al 2001). With the exception of the liver,
background activity was relatively low even at 4 hours
post-injection allowing for PET/CT images with remarkable contrast
(FIGS. 21B and 21C). Strong tumor localization persisted at 19
hours and even 43 hours post-injection (FIG. 21D). The overall
background activity decreased slightly over time but the liver
remained a strong source of activity (FIG. 21D).
[0155] Following the final scan, all animals (n=8 at 19 hours and
n=4 at 43 hours p.i.) were euthanized and selected tissues of
interest (including the positive and negative tumors, blood, liver,
spleen, lungs, and kidneys) were excised, weighed, and measured by
a gamma counter for radioactivity. The biodistribution at 19 hours
post-injection in FIG. 22 showed that CWR22rv1 tumor (Tumor+)
reached an average uptake of 8.23% ID/g compared to 2.69% ID/g for
the PC3 tumor (Tumor-). Localization was significantly higher at
the CWR22rv1 than PC3 tumor (p<0.05). As revealed by the
microPET/CT imaging, uptake in the liver at 19 hours p.i. was
relatively high (21.43% ID/g) whereas the localization was much
less prominent in the other tissues of interest (see FIG. 22).
[0156] At 43 hours postinjection, the biodistribution reveals an
increase in the average uptake at the CWR22rv1 tumor (Tumor+;
13.25% ID/g) compared to 19 hours postinjection (FIG. 22).
Background activity decreased relative to 19 hours post-injection,
particularly the significant decrease in liver activity to 14.37%
ID/g (FIG. 22).
[0157] With the overall decrease in background activity combined
with the increasing accumulation at the CWR22rv1 tumor, the tumor
to background ratios increased dramatically between 19 hours
postinjection to 43 hours postinjection (FIG. 26).
[0158] .sup.124I J591 Minibody.
[0159] As with the .sup.64Cu-DOTA J591 minibody, microPET and
biodistribution experiments were performed with the .sup.124I J591
minibody to evaluate tumor targeting. Both xenograft tumors were
grown to a range in size between 36-192 mg before starting the
imaging experiment. MicroPET images at 4 hours postinjection (p.i.)
showed rapid localization at the CWR22rv1 tumor but high
circulating activity in the thorax, abdomen, and bladder (FIGS. 23A
and 23B). Background activity cleared significantly from the system
by 20 hours postinjection and was almost completely absent by 44
hours while the activity at the positive tumor remained (FIG.
23C).
[0160] For biodistribution analysis, all animals in a group were
euthanized after the final scan (n=6 at 20 hours and n=2 at 44
hours p.i.) and the selected tissues of interest were excised,
weighed, and measured by a gamma counter for radioactivity. The
biodistribution at 20 hours post-injection for the mouse in FIG. 24
showed that CWR22rv1 tumor (Tumor+) uptake reached 8.75% ID/g
compared to 1.8% ID/g for the PC3 tumor (Tumor-). Localization was
significantly higher at the CWR22rv1 than PC3 tumor (p<0.05).
Background activity was relatively low by 20 hours p.i. (FIG.
24).
[0161] By 44 hours post-injection, the CWR22rv1 tumor (Tumor+)
uptake decreased substantially to 3.25% ID/g (FIG. 24). Supporting
the previous results from the in vitro internalization and
retention experiments described above, cell-associated
radioactivity decreased over time from dehalogenation and/or
metabolism of the .sup.124I-J591 minibody. Background activity was
almost entirely cleared from the system by 44 hours p.i. (FIG.
24).
[0162] Although the uptake of activity decreased at the CWR22rv1
tumor over time (FIG. 24), the rapid decrease in background
activity allowed a strong contrast for the images. The
biodistribution ratios reflect this large increase in tumor to
background over time (FIG. 27).
[0163] Upon successful imaging of PSMA positive tumors by the J591
minibody, the biodistribution of the minibody may be investigated
according to embodiments of the disclosure. These biodistribution
studies can investigate the localization of the minibody at the
tumor site versus other selected tissues over time following
injection. These studies may be used to demonstrate high tumor to
background ratios. Use of a J591 minibody would likely produce a
high tumor to background ratio when imaging a tumor that
overexpresses PSMA, such as in prostate cancer. Positive results
from these imaging and biodistribution experiments may lead to
toxicology experiments in preparation for clinical studies.
[0164] Further, the ability of a J591 minibody to target human PSMA
in vivo by PET imaging studies may be demonstrated through clinical
trials in cancer patients. In one embodiment, the clinical trials
may be performed in prostate cancer patients. These clinical trials
in cancer patients may be performed using similar methods as
described above. Briefly, radiolabeled minibody can be injected
intravenously into cancer patients having a form of cancer that is
known to overexpress PSMA. At specific time points post-injection,
each patient may be serially scanned by PET. After the final scan,
patients may be scanned by CT for anatomical reference. The PET and
CT images for each patient may then be analyzed to evaluate tumor
targeting and specificity.
REFERENCES
[0165] The references, patents and published patent applications
listed below, and all references cited in the specification above
are hereby incorporated by reference in their entirety, as if fully
set forth herein. [0166] Bander N H, Trabulsi E J, Kostakoglu L,
Yao D, Vallabhajosula S, Smith-Jones P, Joyce M A, Milowsky M,
Nanus D M, Goldsmith S J. Targeting metastatic prostate cancer with
radiolabeled monoclonal antibody J591 to the extracellular domain
of prostate specific membrane antigen. J Urol, 2003. 170 (5):
1717-1721. [0167] Bander N H, Milowsky M I, Nanus D M, Kostakoglu
L, Vallabhajosula S, Goldsmith S J. Phase I trial of 177
Lutetium-labeled J591, a monoclonal antibody to prostate-specific
membrane antigen, in patients with androgen-independent prostate
cancer. J Clin Oncol, 2005. 23(21): 4591-601. [0168] Hu S, Shively
L, Wu A M. Minibody: A novel engineered anti-carcinoembryonic
antigen antibody fragment (single-chain Fv-CH3) which exhibits
rapid, high-level targeting of xenografts. Cancer Res, 1996.
56(13):3055-61. [0169] Kukis D L, Denardo G L, Denardo S J, Mirick
G R, Miers L A, Greiner D P, Meares C F. Effect of the Extent of
Chelate Substitution on the Immunoreactivity and Biodistribution of
21T-BAT-Lym-1 Immunoconjugates, 1995. 55, 878-884. [0170] Lewis M
R, Kao J Y, Anderson A L, Shively J E, Raubitscheck A. An improved
method for conjugating monoclonal antibodies with
N-hydroxysulfosuccinimidyl DOTA. Bioconjug Chem, 2001. 12: 320-324.
[0171] Leyton J V, Wu A M. Humanized radioiodinated minibody for
imaging of prostate stem cell antigen-expressing tumors. Clin
Cancer Res, 2008. 14(22):7488-96. [0172] Liu H, Rajasekaran A K,
Moy P, Xia Y, Kim S, Navarro V, Rahmati R, Bander N H. Constitutive
and Antibody-induced Internalization of Prostate-specific Membrane
Antigen. Cancer Res, 1998. 58: 4055-4060. [0173] Liu H, Moy P, Kim
S, Xia Y, Rajasekaran A, Navarro V, Knudsen B, Bander N H.
Monoclonal antibodies to the extracellular domain of prostate
specific membrane antigen also react with tumor vasculature
endothelium. Cancer Res, 1997. 57(17): 3629-34. [0174] Milowsky M
I, Nanus D M, Kostakoglu L, Vallabhajosula S, Goldsmith S J, Bander
N H. Phase I trial of Yttrium-90-labeled anti-prostate specific
membrane antigen monoclonal antibody J591 for androgen-independent
prostate cancer. J Clin Oncol, 2004. 22(13): 2522-2531. [0175]
Milowsky M I, Nanus D M, Kostakoglu L, Sheehan C E, Vallabhajosula
S, Goldsmith S J, Ross J S, Bander N H. Vascular targeted therapy
with anti-prostate-specific membrane antigen monoclonal antibody
J591 in advanced solid tumors. J Clin Oncol, 2007. 25(5): 540-547.
[0176] Morris M J, Divgi C R, Pandit-Taskar N, et al. Pilot trial
of unlabeled and indium-1,1-labeled anti-prostate-specific membrane
antigen antibody J591 for castrate metastatic prostate cancer. Clin
Cancer Res, 2005. 11 (20): 7454-7461. [0177] Olafsen T, Tan G J,
Cheung C W, Yazaki P J, Park J M, Shively J E, Williams L E,
Raubitschek A A, Press M F, Wu A M. Characterization of engineered
anti-p185HER-2 (scFv-CH3).sub.2 antibody fragments (minibodies) for
tumor targeting. Protein Eng Des Sel, 2004. 17(4):315-23. [0178]
Olafsen T, Kenanova V E, Wu A M. Tunable pharmacokinetics:
modifying the in vivo half-life of antibodies by directed
mutagenesis of the Fc fragment. Nat Protoc, 2006. 1:2048-60. [0179]
Olafsen T, Betting D, Kenanova V E, Salazar F B, Clarke P, Said J,
Raubitschek A A, Timmerman J M, Wu A M. Recombinant Anti-CD20
Antibody Fragments for Small-Animal PET Imaging of B-Cell
Lymphomas. J Nuc Med, 2009. 50(9):1500-1508. [0180] Olson W C,
Heston W D W, Rajasekaran A K. Clinical Trials of Cancer Therapies
Targeting Prostate Specific Membrane Antigen. Reviews on Recent
Clinical Trials, 2007. 2: 182-190. [0181] Slovin S F. Targeting
novel antigens for prostate cancer treatment: focus on
prostate-specific membrane antigen. Expert Opin Ther Targets, 2005.
9(3): 561-570. [0182] Vaidynathan G, Jestin E, Olafsen T, Wu A M,
Zalutsky M R. Evaluation of an anti-p185(HER2)(scFv-C(H)2-C(H)3)2
fragment following radioiodination using two different
residualizing labels: SGMIB and IB-Mal-D-GEEEK. Nucl Med Biol,
2009. 36(6): 671-80. [0183] Wong J Y, Chu D Z, Williams L E,
Yamauchi D M, Ikle D N, Kwok C S, Liu A, Wilczynski S, Colcher D,
Yazaki P J, Shively J E, Wu A M, Raubitschek A A. Pilot trial
evaluating an 1231-labeled 80-kilodalton engineered
anticarcinoembryonic antigen antibody fragment (cT84.66 minibody)
in patients with colorectal cancer. Clin Cancer Res, 2004.
10(15):5014-21. [0184] Wu A M and Senter P D. Arming antibodies:
prospects and challenges for immunoconjugates. Nat Biotechnol,
2005. 23(9):1137-46. [0185] Wu A M and Olafsen T. Antibodies for
molecular imaging of cancer. Cancer J, 2008. 14(3):191-7. [0186] Wu
A M. Antibodies and Antimatter: The resurgence of ImmunoPET. J Nucl
Med, 2009. 50(1):2-5. [0187] Yazaki P J, Wu A M, Tsai S W, Williams
L E, Ikle D N, Wong J Y C, Shively J E, and Raubitschek A A. Tumor
targeting of radiometal labeled anti-CEA recombinant T84.66 diabody
and T84.66 minibody: Comparison to radioiodinated fragments.
Bioconj Chem, 2001. 12, 220-228.
PATENTS AND PUBLISHED PATENT APPLICATIONS
[0187] [0188] Wu, Anna. Antibody Construct. U.S. Pat. No.
5,837,821, filed Jun. 24, 1994, and issued Nov. 17, 1998. [0189]
Bander, Neil. Treatment and diagnosis of cancer. U.S. Pat. No.
6,649,163, filed Jul. 20, 1999, and issued Nov. 18, 2003. [0190]
Bander, Neil. Treatment and diagnosis of cancer. U.S. Pat. No.
6,770,450, filed Jul. 20, 1999, and issued Aug. 3, 2004. [0191]
Bander, Neil. Carr, Francis. Hamilton, Anita. Modified antibodies
to prostate-specific membrane antigen and uses thereof. U.S. Pat.
No. 7,045,605, filed May 30, 2002, and issued May 16, 2006. [0192]
Bander, Neil. Treatment and diagnosis of prostate cancer. U.S. Pat.
No. 7,112,412, filed Jul. 20, 1999, and issued Sep. 26, 2006.
[0193] Bander, Neil. Treatment and diagnosis of cancer. U.S. Pat.
No. 7,163,680, filed Aug. 13, 2001, and issued Jan. 16, 2007.
[0194] Bander, Neil. Treatment and diagnosis of cancer. U.S. Pat.
No. 6,136,311, filed Jul. 17, 1997, and issued Oct. 24, 2000.
[0195] Bander, Neil. Treatment and diagnosis of prostate cancer
with antibodies to extracellular PSMA. U.S. Pat. No. 6,107,090,
filed Apr. 9, 1997, and issued Aug. 22, 2000. [0196] Bander, Neil.
Carr, Francis. Hamilton, Anita. Methods of treating prostate cancer
with anti-prostate specific membrane antigen antibodies. U.S. Pat.
No. 7,514,078, filed May 30, 2003, and issued Apr. 7, 2009.
Sequence CWU 1
1
1911176DNAArtificial SequenceJ591 Human Composite VHVL Minibody
nucleotide sequence 1atggaaaccg acaccctgct gctgtgggtg ctgctgctct
gggtcccagg ctccaccggt 60gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
ctggcgcctc cgtgaagatc 120tcctgcaaga cctccggcta caccttcacc
gagtacacca tccactgggt gaaacaggcc 180tccggcaagg gcctggaatg
gatcggcaac atcaacccta acaacggcgg caccacctac 240aaccagaagt
tcgaggaccg ggccaccctg accgtggaca agtccacctc caccgcctac
300atggaactgt cctccctgcg gtctgaggac accgccgtgt actactgcgc
cgctggctgg 360aacttcgact actggggcca gggcaccaca gtgacagtct
cgagcggctc tacctctggc 420ggaggctctg ggggaggaag cggcggaggc
ggctcctctg acatcgtgat gacccagtcc 480ccctcctccc tgtctgcctc
cgtgggcgac agagtgacca tcacatgcaa ggcctcccag 540gatgtgggca
ccgccgtgga ctggtatcag cagaagcctg gcaaggcccc taagctgctg
600atctactggg cctccaccag acacaccggc gtgcctgaca gattcaccgg
ctccggctct 660ggcaccgact tcaccctgac catctccagc ctgcagcctg
aggacttcgc cgactacttc 720tgccagcagt acaactccta ccctctgacc
ttcggcggag gcaccaagct ggaaatcaaa 780gagcccaagt cctgcgacaa
gacccacacc tgtccccctt gtggcggcgg atctagtggc 840ggaggatccg
gtggccagcc tcgggagcct caggtgtaca ccctgcctcc ctcccgggac
900gagctgacca agaaccaggt gtccctgacc tgtctggtca agggcttcta
cccttccgat 960atcgccgtgg agtgggagtc caacggccag cctgagaaca
actacaagac cacccctcct 1020gtgctggact ccgacggctc cttcttcctg
tactccaagc tgacagtgga taagtcccgg 1080tggcagcagg gcaacgtgtt
ctcctgttcc gtgatgcacg aggccctgca caaccactat 1140acccagaagt
ccctgtccct gtctcctggc aagtga 117621176DNAArtificial SequenceJ591 2P
VHVL Minibody nucleotide sequence 2atggaaaccg acaccctgct gctgtgggtg
ctgctgctct gggtcccagg ctccaccggt 60gaagtgcagc tggtgcagtc cggccctgaa
gtgaagaagc ctggcgccac cgtcaagatc 120tcttgcaaga cctccggcta
caccttcacc gagtacacca tccactgggt gaaacaggcc 180cctggcaagg
gtctggaatg gatcggcaac atcaacccta acaacggcgg caccacctat
240aaccagaagt tcgaggacaa ggccaccctg accgtggaca agtccaccga
caccgcctac 300atggaactgt cctccctccg gtccgaggac accgcagtgt
attactgcgc cgctggctgg 360aacttcgact actggggcca gggcaccctg
ctgacagtct cgagcggctc cacaagtggc 420ggaggctctg gcggtggatc
tggcggaggc ggctcatccg acatccagat gacccagtcc 480ccctcctccc
tgtccacctc cgtgggcgac agagtgaccc tgacatgcaa ggcctcccag
540gacgtgggca ccgccgtgga ctggtatcag cagaagccag gccagtcccc
taagctgctg 600atctactggg cctccacccg gcacaccggc atcccttccc
ggttctccgg cagtggctct 660ggcaccgact tcaccctgac catctccagc
ctgcagcctg aggacttcgc cgactactac 720tgccagcagt acaactccta
ccctctgacc ttcggcgccg gcacaaaggt ggacatcaaa 780gagcctaagt
cctgcgacaa gacccacaca tgtccccctt gcggcggagg aagcagcgga
840ggcggatccg gtggccagcc tcgggagcct caggtgtaca ccctgcctcc
ctcccgggac 900gagctgacca agaaccaggt gtccctgacc tgtctggtca
agggcttcta cccttccgat 960atcgccgtgg agtgggagtc caacggccag
cctgagaaca actacaagac cacccctcct 1020gtgctggact ccgacggctc
cttcttcctg tactccaagc tgacagtgga taagtcccgg 1080tggcagcagg
gcaacgtgtt ctcctgttcc gtgatgcacg aggccctgca caaccactat
1140acccagaagt ccctgtccct gtctcctggc aagtga 11763115PRTArtificial
Sequenceamino acid sequences of Human Composite J591 V-regions (VH)
3Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu
Tyr 20 25 30 Thr Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr
Tyr Asn Gln Lys Phe 50 55 60 Glu Asp Arg Ala Thr Leu Thr Val Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Asn
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser
115 4115PRTMus musculus 4Glu Val Gln Leu Gln Gln Ser Gly Pro Glu
Leu Lys Lys Pro Gly Thr 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Thr
Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Ile His Trp Val Lys
Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Ile Asn
Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Glu Asp
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Arg Ser Leu Thr Trp Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
Thr 100 105 110 Val Ser Ser 115 5115PRTArtificial Sequenceamino
acid sequences of deimmunized J591 V-regions (VH) 5Glu Val Gly Leu
Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val
Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30
Thr Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys
Phe 50 55 60 Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Asp
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Asn Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Leu Thr 100 105 110 Val Ser Ser 115
6750DNAArtificial SequenceJ591 cys-diabody (CysDB) VH-5-VL
nucleotide sequence 6atggaaaccg acaccctgct gctgtgggtg ctgctcctgt
gggtgcccgg atctaccggt 60gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
ctggcgcctc cgtgaagatc 120tcctgcaaga cctccggcta caccttcacc
gagtacacca tccactgggt gaaacaggcc 180tccggcaagg gcctggaatg
gatcggcaac atcaacccta acaacggcgg caccacctac 240aaccagaagt
tcgaggaccg ggccaccctg accgtggaca agtccacctc caccgcctac
300atggaactgt cctccctgcg gtctgaggac accgccgtgt actactgcgc
cgctggctgg 360aacttcgact actggggcca gggcaccacc gtgacagtct
cgagctccgg tgggggcggc 420gatatcgtga tgacccagtc cccttcctcc
ctgtctgcct ccgtgggcga cagagtgacc 480atcacatgca aggcctccca
ggatgtgggc accgccgtgg actggtatca gcagaagcct 540ggcaaggccc
ctaagctgct gatctactgg gcctccacca gacacaccgg cgtgcctgac
600agattcaccg gctccggctc tggcaccgac ttcaccctga ccatctccag
cctgcagcct 660gaggacttcg ccgactactt ctgccagcag tacaactcct
accctctgac cttcggcgga 720ggcaccaagc tggaaatcaa gggcggttgc
7507759DNAArtificial SequenceJ591 cys-diabody (CysDB) VH-8-VL
nucleotide sequence 7atggaaaccg acaccctgct gctgtgggtg ctgctcctgt
gggtgcccgg atctaccggt 60gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
ctggcgcctc cgtgaagatc 120tcctgcaaga cctccggcta caccttcacc
gagtacacca tccactgggt gaaacaggcc 180tccggcaagg gcctggaatg
gatcggcaac atcaacccta acaacggcgg caccacctac 240aaccagaagt
tcgaggaccg ggccaccctg accgtggaca agtccacctc caccgcctac
300atggaactgt cctccctgcg gtctgaggac accgccgtgt actactgcgc
cgctggctgg 360aacttcgact actggggcca gggcaccacc gtgacagtct
cgagcggcgg agggagtggc 420ggaggcggcg atatcgtgat gacccagtcc
ccttcctccc tgtctgcctc cgtgggcgac 480agagtgacca tcacatgcaa
ggcctcccag gatgtgggca ccgccgtgga ctggtatcag 540cagaagcctg
gcaaggcccc taagctgctg atctactggg cctccaccag acacaccggc
600gtgcctgaca gattcaccgg ctccggctct ggcaccgact tcaccctgac
catctccagc 660ctgcagcctg aggacttcgc cgactacttc tgccagcagt
acaactccta ccctctgacc 720ttcggcggag gcaccaagct ggaaatcaag ggcggttgc
7598750DNAArtificial SequenceJ591 cys-diabody (CysDB) VL-5-VH
nucleotide sequence 8atggaaaccg acaccctgct gctgtgggtg ctgctcctgt
gggtgcccgg atctaccggt 60gatatcgtga tgacccagtc cccttcctcc ctgtctgcct
ccgtgggcga cagagtgacc 120atcacatgca aggcctccca ggatgtgggc
accgccgtgg actggtatca gcagaagcct 180ggcaaggccc ctaagctgct
gatctactgg gcctccacca gacacaccgg cgtgcctgac 240agattcaccg
gctccggctc tggcaccgac ttcaccctga ccatctccag cctgcagcct
300gaggacttcg ccgactactt ctgccagcag tacaactcct accctctgac
cttcggcgga 360ggcaccaagc tggaaatcaa gtccggtggg ggcggcgaag
tgcagctggt gcagtctggc 420gccgaagtga agaaacctgg cgcctccgtg
aagatctcct gcaagacctc cggctacacc 480ttcaccgagt acaccatcca
ctgggtgaaa caggcctccg gcaagggcct ggaatggatc 540ggcaacatca
accctaacaa cggcggcacc acctacaacc agaagttcga ggaccgggcc
600accctgaccg tggacaagtc cacctccacc gcctacatgg aactgtcctc
cctgcggtct 660gaggacaccg ccgtgtacta ctgcgccgct ggctggaact
tcgactactg gggccagggc 720accaccgtga cagtctcgag cggcggttgc
7509759DNAArtificial SequenceJ591 cys-diabody (CysDB) VL-8-VH
nucleotide sequence 9atggaaaccg acaccctgct gctgtgggtg ctgctcctgt
gggtgcccgg atctaccggt 60gatatcgtga tgacccagtc cccttcctcc ctgtctgcct
ccgtgggcga cagagtgacc 120atcacatgca aggcctccca ggatgtgggc
accgccgtgg actggtatca gcagaagcct 180ggcaaggccc ctaagctgct
gatctactgg gcctccacca gacacaccgg cgtgcctgac 240agattcaccg
gctccggctc tggcaccgac ttcaccctga ccatctccag cctgcagcct
300gaggacttcg ccgactactt ctgccagcag tacaactcct accctctgac
cttcggcgga 360ggcaccaagc tggaaatcaa gggcggaggg agtggcggag
gcggcgaagt gcagctggtg 420cagtctggcg ccgaagtgaa gaaacctggc
gcctccgtga agatctcctg caagacctcc 480ggctacacct tcaccgagta
caccatccac tgggtgaaac aggcctccgg caagggcctg 540gaatggatcg
gcaacatcaa ccctaacaac ggcggcacca cctacaacca gaagttcgag
600gaccgggcca ccctgaccgt ggacaagtcc acctccaccg cctacatgga
actgtcctcc 660ctgcggtctg aggacaccgc cgtgtactac tgcgccgctg
gctggaactt cgactactgg 720ggccagggca ccaccgtgac agtctcgagc ggcggttgc
75910391PRTArtificial SequenceJ591 Human Composite VHVL Minibody
amino acid sequence 10Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Ser Val Lys
Ile Ser Cys Lys Thr Ser Gly Tyr Thr 35 40 45 Phe Thr Glu Tyr Thr
Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly 50 55 60 Leu Glu Trp
Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr 65 70 75 80 Asn
Gln Lys Phe Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr 85 90
95 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
100 105 110 Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly
Gln Gly 115 120 125 Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly
Gly Gly Ser Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp
Ile Val Met Thr Gln Ser 145 150 155 160 Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys 165 170 175 Lys Ala Ser Gln Asp
Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys 180 185 190 Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His 195 200 205 Thr
Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe 210 215
220 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Phe
225 230 235 240 Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gly
Gly Thr Lys 245 250 255 Leu Glu Ile Lys Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro 260 265 270 Pro Cys Gly Gly Gly Ser Ser Gly Gly
Gly Ser Gly Gly Gln Pro Arg 275 280 285 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys 290 295 300 Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 305 310 315 320 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 325 330 335
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 340
345 350 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser 355 360 365 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 370 375 380 Leu Ser Leu Ser Pro Gly Lys 385 390
11391PRTArtificial SequenceJ591 2P VHVL Minibody amino acid
sequence 11Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser Gly
Pro Glu Val Lys 20 25 30 Lys Pro Gly Ala Thr Val Lys Ile Ser Cys
Lys Thr Ser Gly Tyr Thr 35 40 45 Phe Thr Glu Tyr Thr Ile His Trp
Val Lys Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Ile Gly Asn
Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr 65 70 75 80 Asn Gln Lys Phe
Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr 85 90 95 Asp Thr
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 100 105 110
Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly 115
120 125 Thr Leu Leu Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly Ser
Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Ile Gln Met
Thr Gln Ser 145 150 155 160 Pro Ser Ser Leu Ser Thr Ser Val Gly Asp
Arg Val Thr Leu Thr Cys 165 170 175 Lys Ala Ser Gln Asp Val Gly Thr
Ala Val Asp Trp Tyr Gln Gln Lys 180 185 190 Pro Gly Gln Ser Pro Lys
Leu Leu Ile Tyr Trp Ala Ser Thr Arg His 195 200 205 Thr Gly Ile Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 210 215 220 Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr 225 230 235
240 Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys
245 250 255 Val Asp Ile Lys Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro 260 265 270 Pro Cys Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly
Gly Gln Pro Arg 275 280 285 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys 290 295 300 Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp 305 310 315 320 Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 325 330 335 Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 340 345 350 Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 355 360
365 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
370 375 380 Leu Ser Leu Ser Pro Gly Lys 385 390 12250PRTArtificial
SequenceJ591 cys-diabody (CysDB) VH-5-VL amino acid sequence 12Met
Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10
15 Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
20 25 30 Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly
Tyr Thr 35 40 45 Phe Thr Glu Tyr Thr Ile His Trp Val Lys Gln Ala
Ser Gly Lys Gly 50 55 60 Leu Glu Trp Ile Gly Asn Ile Asn Pro Asn
Asn Gly Gly Thr Thr Tyr 65 70 75 80 Asn Gln Lys Phe Glu Asp Arg Ala
Thr Leu Thr Val Asp Lys Ser Thr 85 90 95 Ser Thr Ala Tyr Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys
Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly 115 120 125 Thr Thr
Val Thr Val Ser Ser Ser Gly Gly Gly Gly Asp Ile Val Met 130 135 140
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 145
150 155 160 Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala Val Asp
Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr Trp Ala Ser 180 185 190 Thr Arg His Thr Gly Val Pro Asp Arg Phe
Thr Gly Ser Gly Ser Gly 195 200
205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
210 215 220 Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe
Gly Gly 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Gly Gly Cys 245
250 13253PRTArtificial SequenceJ591 cys-diabody (CysDB) VH-8-VL
amino acid sequence 13Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Ser Val Lys
Ile Ser Cys Lys Thr Ser Gly Tyr Thr 35 40 45 Phe Thr Glu Tyr Thr
Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly 50 55 60 Leu Glu Trp
Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr 65 70 75 80 Asn
Gln Lys Phe Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr 85 90
95 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
100 105 110 Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly
Gln Gly 115 120 125 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Ser Gly
Gly Gly Gly Asp 130 135 140 Ile Val Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly Asp 145 150 155 160 Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Val Gly Thr Ala Val 165 170 175 Asp Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 180 185 190 Trp Ala Ser
Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly Ser 195 200 205 Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 210 215
220 Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr
225 230 235 240 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Cys
245 250 14250PRTArtificial SequenceJ591 cys-diabody (CysDB) VL-5-VH
amino acid sequence 14Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Met Thr
Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Val Gly Thr Ala Val
Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu
Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 65 70 75 80 Arg
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90
95 Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn
100 105 110 Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Ser 115 120 125 Gly Gly Gly Gly Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys 130 135 140 Lys Pro Gly Ala Ser Val Lys Ile Ser Cys
Lys Thr Ser Gly Tyr Thr 145 150 155 160 Phe Thr Glu Tyr Thr Ile His
Trp Val Lys Gln Ala Ser Gly Lys Gly 165 170 175 Leu Glu Trp Ile Gly
Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr 180 185 190 Asn Gln Lys
Phe Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr 195 200 205 Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 210 215
220 Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240 Thr Thr Val Thr Val Ser Ser Gly Gly Cys 245 250
15253PRTArtificial SequenceJ591 cys-diabody (CysDB) VL-8-VH amino
acid sequence 15Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu
Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Met Thr Gln Ser
Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp 35 40 45 Val Gly Thr Ala Val Asp Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 65 70 75 80 Arg Phe Thr
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser
Leu Gln Pro Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn 100 105
110 Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
115 120 125 Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Gln Ser
Gly Ala 130 135 140 Glu Val Lys Lys Pro Gly Ala Ser Val Lys Ile Ser
Cys Lys Thr Ser 145 150 155 160 Gly Tyr Thr Phe Thr Glu Tyr Thr Ile
His Trp Val Lys Gln Ala Ser 165 170 175 Gly Lys Gly Leu Glu Trp Ile
Gly Asn Ile Asn Pro Asn Asn Gly Gly 180 185 190 Thr Thr Tyr Asn Gln
Lys Phe Glu Asp Arg Ala Thr Leu Thr Val Asp 195 200 205 Lys Ser Thr
Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu 210 215 220 Asp
Thr Ala Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp 225 230
235 240 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Cys 245 250
1623PRTHomo sapiens 16Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro 20
17107PRTArtificial Sequenceamino acid sequences of Human Composite
J591 V-regions (VL) 17Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Asp Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr
Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 18107PRTMus
musculus 18Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser
Val Gly 1 5 10 15 Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp
Val Gly Thr Ala 20 25 30 Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln
Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Thr Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala
Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe
Gly Ala Gly Thr Met Leu Asp Leu Lys 100 105 19107PRTArtificial
Sequenceamino acid sequences of deimmunized J591 V-regions (VL)
19Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Thr Ser Val Gly 1
5 10 15 Asp Arg Val Thr Leu Thr Cys Lys Ala Ser Gln Asp Val Gly Thr
Ala 20 25 30 Val Asp Trp Tyr Gln Gln Lys Pro Gly Pro Ser Pro Lys
Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Ile Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr
Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Pro Gly
Thr Lys Val Asp Ile Lys 100 105
* * * * *