U.S. patent application number 10/882151 was filed with the patent office on 2005-05-12 for multivalent carriers of bi-specific antibodies.
This patent application is currently assigned to Immunomedics, Inc.. Invention is credited to Hansen, Hans J., McBride, William J., Qu, Zhengxing.
Application Number | 20050100543 10/882151 |
Document ID | / |
Family ID | 34061993 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100543 |
Kind Code |
A1 |
Hansen, Hans J. ; et
al. |
May 12, 2005 |
Multivalent carriers of bi-specific antibodies
Abstract
Provided herein are targetable constructs that are multivalent
carriers of bi-specific antibodies, i.e., each molecule of a
targetable construct can serve as a carrier of two or more
bi-specific antibodies. Also provided are targetable complexes
formed by the association of a targetable construct with two or
more bi-specific antibodies. The targetable constructs and
targetable complexes of the invention are incorporated into
biosensors, kits and pharmaceutical compositions, and are used in a
variety of therapeutic and other methods.
Inventors: |
Hansen, Hans J.; (Picayune,
MS) ; McBride, William J.; (Boonton, NJ) ; Qu,
Zhengxing; (Warren, NJ) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
1717 RHODE ISLAND AVE, NW
WASHINGTON
DC
20036-3001
US
|
Assignee: |
Immunomedics, Inc.
Morris Plains
NJ
|
Family ID: |
34061993 |
Appl. No.: |
10/882151 |
Filed: |
July 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60483832 |
Jul 1, 2003 |
|
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|
Current U.S.
Class: |
424/132.1 ;
424/178.1; 435/188.5; 530/387.3; 530/391.1 |
Current CPC
Class: |
C07K 2317/31 20130101;
C07K 2319/00 20130101; A61P 35/00 20180101; C07K 16/3007 20130101;
C07K 16/2887 20130101; A61K 51/1048 20130101; A61K 51/1027
20130101; A61K 51/109 20130101; B82Y 5/00 20130101; C07K 2319/01
20130101; C07K 2317/622 20130101; A61K 47/6897 20170801; A61K
2039/505 20130101 |
Class at
Publication: |
424/132.1 ;
424/178.1; 530/387.3; 530/391.1; 435/188.5 |
International
Class: |
A61K 039/395; C07K
016/44; C12N 009/00 |
Claims
What is claimed is:
1. A bi-specific antibody comprising the structure
[IgG.sub.1]-[scFv]2; wherein said antibody comprises a pair of
heavy chains and a pair of light chains, wherein each heavy chain
comprises an IgG1 heavy chain and an scFv, wherein said scFv is
fused to the C-terminus of said IgG 1 heavy chain, optionally via a
linker peptide.
2. The antibody according to claim 1, wherein the binding sites
formed by said heavy chain and said light chain specifically binds
to an epitope on a targeted tissue.
3. The antibody according to claim 2, wherein each of said scFv
moieties specifically binds to a carrier epitope.
4. The antibody according to claim 1, wherein said IgG1 is a human,
humanized, chimeric, or CDR-grafted antibody.
5. The antibody according to claim 1, wherein each of said scFv
molecules is human, humanized, or CDR-grafted.
6. The antibody according to claim 5, wherein said antibody further
comprises a bioactive moiety.
7. The antibody according to claim 6, wherein said bioactive moiety
is selected from the group consisting of a drug, a prodrug, an
enzyme, a hormone, an immunomodulator, an oligonucleotide; a
radionuclide, an image enhancing agent and a toxin.
8. The antibody according to claim 1, wherein said antibody is
selected from the group consisting of [hMN14-IgG1]-[734scFv].sub.2
and [hMN14-IgG1.sup.(1253A)]-[734scFv].sub.2.
9. The antibody according to claim 1, wherein said antibody is
selected from the group consisting of [hMN14-IgG1]-[679scFv].sub.2
and [hMN14-IgG1.sup.(1253A)]-[679scFv].sub.2.
10. The antibody according to claim 1, wherein said antibody is
selected from the group consisting of [hA20-IgG1]-[734scFv].sub.2
and [hA20-IgG1.sup.(1253A)]-[734scFv].sub.2.
11. The antibody according to claim 1, wherein said antibody is
selected from the group consisting of [hA20-IgG1]-[679scFv].sub.2
and [hA20-IgG1.sup.(1253A)]-[679scFv].sub.2.
12. The antibody according to claim 1, wherein said antibody is
selected from the group consisting of [hLL2-IgG1]-[734scFv].sub.2
and [hLL2-IgG1.sup.(1253A)]-[734scFv].sub.2.
13. The antibody according to claim 1, wherein said antibody is
selected from the group consisting of [hLL2-IgG1]-[679scFv].sub.2
and [hLL2-IgG1.sup.(1253A)]-[670scFv].sub.2.
14. A binding complex comprising a tetravalent binding molecule
bound to a targetable construct, wherein said tetravalent binding
molecule comprises two binding sites for a carrier epitope and two
binding sites for a target epitope, and wherein said targetable
construct comprises a molecular scaffold and at least two carrier
epitopes.
15. The binding complex according to claim 14, wherein said
targetable construct comprises at least two pairs of carrier
epitopes and wherein at least two of said tetravalent binding
molecules are bound to said targetable construct.
16. The binding complex according to claim 15, wherein said at
least two pairs of carrier epitopes comprise a first pair and a
second pair, wherein said first and second pair are different
epitopes, and wherein a first tetravalent binding molecule is bound
to said first pair of carrier epitopes and a second tetravalent
binding molecule is bound to said second pair of carrier
epitopes.
17. The binding complex according to claim 16, wherein said first
and second pair of carrier epitopes are different epitopes.
18. The binding complex according to claim 17, wherein said first
and second tetravalent binding molecules bind to the same target
epitope.
19. The binding complex according to claim 14, wherein said
targetable construct is selected from the group consisting of IMP
246, IMP 156, IMP 192 and IMP 222.
20. The binding complex according to claim 14, wherein said carrier
epitope is a hapten.
21. The binding complex according to claim 14, wherein said carrier
epitope is a chelator, wherein said chelator optionally is bound to
a metal ion.
22. The binding complex according to claim 21, wherein said
chelator is selected from the group consisting of DTPA, DOTA,
benzyl DTPA, NOTA, and TETA.
23. The binding complex according to claim 14, wherein said
tetravalent binding molecule is a bi-specific antibody comprising
the structure [IgG.sub.1]-[scFv]2; wherein said antibody comprises
a pair of heavy chains and a pair of light chains, wherein each
heavy chain comprises an IgG1 heavy chain and an scFv, wherein said
scFv is fused to the C-terminus of said IgG1 heavy chain,
optionally via a linker peptide.
24. The binding complex according to claim 14, wherein said
molecular scaffold is a peptide or peptide derivative.
25. The binding complex according to claim 14, wherein said target
epitope is an antigen associated with a disease.
26. The binding complex according to claim 25, wherein said disease
is selected from the group consisting of hyperproliferative
disease, pathogenic disease, cancer, cardiovascular disease,
neurodegenerative disease, metabolic disease, and autoimmune
disease
27. The binding complex according to claim 26, wherein said target
epitope is a tumor associated antigen associated with a type of
cancer selected from the group consisting of acute lymphoblastic
leukemia, acute myelogenous leukemia, biliary cancer, breast
cancer, cervical cancer, chronic lymphocytic leukemia, chronic
myelogenous leukemia, colorectal cancer, endometrial cancer,
esophageal, gastric, head and neck cancer, Hodgkin's lymphoma, lung
cancer, medullary thyroid, non-Hodgkin's lymphoma, ovarian cancer,
pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer,
and urinary bladder cancer.
28. The binding complex according to claim 27, wherein said target
epitope is a tumor associated antigen selected from the group
consisting of A3, antigen specific for A33 antibody, BrE3, CD1,
CD1a, CD3, CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30,
CD45, CD74, CD79a, CD80, HLA-DR, NCA 95, NCA90, HCG and its
subunits, CEA, CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu,
KC4, KS-1, KS1-4, Le-Y, MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4, PSA,
PSMA, RS5, S100, TAG-72, p53, tenascin, IL-6, insulin growth
factor-1 (IGF-1), Tn antigen, Thomson-Friedenreich antigens, tumor
necrosis antigens, VEGF, 17-1A, an angiogenesis marker, a cytokine,
an immunomodulator, an oncogene marker, an oncogene product, and
other tumor associated antigens.
29. A method of treating a disease in a subject, comprising
administering to a subject suffering from said disease (i) a
tetravalent binding molecule comprising two binding sites for a
carrier epitope and two binding sites for a target epitope, wherein
said target epitope is an epitope associated with said disease,
(ii) optionally, a clearing agent, and (iii) a targetable construct
comprising a molecular scaffold and at least two carrier
epitopes.
30. The method according to claim 29, wherein said disease is
selected from the group consisting of hyperproliferative disease,
pathogenic disease, cancer, cardiovascular disease,
neurodegenerative disease, metabolic disease, and autoimmune
disease.
31. The method according to claim 29, wherein said targetable
construct further comprises a bioactive moiety.
32. A method of diagnosing/detecting a disease in a subject,
comprising administering to a subject suspected of suffering from
said disease (i) a tetravalent binding molecule comprising two
binding sites for a carrier epitope and two binding sites for a
target epitope, (ii) optionally, a clearing agent, and (iii) a
targetable construct comprising a molecular scaffold and at least
two carrier epitopes, wherein said construct comprises a detectable
label.
33. The method according to claim 32, wherein said target epitope
is comprised within, displayed by or released from one or more
cells, tissues, organs or systems of said subject.
34. A kit, comprising (i) a tetravalent binding molecule comprising
two binding sites for a carrier epitope and two binding sites for a
target epitope, (ii) optionally, a clearing agent, and (iii) a
targetable construct comprising a molecular scaffold and at least
two carrier epitopes.
35. A pharmaceutical composition comprising a bispecific antibody
according to claim 1.
Description
[0001] This application claims priority to provisional application
Ser. No. 60/483,832, filed Jul. 1, 2003, the contents of which are
incorporated herein in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to compounds that are multivalent
carriers of bi-specific antibodies (bsAbs), i.e., each molecule of
the compound can serve as a carrier of two or more bi-specific
antibodies. The invention further relates to complexes formed by
the association of a multivalent compound with two or more
bi-specific antibodies. In preferred embodiments, the compounds of
the invention form complexes that have desirable attributes such as
increased affinity, high stability in vitro and/or in vivo, and
preferred pharmacokinetics. The compounds and complexes of the
invention are useful for therapy and in vitro applications.
BACKGROUND OF THE INVENTION
[0003] The following description of the background of the invention
is provided simply as an aid in understanding the invention and is
not admitted to describe or constitute prior art to the
invention.
[0004] An approach to cancer therapy and diagnosis involves
directing antibodies (Abs) or antibody fragments to disease
tissues, wherein the antibody or antibody fragment can target a
therapeutic agent to the disease site, i.e., a targeted tissue. A
"targeted tissue" is any biological entity (e.g., a system, organ,
tissue, cell, organelle, receptor, surface antigen, transmembrane
protein, secreted polypeptide, or intracellular component) to which
a targetable construct is preferentially delivered. The term
"delivered" encompasses being contacted with, bound to, and/or
internalized by, a targeted tissue. As used herein, the term
"and/or" has the meaning of "and, additionally or alternatively" or
"and, in addition to or in the alternative."
[0005] In therapeutic aspects of the invention, the targeted tissue
is malignant, infected, inflamed (as in certain autoimmune diseased
sites), dysfunctional or displaced or ectopic (e.g., infected
cells, cancer cells, endometriosis, etc.), or otherwise diseased
(e.g., atherosclerosis, ischemia, clots). Antibodies are used to
deliver therapeutic agents to the targeted tissue.
[0006] The use of a bsAb/low molecular weight (MW) hapten system is
not without some limitations. The arm of the bsAb that binds to the
low MW hapten must bind with high affinity, since a low MW hapten
is desirably one that clears the living system rapidly when not
bound by bsAb. The non-bsAb-bound low MW hapten needs to clear the
living system rapidly to avoid non-target tissue uptake and
retention. Moreover, the therapeutic agent must remain associated
with the low MW hapten throughout its application within the bsAb
protocol employed.
[0007] This application incorporates by reference the entirety of
U.S. application Ser. No. 09/337,756 (Docket No. IMM 138; Atty
Docket No. 018733-0884), entitled "Use of bi-specific antibodies
for pre-targeting diagnosis and therapy", which was filed Jun. 22,
1999.
[0008] This application incorporates by reference the entirety of
U.S. application Ser. No. 09/382,186 (Docket No. IMM 138 CIP; Atty
Docket No. 018733-0942), entitled "Use of bi-specific antibodies
for pre-targeting diagnosis and therapy", which was filed Aug. 23,
1999.
[0009] This application incorporates by reference the entirety of
U.S. application Ser. No. 09/823,746 (Docket No. IMM 160; Atty
Docket No. 018733-1015), entitled "Production and use of novel
peptide-based agents for use with bi-specific antibodies", which
was filed Apr. 3, 2001.
[0010] This application incorporates by reference the entirety of
U.S. Provisional Application Ser. No. 60/361,037 (Docket No. IMM
169; Atty Docket No. 018733-1037), entitled "Bispecific antibody
point mutations for enhancing rate of clearance", which was filed
Mar. 1, 2002.
[0011] This application incorporates by reference the entirety of
published PCT Application WO 99/66951 by Hansen et al., entitled
"Use of bi-specific antibodies for pre-targeting diagnosis and
therapy", which describes some of the reagents used in the Examples
herein.
[0012] The synthesis of IMP 192 is described in Karacay et al.,
Experimental pretargeting studies of cancer with a humanized
anti-CEA x murine anti-[In-DTPA] bispecific antibody construct and
a (99m)Tc-/(188)Re-labeled peptide, Bioconjug. Chem. 11:842-854,
2000.
[0013] The radiolabeling of
Ac-Phe-Lys(-DTPA)-Tyr-Lys(-DTPA)-NH.sub.2 with .sup.111In is
described in Boerman et al., Pretargeting of renal cell carcinoma:
improved tumor targeting with a bivalent chelate, Cancer Res.
59:4400-4405, 1999.
SUMMARY OF THE INVENTION
[0014] The present invention provides multimeric targetable
complexes that are multivalent and/or polyspecific. The invention
further provides methods of making such complexes, compositions for
making such complexes, and methods of using the multimeric
targetable complexes of the invention.
[0015] Targetable Complexes
[0016] The present invention relates to multimeric targetable
complexes that are multivalent and/or polyspecific. A non-limiting
example of a multimeric, multivalent targetable complex is a
tetravalent targetable complex, which comprises (a) a targetable
construct and (b) 2 molecules of a bi-specific antibody, each
molecule comprising (i) two arms, each of which binds a carrier
epitope, and (ii) two arms, each of which is capable of binding a
target epitope. The complex is tetravalent because it comprises a
total of 4 arms, each of which is capable of binding a target
epitope (2 molecules of an antibody that has 2 arms capable of
binding the target epitope).
[0017] A targetable complex of the invention may be polyspecific,
multivalent, or both.
[0018] A tetravalent complex is examplary of a multivalent complex
and may be a homodimer or a heterodimer.
[0019] In a homodimer (e.g., a tetravalent targetable complex of
the invention) both of 2 bi-specific antibodies have 2 arms that
bind the same target epitope, and 2 arms that bind a carrier
epitope. The homodimeric complex is bi-specific because its arms
either recognize (a) a carrier epitope or (b) a target epitope, and
is tetravalent because it has 4 arms that recognize the target
epitope.
[0020] A tetravalent targetable complex of the invention has four
arms capable of binding a target epitope, and comprises:
[0021] (a) a targetable construct comprising (i) a molecular
scaffold and (ii) two pairs of a carrier epitope; and
[0022] (b) two molecules of a bi-specific antibody, each antibody
comprising (i) two arms, each arm comprising a binding site for
said carrier epitope, and (ii) two arms, each comprising a binding
site for said target epitope.
[0023] Preferably, a tetravalent complex of the invention has one
or more of the following attributes:
[0024] (I) the targetable complex has a Kd for said target epitope
from about 0.1 nM to about 100 nM,
[0025] (II) mixing the targetable construct and the bi-specific
antibody at relative concentrations ranging from about 10.sup.-3 to
about 10.sup.3 results in a mixture in which greater than about 75%
of the complexes therein have a defined stoichiometry of two
molecules of said bi-specific antibody, and one molecule of said
targetable construct, and
[0026] (III) a pair of carrier epitopes is bound by said
bi-specific antibody in a 1:1 ratio.
[0027] In a heterodimer (e.g., a targetable complex that is
divalent for each of two target epitopes), each of 2 bi-specific
antibodies have two arms that bind different target epitopes, and
two arms that bind a carrier epitope. The heterodimeric complex is
bi-specific (it recognizes two different target epitopes) and
divalent for each target epitope because it has four arms, wherein
two of said arms recognize a first carrier epitope, and wherein the
other two arms recognize a second target epitope.
[0028] A targetable complex of the invention that is divalent for
each of two target epitopes comprises:
[0029] (a) a targetable construct comprising (i) a molecular
scaffold and (ii) two pairs of carrier epitopes, wherein the first
of said two pairs of carrier epitopes is specifically bound by a
first bi-specific antibody, and the second of said two pairs of
carrier epitopes is specifically bound by a second bi-specific
antibody, wherein the targetable construct forms a targetable
complex when combined with
[0030] (b) a first bi-specific antibody, the first bi-specific
antibody comprising (i) two copies of a first arm comprising a
binding site for said carrier epitope, and (ii) two copies of a
second arm comprising a binding site for a first target epitope,
and
[0031] (c) a second bi-specific antibody, the second bi-specific
antibody comprising (i) two copies of a first arm comprising a
binding site for said carrier epitope, and (ii) two copies of a
second arm comprising a binding site for a second target
epitope.
[0032] Preferably, a targetable complex of the invention that is
divalent for each of two target epitopes has one or more of the
following attributes:
[0033] (I) said targetable complexes have a Kd for said target
epitope from about 0.1 nM to about 100 nM,
[0034] (II) mixing said targetable construct and said bi-specific
antibody at relative concentrations ranging from about 10.sup.-3 to
about 10.sup.3 results in a mixture in which greater than about 75%
of the complexes therein have a defined stoichiometry of two
molecules of said bi-specific antibody, and one molecule of said
targetable construct, and
[0035] (III) each pair of carrier epitopes is bound by one of said
bi-specific antibodies in a 1:1 ratio.
[0036] Both homodimeric and heterodimeric tetrameric complexes are
encompassed by targetable complexes that comprise:
[0037] (a) a targetable construct comprising (i) a molecular
scaffold and (ii) two pairs of carrier epitopes, wherein the first
of said two pairs of carrier epitopes is specifically bound by a
first bi-specific antibody, and the second of said two pairs of
carrier epitopes is specifically bound by a second bi-specific
antibody, wherein said targetable construct forms a targetable
complex when combined with
[0038] (b) a first bi-specific antibody, said first bi-specific
antibody comprising (i) two copies of a first arm comprising a
binding site for said carrier epitope, and (ii) two copies of a
second arm comprising a binding site for a first target epitope,
and
[0039] (c) a second bi-specific antibody, said second bi-specific
antibody comprising (i) two copies of a first arm comprising a
binding site for said carrier epitope, and (ii) two copies of a
second arm comprising a binding site for a second target
epitope;
[0040] wherein said first bi-specific antibody and said second
bi-specific antibody can be the same or different, said pairs of
carrier epitopes can be the same or different, and said target
epitopes can be the same or different, and wherein one or more of
the following applies:
[0041] (I) the targetable complex has a Kd for a target epitope
from about 0.1 nM to about 100 nM,
[0042] (II) mixing the targetable construct and the bi-specific
antibody at relative concentrations ranging from about 10.sup.-3 to
about 10.sup.3 results in a mixture in which greater than about 75%
of the complexes therein have a defined stoichiometry of two
molecules of the bi-specific antibody, and one molecule of the
targetable construct, and
[0043] (III) each pair of carrier epitopes is bound by one of said
bi-specific antibodies in a 1:1 ratio.
[0044] In general, multivalent and/or polyspecific complexes
comprise:
[0045] a targetable construct comprising a molecular scaffold and X
pairs of carrier epitopes, wherein each of said pairs of carrier
epitopes is specifically bound by one of X bi-specific antibodies,
each bi-specific antibody comprising (a) two copies of a first arm
comprising a binding site for a carrier epitope, and (b) two copies
of a second arm comprising a binding site for one of Y target
epitopes, wherein
[0046] (i) X is a whole integer .gtoreq.2,
[0047] (ii) Y is a whole integer >1,
[0048] (iii) said X bi-specific antibodies can be the same or a
mixture of different bi-specific antibodies,
[0049] (iv) said X pairs of carrier epitopes can be the same or a
mixture of different carrier epitopes, and
[0050] (v) when Y>2, said Y target epitopes can be the same or a
mixture of different target epitopes, and
[0051] a pair of carrier epitopes is bound by a bi-specific
antibody in a 1:1 ratio.
[0052] Targetable Constructs
[0053] The present invention relates to multivalent targetable
constructs that may be used to form targetable complexes with
bi-specific antibodies prior to and/or after administration to a
subject. The constructs comprise two or more pairs of carrier
epitopes that are bound by an arm of a bi-specific antibody and are
thus multivalent constructs.
[0054] For tetrameric complexes, a tetravalent (i.e., comprising
two pairs of carrier eptiopes) targetable construct comprises a
molecular scaffold, and two pairs of a carrier epitope, wherein the
targetable construct, when combined with a bi-specific antibody
comprising (i) two copies of a first arm comprising a binding site
for the carrier epitope, and (ii) two copies of a second arm
comprising a binding site for a target epitope, forms a targetable
complex.
[0055] A tetrameric targetable complex of the invention that is
divalent for each of two target epitopes comprises a molecular
scaffold and two pairs of carrier epitopes, wherein the first pair
of carrier epitopes is specifically bound by a first bi-specific
antibody, and the second pair of carrier epitopes is specifically
bound by a second bi-specific antibody, wherein the targetable
construct forms a targetable complex when combined with
[0056] (a) a first bi-specific antibody, the first bi-specific
antibody comprising (i) two copies of a first arm comprising a
binding site for a carrier epitope, and (ii) two copies of a second
arm comprising a binding site for a first target epitope, and
[0057] (b) a second bi-specific antibody, said second bi-specific
antibody comprising (i) two copies of a first arm comprising a
binding site for said carrier epitope, and (ii) two copies of a
second arm comprising a binding site for a second target
epitope.
[0058] In general, multivalent and/or polyspecific constructs
comprise a molecular scaffold and X pairs of carrier epitopes,
wherein each of said pairs of carrier epitopes is specifically
bound by one of X bi-specific antibodies, each bi-specific antibody
comprising (a) two copies of a first arm comprising a binding site
for a carrier epitope, and (b) two copies of a second arm
comprising a binding site for one of Y target epitopes, wherein
[0059] (i) X is a whole integer >2,
[0060] (ii) Y is a whole integer >1,
[0061] (iii) said X bi-specific antibodies can be the same or a
mixture of different bi-specific antibodies,
[0062] (iv) said X pairs of carrier epitopes can be the same or a
mixture of different carrier epitopes, and
[0063] (v) when Y>2, said Y target epitopes can be the same or a
mixture of different target epitopes, and
[0064] a pair of carrier epitopes is bound by a bi-specific
antibody in a 1:1 ratio.
[0065] Preferably, a targetable complex formed using a targetable
construct of the invention of the invention has one or more of the
following attributes:
[0066] (a) the targetable complex has a Kd for the target epitope
from about 0.1 nM to about 100 nM, and
[0067] (b) mixing the targetable construct and the bi-specific
antibody at relative concentrations ranging from about 10.sup.-3 to
about 10.sup.3 results in a mixture in which greater than about 75%
of the complexes therein have a defined stoichiometry of two
molecules of the bi-specific antibody, and one molecule of the
targetable construct,
[0068] (c) a pair of carrier epitopes is simultaneously bound by
said two copies of a first arm comprising a binding site for said
carrier epitope, wherein said two copies of a first arm comprising
a binding site for said carrier epitope are part of said
bi-specific antibody.
[0069] Embodiments
[0070] It has been discovered that it is advantageous to prepare
and use multimers of polyspecific antibodies, e.g., antibodies that
have two or more arms that specifically bind a targetable construct
that is capable of carrying one or more therapeutic agents, and two
or more arms that bind a targeted tissue. By utilizing this
technique, the multimerization of bi-specific antibodies and other
polyspecific antibodies, targetable constructs, chelators, metal
chelate complexes, therapeutic agents can be varied to accommodate
differing applications.
[0071] Because a polyspecific antibody must, by definition,
specifically bind at least two different targets, a bispecific
antibody (bsAb) is the simplest type of polyspecific antibody.
Similarly, a multivalent antibody must, by definition, have at
least two binding sites for a single target, and a divalent
antibody is thus the simplest type of multivalent Abs. Thus, at a
minimum, the Abs of the invention are bispecific (i.e., bind 2
different targets) and divalent (comprising 2 copies of an arm that
binds a target).
[0072] An antibody used in in the invention may be a monoclonal
antibody, a humanized antibody, a human antibody, a chimeric
antibody, a single chain antibody, a camelid antibody, a CDR, a
soluble TCR, a fusion protein, a naked antibody, or a fragment of
any of the preceding.
[0073] The targetable construct may comprise a peptide; a
carbohydrate; and/or one or more haptens including but not limited
to a chelator or metal-chelate complex. By way of non-limiting
example, the chelator may be a hard base chelator for a hard acid
cation, and at least one of the chelators is a soft base chelator
for a soft acid cation; or a hard base chelator that comprises
carboxylate and amine groups. Non-limiting examples of hard base
chelators include DTPA, NOTA, DOTA and TETA.
[0074] The invention further relates to methods utilizing
multivalent and/or polyspecific targetable constructs and
complexes. In some embodiments, the targetable construct or complex
comprises a biologically active moiety, such as one that initiates,
enhances, limits or prevents a biochemical process. A bioactive
moiety of the present invention is selected from the group
consisting of a drug, a prodrug, an enzyme, a hormone, an
immunomodulator, an oligonucleotide; a radionuclide, an image
enhancing agent and a toxin. A "biochemical process" is any process
that alters any activity or process of a cell, or of a subcellular
portion. A subcellular portion may be an organelle, e.g., a
mitochondrion, the endoplasmic reticulum, the nucleus, the
nucleolus, or the cell membrane and/or a receptor thereon. By way
of non-limiting example, a biochemical process may be a signaling
cascade, a complement cascade, apoptosis, a biochemical pathway, or
one or more reactions that occur in any of the preceding.
[0075] A biochemical process comprises one or more reactions. A
"reaction" is any response of one or more molecules to being
brought into contact with one or more other molecules, or any
response of one or molecules to a change in the proximity of one or
more other molecules. A chemical reaction, in which a molecule is
split into two or more molecules, and/or two or more molecules are
reacted with each other to form one or more different molecules, is
a non-limiting example of a reaction. A non-covalent association of
two or more molecules with each other is another example of a
reaction. A transfer of an electron or ion from one molecule to
another is another example of reaction. A change in conformation in
response to contact with another molecule is another example of a
reaction. Intracellular and intercellular translocations of
molecules are reactions.
[0076] In some embodiments, the targetable constructs and complexes
are biologically active not because they comprise a bioactive
moiety per se but because the binding of the targetable construct
or complex to its targeted tissue initiates, enhances, limits or
prevents a biochemical process. For example, in some embodiments,
the bi-specific antibodies of the targetable complex may be naked
antibodies. A "naked antibody" is, generally, an antibody that
lacks the Fc portion of an antibody. The Fc portion of the antibody
molecule provides effector functions, such as complement fixation
and ADCC (antibody dependent cell cytotoxicity), which set
mechanisms into action that may result in cell lysis. However, the
Fc portion may nor be required for therapeutic function in every
instance, with other mechanisms, such as apoptosis, coming into
play.
[0077] The targetable constructs and complexes may comprise one or
more agents useful for killing or slowing the growth of diseased
tissue. By way of non-limiting example, the agent may be a
radioactive isotope, particularly the therapeutically useful
therapeutic radionuclides set forth herein. The agent may also be a
toxin; one or more drugs; and/or one or more prodrugs. By way of
non-limiting example, the targetable construct or complex may
comprise doxorubicin, CPT-11 or SN38.
[0078] One embodiment of the invention involves using compositions
and methods of the disclosure in boron neutron capture therapy
(BNCT). In BNCT, the targetable constructs comprise boron atoms, in
which case the method further comprise the step of irradiating the
boron atoms localized at the diseased tissue, thereby effecting
BNCT.
[0079] Various embodiments of the invention provide pre-targeting
methods and compositions using pre-formed targetable complexes of
the invention. A targetable complex comprises a multivalent
targetable construct, which optionally carries one or more
bioactive agents; and one or more pairs of a bi-specific antibody
comprising a pair of arms that specifically bind the multivalent
targetable construct, and two or more arms that bind a targeted
tissue.
[0080] A further embodiment of the invention involves a kit
comprising the targetable complexes of the invention, which may
further comprise one or more compounds selected from the group
consisting of one or more radioactive isotopes useful for killing
or slowing the growth of diseased tissue, one or more toxins, one
or more drugs, and one or more prodrugs.
[0081] In a further embodiment, the invention provides compositions
and methods for targeting cardiovascular lesions such as
atherosclerotic plaques, vascular clots including thrombi and
emboli, myocardial infarction, and other organ infarcts.
[0082] The invention also provides compositions and methods for
targeting metabolic disease, such as amyloid in amyloidosis, as
well as a neurodegenerative disease such as Alzheimer's
disease.
[0083] In a further embodiment, the invention provides compositions
and methods for treating a mammal having a hypoplastic, absent,
anatomically displaced or ectopic tissue or organ.
[0084] In a further embodiment, the invention provides compositions
and methods for treating diseases, including, for example,
pathogenic diseases, cancer, cardiovascular diseases,
neurodegenerative diseases, metabolic diseases, and autoimmune
diseases.
[0085] In a further embodiment, the invention provides
pharmaceutical compositions and kits comprising the compositions of
the invention.
[0086] In a further embodiment, the invention provides compositions
and methods for making a biosensor that may be used to detect
substances in samples or in the environment.
[0087] In a further embodiment, the invention provides compositions
and methods for in vitro immunochemical methods, including but not
limited to immunoassays and immunoaffinity purification. In these
and other embodiments, the compositions of the invention may be
attached to solid supports. Representative solid supports include
dipsticks, beads, multititer plates, the interior surface of wells
in a multiwell/microtiter plate, and membranes.
[0088] In a further embodiment, the invention provides compositions
and methods for separating a compound of interest from undesirable
substances in a composition. In this embodiment, a targetable
construct or complex is attached to a solid support, to which the
composition is contacted. A compound of interest or an undesirable
substance is bound by the targetable construct or complex that is
attached to the solid support. In a further step, the compound of
interest or the undesirable substance substance are separated from
each other as either the compound of interest or the undesirable
substance is retained by the immobilized targetable construct or
complex. The compositions and methods of the invention can be used
in a dialysis machine or system, as well as in a manufacturing
process.
[0089] In addition, the present invention provides a bi-specific
antibody having the structure [IgG.sub.1]-[scFv]2; where the
antibody has a pair of heavy chains and a pair of light chains,
where each heavy chain has an IgG1 heavy chain and an scFv, and
where the scFv is fused to the C-terminus of the IgG1 heavy chain,
optionally via a linker peptide. The antibody binding sites formed
by the heavy chain and the light chain may specifically bind to an
epitope on a targeted tissue. Each of the scFv moieties may
specifically bind to a carrier epitope. The IgG1 and/or the scFv
molecules may be human, humanized, chimeric, or CDR-grafted. The
antibody further contain a bioactive moiety. The bioactive moiety
may be, for example, a drug, a prodrug, an enzyme, a hormone, an
immunomodulator, an oligonucleotide; a radionuclide, an image
enhancing agent and/or a toxin. The bi-specific antibody may be
formulated into a pharmeceutical composition.
[0090] Specific examples of such bi-specific antibodies include,
but are not limited to [hMN14-IgG1]-[734scFv].sub.2 and
[hMN14-IgG1.sup.(1253A)]-- [734scFv].sub.2,
[hMN14-IgG1]-[679scFv].sub.2 and [hMN14-IgG1.sup.(1253A)]-
-[679scFv].sub.2, [hA20-IgG1]-[734scFv].sub.2 and
[hA20-IgG1.sup.(1253A)]-- [734scFv].sub.2,
[hA20-IgG1]-[679scFv].sub.2 and [hA20-IgG1.sup.(1253A)]-[-
679scFv].sub.2, [hLL2-IgG1]-[734scFv].sub.2 and
[hLL2-IgG1.sup.(1253A)]-[7- 34scFv].sub.2, and
[hLL2-IgG1]-[679scFv].sub.2 and [hLL2-IgG1.sup.(1253A)]-
-[670scFv].sub.2.
[0091] The invention further provides a binding complex having a
tetravalent binding molecule bound to a targetable construct, where
the tetravalent binding molecule has two binding sites for a
carrier epitope and two binding sites for a target epitope, and
where the targetable construct has a molecular scaffold and at
least two carrier epitopes. The targetable construct may have at
least two pairs of carrier epitopes and in the complex at least two
of the tetravalent binding molecules may be bound to the targetable
construct. The targetable construct may contain at least two pairs
of different carrier epitopes and in the complex the first
tetravalent binding molecule may be bound to one pair of carrier
epitopes and a second tetravalent binding molecule may be bound to
a second pair of carrier epitopes. The pairs of carrier epitopes
may be different epitopes. The first and second tetravalent binding
molecules may bind to the same target epitope. The targetable
construct may be selected from the group consisting of IMP 246, IMP
156, IMP 192 and IMP 222. The carrier epitope may be a hapten. The
carrier epitope may be a chelator, where the chelator optionally is
bound to a metal ion. The chelator may be, for example, DTPA, DOTA,
benzyl DTPA, NOTA, or TETA. The tetravalent binding molecule may be
a bi-specific antibody having the structure [IgG.sub.1]-[scFv]2,
where the antibody has a pair of heavy chains and a pair of light
chains, and where each heavy chain has an IgG1 heavy chain and an
scFv, where the scFv is fused to the C-terminus of the IgG1 heavy
chain, optionally via a linker peptide. In the binding complex the
molecular scaffold may be, for example, a peptide or peptide
derivative.
[0092] In each of these examples, the target epitope may be an
antigen associated with, for example, a disease, such as a
hyperproliferative disease, pathogenic disease, cancer,
cardiovascular disease, neurodegenerative disease, metabolic
disease, or autoimmune disease. The cancer may be, for example, a
cancer such as acute lymphoblastic leukemia, acute myelogenous
leukemia, biliary cancer, breast cancer, cervical cancer, chronic
lymphocytic leukemia, chronic myelogenous leukemia, colorectal
cancer, endometrial cancer, esophageal, gastric, head and neck
cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid,
non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, glioma,
melanoma, liver cancer, prostate cancer, and/or urinary bladder
cancer. The target epitope may be, for example, a tumor associated
antigen selected from the group consisting of A3, antigen specific
for A33 antibody, BrE3, CD1, CD1a, CD3, CD5, CD15, CD19, CD20,
CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, NCA
95, NCA90, HCG and its subunits, CEA, CSAp, EGFR, EGP-1, EGP-2,
Ep-CAM, Ba 733, HER2/neu, KC4, KS-1, KS1-4, Le-Y, MAGE, MUC1, MUC2,
MUC3, MUC4, PAM-4, PSA, PSMA, RS5, S100, TAG-72, p53, tenascin,
IL-6, insulin growth factor-1 (IGF-1), Tn antigen,
Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF,
17-1A, an angiogenesis marker, a cytokine, an immunomodulator, an
oncogene marker, an oncogene product, and other tumor associated
antigens.
[0093] The present invention also provides a method of treating a
disease in a subject, by administering to a subject suffering from
the disease (i) a tetravalent binding molecule having two binding
sites for a carrier epitope and two binding sites for a target
epitope, where the target epitope is an epitope associated with the
disease, (ii) optionally, a clearing agent, and (iii) a targetable
construct having a molecular scaffold and at least two carrier
epitopes. The disease may be, for example, a hyperproliferative
disease, pathogenic disease, cancer, cardiovascular disease,
neurodegenerative disease, metabolic disease, or autoimmune
disease. The targetable construct used in the method may contain a
bioactive moiety.
[0094] The present invention also provides a method of
diagnosing/detecting a disease in a subject, by administering to a
subject suspected of suffering from the disease (i) a tetravalent
binding molecule having two binding sites for a carrier epitope and
two binding sites for a target epitope, (ii) optionally, a clearing
agent, and (iii) a targetable construct having a molecular scaffold
and at least two carrier epitopes, where the construct has a
detectable label. The target epitope may, for example, be contained
within, displayed by or released from one or more cells, tissues,
organs or systems of the subject.
[0095] The present invention also provides a kit, having (i) a
tetravalent binding molecule having two binding sites for a carrier
epitope and two binding sites for a target epitope, (ii)
optionally, a clearing agent, and (iii) a targetable construct
having a molecular scaffold and at least two carrier epitopes.
[0096] Additional aspects, features and advantages of the invention
will be set forth in the description which follows, and in part
will be obvious from the description, or may be learned by practice
of the invention. The embodiments and advantages of the invention
may be realized and obtained by means of the instrumentalities and
combinations particularly pointed out in the appended
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0097] FIG. 1 schematically illustrates hMN14-734scFv, an example
of a bispecific and divalent antibody of the invention. See
published PCT Application WO 99/66951 for details of the structure
and prepartion of hMN14-734scFv. Symbols: solid black rectangle,
molecular scaffold; .fwdarw.carrier epitopes, open circle, optional
biologically active moiety; filled diamonds, target epitopes; and
ovals, antibody domains. Each pair of filled and open ovals
(bottom) represent an arm of the bi-specific antibody that binds a
carrier epitope, and each pair of striped and cross-hatched ovals
(top) represent an arm of the bi-specific that binds a target
epitope.
[0098] FIG. 2 schematically illustrates a tetravalent targetable
complex of the invention. Two bi-specific and divalent antibody
molecules are shown bound to a targetable construct. Symbols are
the same as in FIG. 1.
[0099] FIGS. 3-7 show HPLC traces of peptide/antibody
complexes.
[0100] FIG. 8 shows HPLC traces of targetable complexes formed by
mixing IMP 246 with hMN-14 IgG.sup.(1253A)(734scFV).sub.2 (panel A)
or with m734 IgG (panel B) mixed in a ratio of 1:10
(peptide:antibody).
DETAILED DESCRIPTION OF THE INVENTION
[0101] The present invention provides targetable complexes
comprising multivalent and polyspecific (e.g., bi-specific)
antibodies. Bi-specific antibodies have at least one arm that
specifically binds a targeted tissue and at least one other arm
that specifically binds a targetable construct.
[0102] A "targeted tissue" is a system, organ, tissue, cell,
intracellular component, receptor, or organelle to which a
targetable construct is preferentially delivered. In the
therapeutic aspects of the invention, the targeted tissue is
infected and/or misfunctioning (e.g., cancer cells, infected cells,
ectopic cells, etc.).
[0103] In addition to antibodies, the complexes comprise at least
one targetable construct. A "targetable construct" comprises a
molecular scaffold which comprises or bears at least two pairs of
carrier epitopes recognized by the arm of the bi-specific antibody.
As used herein, a "molecular scaffold" (or simply "scaffold") is
any chemical structure to which epitopes and other moieties can be
attached at a variety of positions, and/or with a variety of
orientations, relative to the scaffold and/or other moieties.
Non-limiting examples of molecular scaffolds include polymers such
as peptides or peptide derivatives, oligopeptides and
oligonucleotides. See Skerra, Engineered protein scaffolds for
molecular recognition, J. Mol. Recog. 13:167-187, 2000; Erratum in:
J. Mol. Recog. 14:141, 2001. The oligonucleotides can be antisense
oligonucleotide molecules or genes that correspond to p53. Also, an
oligonucleotide, such as an antisense molecule inhibiting bcl-2
expression is described in U.S. Pat. No. 5,734,033 (Reed).
[0104] The epitopes of a targetable construct are called "carrier
eptiopes" herein. As used herein, the term "epitope" (also known as
"immunogenic recognition moiety") encompasses any molecule or
moiety that is specifically bound by a recognition moiety or
molecule. Non-limiting examples of recognition moieties and
molecules include antibodies, antibody derivatives, antigen-binding
regions and minimal recognition units of antibodies, and
receptor-specific ligands.
[0105] Non-limiting examples of recognizable haptens include, but
are not limited to, chelators, such as
diethylenetriaminepentaacetic acid (DTPA), histamine-succinyl
glycine (HSG), fluorescein isothiocyanate (FITC), vitamin B-12 and
other moieties to which specific antibodies can be raised, with
scFv being preferred. Antibodies raised to the HSG or DTPA hapten
are known and the scFv portion of the antibody can be used as a
carrier epitope binding arm of a bi-specific antibody. Binding of
the carrier eptiopes is highly specific for each scFv
component.
[0106] The targetable construct is multivalent, with bivalent
peptides being the preferred peptide. The targetable construct may,
but need not, be linked or conjugated to a variety of agents useful
for treatment. Alternatively, the targetably construct may be
administered in combination with such agents. Examples of such
agents include, but are not limited to, metal chelate complexes,
folate moieties, drugs, toxins and other effector molecules, such
as cytokines, lymphokines, chemokines, immunomodulators, enzymes,
radiosensitizers, asparaginase, RNAse, DNAse, carboranes, receptor
targeting agents and radioactive halogens. Additionally, enzymes
useful for activating a prodrug or increasing the target-specific
toxicity of a drug can be conjugated to the carrier. Thus, the use
of a bi-specific antibody which have at least one arm that
specifically binds a targetable construct allows a variety of
applications to be performed without raising new bsAb for each
application.
[0107] The term "antibody fragment" (also known as "antibody
derivative") encompasses any synthetic or genetically engineered
protein that acts like an antibody by binding to a specific antigen
to form a complex. For example, antibody fragments include isolated
fragments, "Fv" fragments, consisting of the variable regions of
the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy chain variable regions are
connected by a peptide linker ("scFv proteins"), and minimal
recognition units consisting of the amino acid residues that mimic
the hypervariable region. Antibodies and antibody fragments are
described in more detail below.
[0108] I. Biological Activity of Targetable Constructs and
Complexes
[0109] A targetable construct may be biologically active due to the
activity of the molecular scaffold, or the construct may optionally
comprise a biologically active moiety. A targetable complex may be
biologically active due to the activity of the targetable
construct, or the complex may optionally comprise a biologically
active moiety or molecule.
[0110] I.A. Definitions
[0111] The term "biologically active" (synonymous with "bioactive")
indicates that a composition or compound itself has a biological
effect, or that it modifies, causes, promotes, enhances, blocks,
reduces, limits the production or activity of, or reacts with or
binds to an endogenous molecule that has a biological effect. A
"biological effect" may be but is not limited to one that
stimulates or causes an immunreactive response; one that impacts a
biological process in an animal; one that impacts a biological
process in a pathogen or parasite; one that generates or causes to
be generated a detectable signal; and the like. Biologically active
compositions, complexes or compounds may be used in therapeutic,
prophylactic and diagnostic methods and compositions. Biologically
active compositions, complexes or compounds act to cause or
stimulate a desired effect upon an animal. Non-limiting examples of
desired effects include, for example, preventing, treating or
curing a disease or condition in an animal suffering therefrom;
limiting the growth of or killing a pathogen in an animal infected
thereby; augmenting or altering the phenotype or genotype of an
animal; and stimulating a prophylactic immunoreactive response in
an animal.
[0112] In the context of therapeutic applications of the invention,
the term "biologically active" indicates that the composition,
complex or compound has an activity that impacts an animal
suffering from a disease or disorder in a positive sense and/or
impacts a pathogen or parasite in a negative sense. Thus, a
biologically active composition, complex or compound may cause or
promote a biological or biochemical activity within an animal that
is detrimental to the growth and/or maintenance of a pathogen or
parasite; or of cells, tissues or organs of an animal that have
abnormal growth or biochemical characteristics, such as cancer
cells, or cells affected by autoimmune or inflammatory
disorders.
[0113] In the context of prophylactic applications of the
invention, the term "biologically active" indicates that the
composition or compound induces or stimluates an immunoreactive
response. In some preferred embodiments, the immunoreactive
response is designed to be prophylactic, i.e., prevents infection
by a pathogen. In other preferred embodiments, the immunoreactive
response is designed to cause the immune system of an animal to
react to the detriment of cells of an animal, such as cancer cells,
that have abnormal growth or biochemical characteristics. In this
application of the invention, compositions, complexes or compounds
comprising antigens are formulated as a vaccine.
[0114] It will be understood by those skilled in the art that a
given composition, complex or compound may be biologically active
in therapeutic, diagnostic and prophylactic applications. A
composition, complex or compound that is described as being
"biologically active in a cell" is one that has biological activity
in vitro (i.e., in a cell culture) or in vivo (i.e., in the cells
of an animal). A "biologically active portion" of a compound or
complex is a portion thereof that is biologically active once it is
liberated from the compound or complex. It should be noted,
however, that such a component may also be biologically active in
the context of the compound or complex.
[0115] In order to achieve a biological effect, invention
constructs may comprise an additional moiety to facilitate
internalization and/or uptake by a target cell. For aspects of the
present invention that involve an internalization moiety,
internalization can be accomplished in various ways. In particular
embodiments, the internalization moiety binds to a recycling
receptor, such as a folate receptor. For binding to a folate
receptor, the internalization moiety can, for example, include
folate or methotrexate, or a folate analog binding to a folate
receptor. In other embodiments, the internalization moiety includes
a peptide that enhances non-receptor mediated internalization.
[0116] Likewise, a number of internalization mechanisms can be
utilized in place of the folate receptor with folate or
methotrexate. For example, hormone or hormone analog/hormone
receptor pairs such as steroid hormones; specific peptide/peptide
receptor; and non-receptor mediated peptide internalization.
[0117] A variety of different species that enhance internalization
are known and can be utilized. Examples include folic acid (folate)
or methotrexate with internalization via folate receptor; steroid
hormones and their respective receptors; receptor-recognized
peptides, e.g., somatostatin, LHRH bombesin/CCKB, substance P, VIP.
In addition, antibodies that cross-link the targetable construct to
a rapidly internalizing membrane protein can also be used to
enhance internalization.
[0118] I.B. Therapy of Tissues and Organs
[0119] I.B.1. Cardiovascular Lesions, Atherosclerotic Plaques and
Vascular Clots
[0120] When there is an insult to vascular endothelium, circulating
blood cells, particularly leukocytes, accumulate. Granulocytes tend
to concentrate in the largest numbers, but monocytes and
lymphocytes also accumulate to a lesser degree. These cells wander
through the vascular endothelium to congregate in the areas of
injury. The granulocytes survive in the extravascular space for up
to about three days, after which the mononuclear cells, monocytes
and lymphocytes, become the dominant population.
[0121] Two phases are associated with a vascular insult. The first
phase involves a brief early increase in vascular permeability. The
more prolonged second phase involves increased permeability,
attachment of leukocytes, mainly granulocytes, to the vessel wall,
diapedesis of predominately leukocytes through the vessel wall,
accumulation of leukocytes in the injured area, leukocyte
phagocytosis, leakage of fibrinogen and platelets from the vessel,
fibrin deposition in the injured area, intravascular clotting with
vessel destruction, macrophage engulfment of necrotic debris,
migration of fibroblasts and formation of connective tissue, and
the neovascularization by ingrowth of capillaries. Infiltration by
leukocytes, particularly granulocytes, is a relatively early and
significant event in the response to vascular insult.
[0122] The well-developed atherosclerotic plaque is a result of the
interplay of inflammatory and repair events, resulting in a lesion
consisting of extracellular calcium salts, cholesterol crystals,
glycosaminoglycans, and blood cells and plasma components.
Endothelial permeability of arterial walls is induced in early
stages of atherosclerosis, allowing the afflux of circulating
macromolecules and blood cells, particularly leukocytes (and mainly
granulocytes). Secondary changes may involve reduction in
permeability of the arterial intima, and the later deposition of
platelets and/or fibrin, proliferative, degenerative, necrotic, and
repair processes that result in atheromatous lesions. Here again,
an early component is the concentration and extravasation of
leukocytes in the injured area.
[0123] With regard to clots, when vessels are injured, plugging may
occur by the formation of fibrin, the aggregation of platelets, and
combinations of both. During these events, leukocyte sticking and
aggregation, independent of platelet aggregation, occurs. Very
early, even before fibrin formation, extravasation of leukocytes
takes place.
[0124] Deep vein thrombosis (DVT) and pulmonary embolism are very
common in the general population, affecting 30% to 60% of otherwise
healthy men and women, and up to 80% in high-risk patients. It has
been estimated that as much as 20% of all hospital patients are
affected with thromboembolic events. In the U.S. alone, it has been
estimated that 2.5 million cases occur each year (Sherry, Sem.
Nucl. Med. 7: 205-211, 1977).
[0125] The majority of commonly used nuclear medicine tests for
deep vein thrombosis (DVT) involve nonspecific radiopharmaceuticals
employed for radionuclide venography. There is thus an ongoing need
for a thrombosis-specific radiopharmaceutical for specific,
sensitive, and rapid disclosure of thrombi by non-invasive external
scintigraphy. Contrast venography, a common radiological method,
has been the "gold standard" for DVT, but it has a high incidence
of side effects which limit its repeated use (Rabinov and Paulin,
Arch. Surg. 104:134-144, 1972). Compression B-mode ultrasound is
also of use for diagnosing the presence of thrombi in the legs, but
this is region-limited and, again, not lesion-specific (Lensing et
al., N. Engl. J. Med. 320:342-345, 1989). Hence,
radiopharmaceuticals are being sought to achieve simplicity,
rapidity, and specificity for the detection and diagnosis of
DVT.
[0126] Where the aforementioned imaging agents may be useful for
DVT, they may fail to disclose pulmonary emboli, which are
life-threatening lesions. Different thrombi may require different
agents. Venous thrombi consist primarily of polymers of fibrin with
entrapped cells, alternating with layers of platelets, whereas
arterial thrombi are made up primarily of aggregated platelets with
less fibrin (Freiman, in: Coleman et al., eds, Hemostasis and
Thrombosis--Basic Principles and Clinical Practice. New York, N.Y.,
Lippincott, 56: 766-780, 1982).
[0127] For the most part, the agents available appear to be
restricted to either fibrin-directed or platelet-directed
pharmaceuticals, as reviewed by Knight, Sem. Nucl. Med. 20:52-67,
1990. Fibrin-specific radiopharmaceuticals include radiolabeled
fibrinogen, soluble fibrin, antifibrin antibodies and antibody
fragments, fragment E.sub.1 (a 60 kDa fragment of human fibrin made
by controlled plasmin digestion of crosslinked fibrin), plasmin (an
enzyme in the blood responsible for dissolution of fresh thrombi),
plasminogen activators (e.g., urokinase, streptokinase and tissue
plasminogen activator), heparin, and fibronectin (an adhesive
plasma glycoprotein of 450 kDa).
[0128] Platelet-directed pharmaceuticals include radiolabeled
platelets, antiplatelet antibodies and antibody fragments,
anti-activated-platelets, and anti-activated-platelet factors,
which have been reviewed by Knight (Id.), as well as by Koblik et
al., Sem. Nucl. Med. 19:221-237 1989, all of which are included
herein by reference. Platelet imaging is most useful during the
acute phase of thrombosis, when active platelet aggregation occurs,
so that these platelet-based imaging methods have difficulty in
disclosing clots that are older than about 24 to about 48 hours
(Oster et al., Proc. Natl. Acad. Sci. USA 82:3465-3468, 1985).
Another concern is that platelet imaging may be inhibited by
concurrent heparin administration in the treatment of these
patients (Seabold et al., J. Nucl. Med. 29:1169-1180, 1988).
Heparinization can also reduce the total number of lesions found
with anti-fibrin antibodies (Alavi et al., J Nucl. Med. 29:825,
1988). In comparison to antifibrin antibodies, fragment El that is
radiolabeled appears to demonstrate clots earlier (Koblik et al.,
supra). However, the fragment E.sub.1 is difficult to isolate and
prepare, and its binding to blood clots is transient (Knight et al,
Radiology 156:509-514, 1985).
[0129] Inadequate blood and oxygen supply to the myocardium,
inducing symptoms of myocardial ischemia or ischemic heart disease,
are the usual events resulting from stenotic coronary
atherosclerosis. Acute and total coronary artery occlusion results
in severe ischemia and, consequently, myocardial infarction.
Chronologically, in the first hour, subcellular changes of ischemic
heart muscle manifest as mitochondrial granules, reduction of
glycogen and respiratory enzymes. Thereafter, from about 1 to about
6 hours, margination and clumping of nuclear chromatin, loss of
nuclear and myofilament architecture, and infiltration with
granulocytes, are observed. In the next phase, from about 6 to 12
hours, typical ischemic necrosis is seen. After 24 hours, severe
histological changes are easily seen, leading to focal hemorrhage
of different size and dilated capillaries by days 2-4.
[0130] Accordingly, the present invention provides compositions and
methods for the detection and/or treatment of cardiovascular
disorders, including fibrin clots, deep vein thrombosis, emboli,
ischemia, and atherosclerotic plaques.
[0131] I.B.2. Anatomically Displaced or Ectopic Tissues and
Organs
[0132] The invention provides compositions and methods for treating
a mammal having a hypoplastic, absent, anatomically displaced or
ectopic tissue or organ. Where normal organs or tissues are
developed abnormally or are displaced in the body, or are
insufficiently removed during ablative surgery, the
tissue/organ-associated antibodies may be used as tissue-targeted
vehicles for delivering therapeutic agents to the tissues in order
to induce their involution or chemical and/or isotopic ablation.
The antibodies or their fragments, or recognition moities, can be
conjugated to or administered in combination with therapeutic
modalities including, but not limited to, isotopes, drugs, toxins,
photodynamic therapy agents, cytokines, hormones, autocrines, etc.,
which are used as cytotoxic or modulating agents, and which have
hitherto been employed principally as toxic conjugates to
cancer-targeting antibodies, as described in reviews by Waldmann,
Science 252:1657, 1991; Koppel, Bioconjug. Chem. 1:13, 1990;
Oeltmann and Frankel, FASEB J. 5:2334, 1991; and van den Bergh,
Chemistry in Britain, May 1986, 430-439, each of which is
incorporated by reference herein in its entirety.
[0133] The method comprises the steps of (a) parenterally injecting
a mammalian subject, at a locus and by a route providing access to
the tissue or organ, with an amount of a scintigraphic imaging
agent or magnetic resonance image enhancing agent sufficient to
permit a scintigraphic image or an enhanced magnetic resonance
image of the structure to be effected; and (b) obtaining a
scintigraphic image or an enhanced magnetic resonance image of the
structure, at a time after injection of the agent sufficient for
the agent to accrete in the structure. The targetable complex
comprises an antibody or antibody fragment that specifically binds
to the organ or tissue, and further comprises a bioactive
(therapeutic) agent. In the case of naked antibodies, the complex
itself may be biologically active and induce processes such as
apoptosis.
[0134] Tissues, organs, and conditions of interest include but are
not limited to:
[0135] (1) hypoplastic or absent tissue or organs, in conditions
such as, juvenile diabetes, wherein the islet cells of the pancreas
can be atrophic or significantly reduced; thymic aplasia or
agenesis; DiGeorge's Syndrome wherein there is a hypoplasia or
absence of parathyroid and the thymus;
[0136] (2) ectopic tissue and organs, such as, implants of
endometrial glands and stroma;
[0137] (3) retained tissue, such as, retained placental tissue
after pregnancy, and organ remnants after surgical removal of the
organ;
[0138] (4) the condition of organs adjacent to a surgically removed
organ; and
[0139] (5) ablation of certain normal organs and tissues for other
therapeutic purposes, such as the spleen in patients with immune
disease or lymphomas, the bone marrow in patients requiring bone
marrow transplantation, or normal cell types involved in
pathological processes, such as certain T-lymphocytes in particular
immune diseases.
[0140] The above methods of the invention include the use of a
growth factor receptor antibody or a hormone receptor antibody to
target to end-organs bearing such receptor(s), the functions of
which can be blocked with said antibodies. An isotopic or drug
conjugate of these antibodies can also be used to deliver a
therapeutic agent to said tissues and organs, in order to affect
diseases of tissues which bear such receptors. For example, in
endometriosis, involving ectopic endometrial tissue, the current
standard drug therapy involves administration of a synthetic
steroid derived from ethisterone (DANOCRINE brand of danazol),
which is chemically a 17-alpha-Pregna-2,3-dien-20-yno[-
2,3,3-d]-isoxazol-17-ol. This probably acts, at least in part, on
sex steroid metabolism and with sex hormone receptors, particularly
follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at
the target organ. It is now possible to use an antibody against
these gonadal steroid receptors, alone or as an immunoconjugate
with isotopes, drugs, toxins, hormone antagonists, cytokines,
autocrines, etc., to inactivate and make the ectopic endometrium
atrophic.
[0141] The above methods of the invention include providing an
immunological method of affecting ovarian and other hormone
end-organ function, such as to induce amenorrhea or sterility. By
use of an ovarian-targeting antibody or an antibody to an
ovarian-related hormone receptor, such as FSH receptor, either as
unconjugated antibodies or as antibodies conjugated with a
therapeutic principle, a relatively convenient and safe method of
blocking ovarian function and inducing atrophy at the end-organ can
be achieved.
[0142] Many hormone and growth factor receptors are known, and
frequently show sufficient organ and tissue proclivity to allow
these to serve as targets for antibodies which, when bound to said
receptors, affect the function of the tissues and result in an
immunological or, by the use of conjugates with drugs, a chemical
ablation, or a radiation ablation when used as a conjugate with
therapeutic isotopes.
[0143] Another application is in the treatment of fibrocystic
breast disease. An antibody to FSH receptor or to estrogen receptor
can be given alone or as an immunoconjugate with a therapeutic
principle to decrease the fibrocystic disease and to control its
symptoms.
[0144] Still another indication is in benign prostatic hyperplasia
or prostatic cancer, where the use of an antibody against an
androgen receptor can alone, or as a conjugate with a therapeutic
principle (hormone end-organ antagonist, cytotoxic drug, toxin, or
isotope), can decrease the prostatic tissue proliferation.
[0145] Another therapeutic application for such organ- and
tissue-targeting antibodies conjugated with a toxic agent is for
the ablation of certain normal cells and tissues as part of another
therapeutic strategy, such as in bone marrow ablation with
antibodies against bone marrow cells of particular stages of
development and differentiation, and in the cytotoxic ablation of
the spleen in patients with lymphoma or certain immune diseases,
such as immune thrombocytopenic purpura, etc.
[0146] Another therapeutic application for such organ- and
tissue-targeting antibodies or fragments is to link them to a
cytoprotective agent to form therapuetic conjugates. The conjugate
is administered to a patient undergoing chemotherapy or radiation
therapy so that the targeted normal organs and tissues are
protected during the therapy.
[0147] I.B.3. Cancer
[0148] The present invention further provides compositions and
methods for treating a disease state selected from the group
consisting of a carcinoma, a melanoma, a sarcoma, a neuroblastoma,
a leukemia, a glioma, a lymphoma and a myeloma. Specific
tumor-associated antigens may be associated with a type of cancer
selected from the group consisting of acute lymphoblastic leukemia,
acute myelogenous leukemia, biliary, breast, cervical, chronic
lymphocytic leukemia, chronic myelogenous leukemia, colorectal,
endometrial, esophageal, gastric, head and neck, Hodgkin's
lymphoma, lung, medullary thyroid, non-Hodgkin's lymphoma, ovarian,
pancreatic, glioma, melanoma, liver cancer, prostate, and urinary
bladder. A tumor-associated antigen may be selected from the group
consisting of A3, the antigen specific for the A33 antibody, BrE3,
CD1, CD1a, CD3, CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD25,
CD30, CD33, CD45, CD74, CD79a, CD80, NCA90, NCA 95, HLA-DR, CEA,
CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, KC4, KS-1,
KS1-4, Le-Y, S100, MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4, PSA, PSMA,
AFP, HCG and ist subunits, RS5, TAG-72, tenascin, IL-6, insulin
growth factor-1 (IGF-1), Tn antigen, Thomson-Friedenreich antigens,
tumor necrosis antigens, VEGF, 17-1A, an angiogenesis marker, a
cytokine, an immunomodulator, an oncogene marker (e.g., p53), and
an oncogene product.
[0149] Tumor-associated markers have been categorized by Herberman
(see, e.g., Immunodiagnosis of Cancer, in THE CLINICAL BIOCHEMISTRY
OF CANCER, Fleisher ed., American Association of Clinical Chemists,
1979) in a number of categories including oncofetal antigens,
placental antigens, oncogenic or tumor virus associated antigens,
tissue associated antigens, organ associated antigens, ectopic
hormones and normal antigens or variants thereof. Occasionally, a
sub-unit of a tumor-associated marker is advantageously used to
raise antibodies having higher tumor-specificity, e.g., the
beta-subunit of human chorionic gonadotropin (HCG) or the gamma
region of carcinoembryonic antigen (CEA), which stimulate the
production of antibodies having a greatly reduced cross-reactivity
to non-tumor substances as disclosed in U.S. Pat. Nos. 4,361,644
and 4,444,744. Markers of tumor vasculature (e.g., VEGF), of tumor
necrosis, of membrane receptors (e.g., folate receptor, EGFR), of
transmembrane antigens (e.g., PSMA), and of oncogene products can
also serve as suitable tumor-associated targets for antibodies or
antibody fragments. Markers of normal cell constituents which are
overexpressed on tumor cells, such as B-cell complex antigens, as
well as cytokines expressed by certain tumor cells (e.g., IL-2
receptor in T-cell malignancies) are also suitable targets for the
antibodies and antibody fragments of this invention.
[0150] The BrE3 antibody is described in Couto et al., Cancer Res.
55:5973s-5977s, 1995. The EGP-1 antibody is described in U.S.
Provisional Application Ser. No. 60/360,229, some of the EGP-2
antibodies are cited in Staib et al., Int. J. Cancer 92:79-87,
2001; and Schwartzberg et al., Crit. Rev. Oncol. Hematol. 40:17-24,
2001. The KS-1 antibody is cited in Koda et al., Anticancer Res.
21:621-627, 2001; the A33 antibody is cited in Ritter et al.,
Cancer Res. 61:6854-6859, 2001; Le(y) antibody B3 is described in
Di Carlo et al., Oncol. Rep. 8:387-392, 2001; and the A3 antibody
is described in Tordsson et al., Int. J. Cancer 87:559-568,
2000.
[0151] Also of use are antibodies against markers or products of
oncogenes, or antibodies against angiogenesis factors, such as
VEGF. VEGF antibodies are described in U.S. Pat. Nos. 6,342,221,
5,965,132 and 6,004,554, and are incorporated by reference in their
entirety. Antibodies against certain immune response modulators,
such as antibodies to CD40, are described in Todryk et al., J.
Immunol. Meth. 248:139-147, 2001 and Turner et al., J. Immunol.
166:89-94, 2001. Other antibodies suitable for combination therapy
include anti-necrosis antibodies as described in Epstein et al.,
see e.g., U.S. Pat. Nos. 5,019,368; 5,882,626; and 6,017,514.
[0152] I.B.4. Autoimmune Diseases
[0153] The present invention further provides compositions and
methods for treating an autoimmune disease or disorder.
Immunothereapy of autoimmune disorders using antibodies which
target B-cells is described in PCT Application Publication No. WO
00/74718, which claims priority to U.S. Provisional Application
Ser. No. 60/138,284, the contents of each of which is incorporated
herein in its entirety. Exemplary autoimmune diseases are acute
idiopathic thrombocytopenic purpura, chronic idiopathic
thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,
myasthenia gravis, systemic lupus erythematosus, lupus nephritis,
rheumatic fever, polyglandular syndromes, bullous pemphigoid,
diabetes mellitus, Henoch-Schonlein purpura,
post-streptococcalnephritis, erythema nodosurn, Takayasu's
arteritis, Addison's disease, rheumatoid arthritis, multiple
sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme,
IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis,
Goodpasture's syndrome, thromboangitisubiterans, Sjogren's
syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,
thyrotoxicosis, scleroderma, chronic active hepatitis,
polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,
Wegener's granulomatosis, membranous nephropathy, amyotrophic
lateral sclerosis, tabes dorsalis, giant cell
arteritis/polymyalgia, pemiciousanemia, rapidly progressive
glomerulonephritis and fibrosing alveolitis.
[0154] I.C. Therapeutic Applications
[0155] I.C.1. Photodynamic Diagnosis or Therapy (PDT)
[0156] The present mutant bsAb can be used in a method of
photodynamic therapy (PDT) as discussed in U.S. Pat. Nos.
6,096,289; 4,331,647; 4,818,709; 4,348,376; 4,361,544; 4,444,744;
5,851,527.
[0157] In PDT, a photosensitizer, e.g., a hematoporphyrin
derivative such as dihematoporphyrin ether, is administered to a
subject. Anti-tumor activity is initiated by the use of light,
e.g., 630 nm. Alternate photosensitizers can be utilized, including
those useful at longer wavelengths, where skin is less
photosensitized by the sun. Examples of such photosensitizers
include, but are not limited to, dihematoporphyrin, benzoporphyrin
monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated
aluminum phthalocyanine (AISPc) and lutetium texaphyrin
(Lutex).
[0158] Radionuclides useful in therapeutic agents, which
substantially decay by beta-particle emission include, but are not
limited to: P-32, P-33, Sc-47, Fe-59, Cu-64, Cu-67, Se-75, As-77,
Sr-89, Y-90, Mo-99, Rh-105, Pd-109, Ag-111, I-125, I-131, Pr-142,
Pr-143, Pm-149, Sm-153, Th-161, Ho-166, Er-169, Lu-177, Re-186,
Re-188, Re-189, Ir-194, Au-198, Au-199, Pb-211, Pb-212, and Bi-213.
Maximum decay energies of useful beta-particle-emitting nuclides
are preferably 20-5,000 keV, more preferably 100-4,000 keV, and
most preferably 500-2,500 keV.
[0159] Radionuclides useful in therapeutic agents which
substantially decay with Auger-emitting particles include, but are
not limited to: Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,
In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192. Decay energies
of useful beta-particle-emitting nuclides are preferably <1,000
keV, more preferably <100 keV, and most preferably <70
keV.
[0160] Radionuclides useful in therapeutics and which substantially
decay with generation of alpha-particles include, but are not
limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211,
Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of useful
alpha-particle-emitting radionuclides are preferably 2,000-9,000
keV, more preferably 3,000-8,000 keV, and most preferably
4,000-7,000 keV.
[0161] Metals useful, as complexes, as part of a photodynamic
therapy procedure include, but are not limited to zinc, aluminum,
gallium, lutetium and palladium.
[0162] Therapeutically useful immunoconjugates can be obtained by
conjugating photoactive agents or dyes to an antibody composite.
Fluorescent and other chromogens, or dyes, such as porphyrins
sensitive to visible light, have been used to detect and to treat
lesions by directing the suitable light to the lesion. In therapy,
this has been termed photoradiation, phototherapy, or photodynamic
therapy (Jori et al., eds., Photodynamic Therapy of Tumors and
Other Diseases (Libreria Progetto 1985); van den Bergh, Chem.
Britain 22:430, 1986). Moreover, monoclonal antibodies have been
coupled with photoactivated dyes for achieving phototherapy. Mew et
al., J. Immunol. 130:1473, 1983; idem., Cancer Res. 45:4380, 1985;
Oseroffet al., Proc. Natl. Acad. Sci. USA 83:8744, 1986; idem.,
Photochem. Photobiol. 46:83, 1987; Hasan et al., Prog. Clin. Biol.
Res. 288:471, 1989; Tatsuta et al., Lasers Surg. Med. 9:422, 1989;
Pelegrin et al., Cancer 67:2529, 1991. However, these earlier
studies did not include use of endoscopic therapy applications,
especially with the use of antibody fragments or subfragments.
Thus, the present invention contemplates the therapeutic use of
immunoconjugates comprising photoactive agents or dyes.
[0163] I.C.2. Boron Neutron Capture Therapy (BNCT)
[0164] BNCT is a binary system designed to deliver ionizing
radiation to tumor cells by neutron irradiation of tumor-localized
boron-10 atoms. BNCT is based on the nuclear reaction which occurs
when a stable isotope, isotopically enriched B-10 (present in 19.8%
natural abundance), is irradiated with thermal neutrons to produce
an alpha particle and a Li-7 nucleus. These particles have a path
length of about one cell diameter, resulting in high linear energy
transfer. Just a few of the short-range 1.7 MeV alpha particles
produced in this nuclear reaction are sufficient to target the cell
nucleus and destroy it. Barth et al., Cancer 70:2995-3007,
1992.
[0165] Historically, BNCT was first employed for the treatment of
glioblastoma (a fatal form of brain tumor) and other brain tumors
at a time when tumor specific substances were almost unknown.
Hatanaka et al., in Boron Neutron Capture Therapy for Tumors,
pp.349-78 (Nishimura Co., 1986). One of the first boronated
compounds employed, a sulfhydryl-containing boron substance called
sodium borocaptate or BSH (Na.sub.2, B.sub.12H.sub.11 SH), crosses
the blood-brain barrier to localize in brain, and this has been the
anatomical basis for neutron capture therapy of brain tumors.
Clinical trials have been carried out, or are scheduled, for the
treatment of gliomas in Japan, the US and Europe. Barth et al.,
Cancer, supra. Problems with previous inorganic boron therapy
methods was that the boron reached both targeted and non-target
areas. Accordingly, when the boron was irradiated, healthy cells as
well as cancerous cells were destroyed.
[0166] The BNCT concept has been extended to other cancers, spurred
on by the discovery of a number of tumor-localizing substances,
including tumor-targeting monoclonal antibodies. For instance,
boronated amino acids such as p-boronophenylalanine accumulated in
melanoma cells. The potential of using boronated monoclonal
antibodies directed against cell surface antigens, such as CEA, for
BNCT of cancers has been demonstrated. Ichihashi et al., J. Invest.
Dermatol. 78:215-18, 1982; Goldenberg et al., Proc. Natl. Acad.
Sci. USA 81:560-63, 1984; Mizusawa et al., Proc. Natl. Acad. Sci.
USA 79:3011-14, 1982; Barth et al., Hybridoma 5(supp. 1):543-5540,
1986; Ranadive et al., Nucl. Med. Biol. 20: 663-68, 1993.
[0167] Success with BNCT of cancer requires methods for localizing
a high concentration of boron-10 at tumor sites, while leaving
non-target organs essentially boron-free. Compositions and methods
for treating tumors in patients using pre-targeting bsAb for BNCT
are described in U.S. application Ser. No. 09/205,243 and can
easily be modified in accordance with the present invention.
Additionally, other elements are suitable for neutron capture
reactions. Nuclides useful in therapies based on neutron capture
procedures include, but are not limited to: B-10, Gd-157 and U-235.
Uranium, in large amounts, can be bound by naturally occurring
chelating agents such as ferritin.
[0168] II. Pre-Formed Targetable Complexes
[0169] In therapeutic embodiments of the present invention, the
bi-specific antibodies may be adminstered at some time prior to
administration of the targetable construct. However, it is also
possible to mix targetable constructs and bi-specific antibodies
prior to administration, and thus to form "pre-formed" targetable
complexes that are then administered to a subject. Targetable
complexes are also useful in ex vivo and in vitro modalities.
[0170] II.A. Pre-Targeting Applications
[0171] In an exemplary method that does not involve pre-targeting,
the targetable construct comprises a bioactive moiety. In this
case, the targetable construct is administered following
administration of the bsAb. The bioactive agent is targeted to the
target site because the targetable construct is recognized by and
binds to the bsAb, which is itself bound to a targeted tissue.
[0172] In an alternative method, a targetable construct comprising
a bioactive agent is mixed with its cognate bsAb prior to
administration to the patient, thus forming a targetable complex
comprising a bioactive agent. A targetable complex formed in this
fashion is administered and binds its targeted tissue, thereby
effecting direct delivery of the bioactive agent as a part of the
targetable complex comprising the agent.
[0173] The latter or pre-targteing modality has several potential
advantages over methods in which the targetable constructs and bsAb
are separately administered. The total amount of targetable
construct and bsAb that needs to be administered in order to be
effective may be less than in non-pre-targeting modalities,
particuarly if the targetable complex is relatively stable under
physiological conditions. In addition, a targetable complex
according to the invention may have a higher affinity for the
targeted tissue that the targetable construct per se, thereby
providing compositions and methods for more effective delivery of
the bioactive agent to the targeted tissue.
[0174] Pre-formed targetable complexes may be used in any of the
compositions and methods of the invention. One skilled in the art
will be able to determine what site of complex formation (i.e., in
vitro or in situ) is appropriate for any given application.
[0175] II.B. Immunoaffinity-Based Applications
[0176] II.B.1. Immunoaffinity
[0177] Immunoaffinity is known in the art and generally involves
the immobilization of antibodies to a solid support, often in the
form of beads, that are then packed into a column. A sample
containing an antigen recognized by the antibody is passed through
the column, wherein the antigen is bound and retained by the
immobilized antibodies. The antigen is then washed off the column
using any of a variety of methods known in the art. Depending on
the particular circumstances, the antigen may be a substance that
is being purified, or the antigen can be a contaminant that is
being removed. For further details and reviews, see Springer,
Section 10.11, Immunoaffinity Chromatography, Chapter 10 in: Short
Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John
Wiley and Sons, New York, 1992, pages 10-43 to 10-45); Affinity
Chromatography: A Practical Approach, edited by Dean P. D. G.,
Johnson, W. S., Middle, F. A., IRL Press,1985; Immunoaffinity
Purification: Basic Principles and Operational Considerations,
Yarmush et al., Biotech Adv. 10:412-446, 1992.
[0178] Immunoaffinity comprises three general steps: adsorption,
washing and elution. In the first step, absorption, a substance of
interest is bound by an antibody. Absorption is accomplished by,
e.g., contacting a sample containing the substance of interest with
an antibody bound to a solid support matrix in a suitable medium
within a column. The next step is a washing step wherein impurities
present in the fluid volume of the column, as well as those bound
nonspecifically to the antibody, solid support or column walls, are
removed. Washing is accomplished by passing a volume of a wash
solution, e.g., buffer, such as phosphate buffered saline (PBS)
through the column. The volume of wash solution used in the washing
step should not be so great as to result in loss of the substance
of interest but not so limited so as not to remove impurities. In
the elution step, the target molecule is removed from the column
by, e.g., addition of a solvent or other solution, or change in
conditions such as temperature or pressure, that reduces the
affinity of the substance of interest to the antibody or the
affinity of the complex formed between an antibody and molecules of
the substance of interest to the solid support. Elution of an
antibody coupled to the substance of interest may be accomplished
by either a salt gradient, to change the pH; buffered
step-gradient, to change the ionic strength; or other methods known
in the art.
[0179] Elution of the target molecule may be accomplished by a
number of methods. There are no covalent bonds involved in the
interaction between antibody and the substance of interest. Thus,
the conditions of the buffer may be changed such that the affinity
of the antibody:substance complex falls sufficiently to reduce the
amount of effective binding to each other or to the solid support.
This may be achieved by altering the pH or the ionic strength of
the buffer, or both, or by chaotropic ions, e.g., cyanates.
Increased separation may be obtained by gradient elution. In the
case of immunosorption, the binding of a substance of interest to
its antibody may be so strong that more harsh elution conditions
are necessary, such as the use of buffers which are very strongly
acidic or basic. Other methods of elution include use of chaotropic
agents such as KSCN; organic solvents, e.g., ethylene glycol, DMSO,
or acetonitrile; denaturing agents, e.g., 8 M urea or 6 M guanine;
electrophoretic elution; pressure induced elution and metal ion
elution. Preferably, the elution conditions allows for complete or
mostly complete elution of the product after one or two column
volumes have passed through the column.
[0180] Various impurities can be present and may have an
unpredictable and adverse affect on the composition as it is used
in the pharmaceutical industry. In the case of biological samples,
typical impurities are blood clots, tissue debris, hair, foreign
particles, activated coagulation factors, denatured proteins,
plasma-free hemoglobin (e.g., irrigation fluid) added into a wound
site, human viruses, antigens and antibiotics.
[0181] By way of non-limiting example, a sample may be passed
through an immunoaffinity column having an immobilized antibody
directed against the substance of interest. The immobilized
antibodies react with and bind molecules of the substance of
interest in the sample, thereby absorbing them and removing them
from the solution. Although the substance of interest is retained
on the column, impurities pass through the column. The column can
then be washed with a buffer solution to remove any impurities
remaining on the column, e.g., impurities retained by non-specific
binding. The column is washed free of impurities and any substance
bound to the column is eluted with a solvent. This process is known
as positive immunoabsorption. In negative immunoabsorption, in
contrast, the substances of interest present in the crude
preparation pass freely through the column while the antigenic
impurities bind with antibodies and are held by the column.
[0182] II.B.2. Immobilization of Antibodies
[0183] A solid support or matrix is used to immobilize antibodies.
The matrix may possess desirable characteristics including,
macroporosity, mechanical stability, ease of activation,
hydrophilicity, and inertness, i.e., low nonspecific adsorption.
Matrices commonly used by those skilled in the art include
cross-linked dextran, agarose, polyacrylamide, cellulose, silica
and poly(hydroxyethylmethacrylate). For immuno-adsorbents, beaded
agarose is a preferred solid support by those skilled in the art
due to its high adsorptive capacity for proteins, high porosity,
hydrophilicity, chemical stability, lack of charge and relative
inertness toward nonspecific adsorption.
[0184] Antibodies may be physically adsorbed to matrices or
covalently attached to polymeric matrices containing hydroxylic or
amino groups by means of bifunctional reagents, such as those
disclosed herein. Attachment typically requires two steps,
activation of the matrix and coupling of the ligand to the
activated matrix. Activated matrices are available commercially.
The selection method for coupling the ligand to the matrix is
dictated in part by the choice of matrix and, in part, by the
choice of antibody. Most methods commonly used to immobilize
peptide or polypeptide ligands, such as antibodies, are based on
coupling of amino groups. The polypeptide ligand must be coupled in
a manner that will not interfere with its ability to be recognized
by the target molecule. Methods for activation and coupling are
commonly used by those skilled in the art.
[0185] For successful use of affinity chromatography, the
polymer-bound ligand must be sufficiently distant from the polymer
surface to minimize steric interference. This is accomplished by
inserting an interconnecting link or spacer between the antibody
and the matrix. The spacer may be bound directly to the matrix so
that the antibody can be attached directly to these spacers. Types
of spacers commonly used by those skilled in the art include but
are not limited to cystamine, p-aminobenzoic acid, tyramine and
p-hydroxy-mercuribenzoate.
[0186] II.B.3. Beads
[0187] In embodiments wherein the solid support is a bead, the bead
may be any of a variety of types, depending upon the application.
For immunopurification, porous beads are often used. The beads may
be prepared from commercially available beads that are derivatized
with amino or carboxyl groups that are available for linkage to a
protein or other capture moiety using, for example, glutaraldehyde,
carbodiimide, diazoto compounds, or any other suitable crosslinking
reagent.
[0188] II.B.3.a. Magnetic Beads
[0189] Targetable complexes may be attached to magnetic particles
via functional groups that coat the particles. In a purification
application, a sample containing an antigen comprising a target
epitope binds to the attached targetable complex, and the
conjugated magnetic particle is removed from suspension by the
application of a magnetic field.
[0190] Magnetic beads or particles, such as magnetic latex beads
and iron oxide particles, to which the targetable complexes of the
invention may be attached, are known in the art. For example,
magnetic particles are described in U.S. Pat. No. 4,672,040.
Coupling of capture moieties to magnetic beads can be accomplished
using known methods. For example, beads are commercially available
that are derivatized with amino or carboxyl groups that are
available for linkage to a protein or other capture moiety using,
for example, glutaraldehyde, carbodiimide, diazoto compounds, or
other suitable crosslinking reagent. Silanization of magnetically
responsive particles provides one method of obtaining reactive
groups on the surface of the particles (see, e.g., U.S. Pat. No.
4,672,040 for a description of silanization and silane coupling
chemistry). Linking bonds can include, for example, amide, ester,
ether, sulfonalmide, disulfide, azo, and others known to those of
skill in the art.
[0191] Superparamagnetic particles, which can be made from a number
of substances such as polystyrene or iron oxide and
polysaccharides, are magnetic when placed in a magnetic field, but
retain no residual magnetism when removed from the magnetic field.
This lack of residual magnetism ensures that the particles can be
repeatedly separated and resuspended without magnetically induced
aggregation.
[0192] II.B.3.b. Beads for Immunoaffinity Purification
[0193] Beads may be coated with the targetable complexes of the
invention for use in immunoaffinity purification. Generally, such
beads are of a size, composition and structure suitable for use in
flow-through columns. Porous beads may be used. By way of
non-limiting example, such beads can be made of Sephadex.RTM.,
Sepharose, agarose, glass, and polystyrene.
[0194] II.B.4. Preparation of an Immunoaffinity Column
[0195] In column immunoaffinity, a column comprising a solid
support onto which the construct or complex is immobilized is
prepared. The preparation of the column depends on the type of
solid support used, the chemical or physical nature of the samples
to be processed through the column, reagents, e.g., washing and
elution solutions, and the like.
[0196] It may be desirable to equilibrate the column before
application of a sample. The buffering conditions used for
equilibrating the affinity column in preparation for sample
application will reflect the specific properties of the interacting
system being used. The nature of the buffer used, including its pH
and ionic strength, are adjusted for the particular antibody and
substance of interest. The sample comprising the substance of
interest that is applied to the column typically contained in the
same buffer used to equilibrate the column. After sample
application and absorption, the column is washed with the starting
buffer to remove any unbound sample and any impurities. It may also
be preferable in some instances to wash the column with buffers
different from the starting buffer in order to remove
nonspecifically adsorbed substances.
[0197] II.C. Manufacturing Embodiments
[0198] In addition to being useful for purifying compounds of
interest from mixtures of compounds, immunoafinity can be used to
remove undesirable substances from mixtures in manufacturing and
other applications. Exemplary undesiarble substances include, but
are not limited to, contaminants, undesirable reaction products
and/or catalysts including but not limited to enzymes, that are
used during manufacturing processes.
[0199] The immunoaffinity aspects of the invention may also be
applied to manufacturing processes. A manufacturing process can be
a "continuous process," in which the substance of interest is
continually produced and harvested from an ongoing manufacturing or
production process. In contrast, in a "batch" approach in
manufacturing, multiple reparations are combined and then
harvested. Regardless of the type of manufacturing process, the
compositions of the invention can be used at any of a variety of
steps in the process.
[0200] A sample or manufacturing preparation may be "clarified"
prior to further preparation in order to remove contaminants
(including without limitation, in chemical syntheses, reaction
byproducts and unreacted compounds) produce a sample containing
only, or enriched for, the substance of interest. Additionally or
alternatively, a substance of interest may be partially purified,
substantially purified or purified. A substance is said to be
"partially purified" when it comprises .gtoreq.50% w/w of a
composition; "substantially purified" when it comprises .gtoreq.75%
of a composition, and "purified" when it comprises .gtoreq.90%,
preferably .gtoreq.95%, more preferably .gtoreq.99% and most
preferably .gtoreq.99.9% of a composition. Generally, clarification
of a sample removes a limited number of undesirable compounds from
a preparation without changing the concentration of the substance.
In contrast, the purification of a substance generally refers to a
process by which the substance is preferentially removed from a
sample, leaving behind a variety of contaminants; the separated
substance may be moved to a new solution in which its concentration
is higher.
[0201] Impurities can be removed from a preparation of a substance
of interest by negative or positive immunoabsorption techniques. A
preparation so treated is said to be enriched for the substance of
interest, and the substance of interest in the preparation is said
to be partially purified, substantially purified or purified. The
substance purified in this manner may be an antibody that
specifically binds the carrier eptitope of the targetable
construct, or a [target epitope]:[bi-specific antibody]
complex.
[0202] When the latter type of complex is prepared, the target
epitope may be further purified by treatment with agents or
conditions that reduce the affinity of the bi-specific antibody for
the target epitope. In instances where a targetable construct
comprising a carrier epitope is attached to a solid support, and a
bi-specific antibody is bound to the carrier epitope, the [target
epitope]:[bi-specific antibody] complex can be separated from the
bound targetable construct by addition of an excess amount of the
carrier epitope. By way of non-limiting example, in the case of IMP
246 bound to a solid support, a complex comprising a bispecific
antibody that is bound thereto may be removed from the bound IMP
246 by the addition of an excess amount of the chelator
corresponding to the chelator moiety present on IMP 246, i.e.,
DTPA.
[0203] For example, a sample may be passed through an
immunoaffinity column having an immobilized antibody directed
against the substance of interest. The immobilized antibodies react
with and bind molecules of the substance of interest in the sample,
thereby absorbing them and removing them from the solution.
Although the substance of interest is retained on the column,
impurities pass through the column. The column can then be washed
with a buffer solution to remove any impurities remaining on the
column, e.g., impurities retained by non-specific binding. The
column is washed free of impurities and any substance bound to the
column is eluted with a solvent. This process is known as positive
immunoabsorption. In negative immunoabsorption, in contrast, the
substances of interest present in the crude preparation pass freely
through the column while the antigenic impurities bind with
antibodies and are held by the column.
[0204] II.D. Immunoassays and Other In vitro Immunochemical
Methods
[0205] The targetable complexes of the present invention may be
used as reagents in a variety of in vitro immunochemical methods.
Immunochemical methods include, but are not limited to, Western
blotting, immunoaffinity purification, immunoprecipitation, ELISA,
dot or slot blotting, radioimmunoassay (RIA), immunohistochemical
staining, immunocytochemical staining, and flow cytometry.
[0206] Such methods may, but need not in every instance, involve
the attachment of a targetable complexes to a solid support. The
term "solid support" refers to a material having a solid surface to
which a targetable complex is immobilized. By "immobilized" it is
meant bound covalently, or bound by noncovalent means such as
hydrophobic adsorption. By way of non-limiting example, a solid
support may be the surface of a multiwell (microtiter) plate well,
a bead, a membrane or a dipstick. Methods and means for covalently
or noncovalently binding proteins to solid supports are known in
the art.
[0207] Suitable solid supports include, by way of illustration and
not limitation, latex, glass particles, including porous glass
particles; polyacrylamide particles; agarose; SEPHADEX.RTM.
(Pharmacia Fine Chemicals, Inc.); sepharose; bibulous materials
such as glass or cellulose paper; plastics and polymers (e.g., in
sheets, beads or microtiter wells) such as polystyrene, polyvinyl
chloride, polystyrene latex, or polyvinylidine fluoride (known as
Immulon.quadrature.); nylon; polymethacrylate; etc.; silicons;
metals such as gold and indium; nitrocellulose nitrocellulose
(e.g., in membrane or microtiter well form); activated beads;
Protein A beads; diazotized paper; and the like.
[0208] The nature of the solid surface varies depending upon the
intended use or method. For assays carried out in microtiter wells,
e.g., in multiwell (microtiter) plates, the solid surface is the
wall of the well or cup. For assays using beads, the solid surface
is the surface of the bead. In assays using a dipstick (i.e., a
solid body made from a porous or fibrous material such as fabric or
paper) the surface is the surface of the material from which the
dipstick is made. In agglutination assays the solid surface may be
the surface of latex or gelatin particles. When individual antigens
are bound to a solid surface they may be distributed homogeneously
on the surface or distributed thereon in a pattern, such as bands
so that a pattern of antigen binding may be discerned.
[0209] II.D.1. Immunoassays
[0210] The design of immunoassays is subject to a great deal of
variation, and many formats are known in the art. Immunochemical
Protocols, Humana Press, Totowa, N.J., 1998; Current Protocols in
Immunology, Greene Pub. Associates and Wiley-Interscience, New
York, N.Y., 1997. Protocols may, for example, use solid supports,
or immunoprecipitation. Most assays involve the use of labeled
targetable complex or antigen. As used in this section, an
"antigen" is a substance that is or comprises a targetable epitope.
The labels may be, for example, enzymatic, fluorescent,
chemiluminescent, radioactive, or dye molecules. Assays which
amplify the signals are known; examples of which are assays which
utilize biotin and avidin, enzyme-labeled and mediated
immunoassays, such as ELISA, RIA, immunofluorescence,
chemiluminescence and nephelometry.
[0211] Typically, standard ELISA techniques are employed using
labelled antibody or antigen. The label can be an enzyme,
fluorophore, chemiluminescent material, radioisotope, or coenzyme.
Generally enzyme labels such as alkaline phophatase, or beta
galactosidase are employed together with their appropriate
substrates. The enzyme/substrate reaction can be detected by any
suitable means such as spectrophotometry.
[0212] The immunoassay may be, without limitation, in a
heterogenous or in a homogeneous format, and of a standard or
competitive type. In a heterogeneous format, the targetable
construct or complex is typically bound to a solid support to
facilitate separation of the sample therefrom after incubation. The
solid support containing the targetable construct or complex is
typically washed after separating it from the test sample, and
prior to detection of bound antigens. In a homogeneous format, the
test sample is incubated with the combination of targetable
constructs or complexes in solution. For example, it may be under
conditions that will precipitate any targetable complex/antigen
assemblages that are formed. Both standard and competitive formats
for these assays are known in the art.
[0213] In a standard format, the amount of antigen bond to the
immobilized targetable construct or complex is directly monitored.
This may be accomplished, for example, by detecting labeled
anti-xenogenic (e.g., anti-human) antibodies that recognize an
epitope on the bsAbs or targetable construct. In a competitive
format, the amount of antigens in a sample is deduced by monitoring
the competitive effect on the binding of a known amount of labeled
antigen (or other competing ligand) added to the sample before or
during the assay.
[0214] Targetable constructs or complexes may be immobilized to the
inner surface of microtiter wells and the test sample and
prelabeled target epitopes added to the wells. After a select
period, the wells are washed and the color developed on the floor
of the wells from the antibody-antigen reaction examined. By use of
an automatic reader, the results of numerous tests can be
determined in a few minutes.
[0215] II.D.2. Immunoassay Kits
[0216] The targetable complexes may be packaged in the form of a
kit for use in immunoassays. The kit contain in separate containers
the separate combination of targetable constructs, targetable
complexes and bsAbs (either already bound to a solid matrix or
separate with reagents for binding them to the matrix), control
antibody formulations (positive and/or negative), labeled antibody
when the assay format requires same and signal generating reagents
(e.g., enzyme substrate) if the label does not generate a signal
directly. Instructions (in any of a number of formats, e.g.,
written, tape, VCR, CD-ROM, etc.) for carrying out the assay may be
included in the kit.
[0217] Test kits according to the invention for comprising a solid
support that is an immunoassay contain, for example, a suitable
container, coated with a targetable construct or targetable complex
of the invention, optionally freeze-dried or concentrated solutions
of a targeted epitope and/or a labelled derivative thereof,
standard solutions of this protein, buffer solutions and,
optionally, polypeptides and detergents for preventing non-specific
adsorption and aggregate formation, pipettes, reaction vessels,
calibration curves, instruction manuals and the like.
[0218] II.D.3. Dipsticks
[0219] The targetable constructs and complexes may be used in a
"dipstick" or sheet which is capable of being inserted into and
withdrawn from a sample. The dipstick is dipped into a well mixed
urine sample, and after a time period of thirty seconds to two
minutes, the various reagent bands are visually or optically
examined for color changes. The bands can be visually compared to a
preprinted color chart in order to determine the amount of each of
the constituents or parameters being measured.
[0220] In a typical dipstick based analytical assay, a ligand,
which specifically binds to the analyte of interest, is bound to a
solid support on the dipstick. The dipstick is contacted with a
sample in which the presence of the analyte of interest is to be
determined. Frequently, steps are employed to aid in the removal of
non-specifically bound material from the dipstick. Finally, the
dipstick is processed to determine the presence of the analyte. The
dipstick generally comprises a solid material, which is planar or
columnar in geometry.
[0221] II.E. Ex vivo Therapeutic Modalities
[0222] The compositions and devices of the invention, and methods
of use thereof, may be used in ex vivo modalities. An "ex vivo
modality" is one in which a biological sample, such as a body
fluid, is temporarily removed from an animal, altered through in
vitro manipulation designed to remove or inactivate one or more
undesirable substances, and then returned to the body. One way in
which undesirable substances may be removed from the sample is by
contacting the sample with an agent that binds the undesirable
substance. In an ex vivo modality of the invention, a sample that
has been temporarily removed from a patient is contacted with a
solid support comprising a targetable complex of the invention. In
this embodiment, the undesirable substance is or comprises a target
epitope that is recognized by the targetable complex of the solid
support. The undesirable substance is, or is part of, e.g., a
toxin, a hyperproliferative cell, an infected cell or a pathogen.
For example, for the treatment of viremia, a virus that comprises a
target epitope is cleared from blood by contacting the blood with a
solid support comprising a targetable complex of the invention. The
targetable complex recognizes and binds the virus, which is
retained in the dialysis system but not in the blood that is
returned to the patient.
[0223] An exemplary ex vivo modality of the invention is a
hemodialysis system, which comprises a dialysis machine. A
"dialysis machine" is a device in which a fluid such as blood of an
animal is temporarily removed therefrom and processed through one
or more physical, chemical, biochemical or other types of processes
designed to remove or inactivate undesirable substances. Bodily
waste products, toxins, venoms, overexpressed or overactive
endogenous agents, molecules derived from any of the preceding, and
pathogens comprising any of the preceding, are non-limiting
examples of undesirable substances. In an exemplary mode, a human
is treated by a dialysis machine that augments or substitutes for
the natural kidney functions of a human body. Blood is removed from
the body, passed through the dialysis machine, which separates the
wastes from the blood extracorporeally. The separated wastes are
discharged and disposed of, whereas the treated blood is returned
to the body.
[0224] The transfer of blood between the patient and the dialyzer
occurs within a blood tubing set that is usually disposable. The
blood tubing set and the dialyzer represent a closed extracorporeal
path through which a patient's blood travels. The blood tubing set
includes an arterial line connected to an arterial reservoir for
drawing blood from a patient, a venous line connected to a venous
reservoir for returning blood to the patient, and a number of other
lines for connecting a pump and the dialyzer between the arterial
and venous reservoirs. Before the blood tubing set and the dialyzer
can be used in a dialysis treatment, both must be primed with a
sterile saline solution to remove air from the extracorporeal
circuit. Once primed, the saline solution is recirculated through
the blood tubing set and the dialyzer to produce a stabilized flow
and remove additional trapped air from within the extracorporeal
circuit. The priming and recirculating process also serves to clean
the dialyzer and flush the dialyzer membrane of any debris or
chemicals remaining from a prior use.
[0225] A commonly used method of creating blood access for
hemodialysis is by means of an arteriovenous fistula. For each
dialysis session, the fistula must be punctured with large bore
needles to deliver blood into, and return blood from, the
artificial kidney (dialyzer). Even with the use of anesthetics, the
punctures with these large bore needles are painful. Patients
undergoing dialysis thus benefit if the punctures can be done as
infrequently as possible. Moreover, frequent punctures may be
detrimental to the longevity of the fistula.
[0226] Existing hemodialysis systems consist fundamentally of two
halves; one comprising the extracorporeal blood circuit (the blood
flow path) and the other comprising the dialysate circuit or flow
path. Typically, the entire blood circuit is disposable and
comprises: (1) an arterial and venous fistula needle, (2) an
arterial (inflow) and venous (outflow) blood line, (3) a
hemodialyzer, (4) one or more physiologic priming solutions (e.g.,
saline), and (5) one or more anticoagulants (e.g., heparin or
citrate).
[0227] The arterial fistula needle accesses blood from the
patient's fistula and is connected to the arterial blood tubing
set, which conveys blood to the dialyzer. The arterial line
comprises a pumping segment with interfaces to a blood pump (which
may be, e.g., a rotary or peristaltic pump) on the dialysis
machine, pressure or flow monitoring chambers including tubing
which interfaces to pressure or flow transducers on the machine to
monitor the pressure and flow pre-pump and/or post pump, inlet
ports for saline and anticoagulant, and one or more injection sites
for, e.g., drawing blood or injecting drugs.
[0228] The hemodialyzer typically comprises a case which encloses a
bundle of hollow fiber semi-permeable membranes, which are usually
made from cellulose or synthetic polymers. The blood is circulated
on one side of a semipermeable membrane, and the dialysis solution
is circulated on the other side, so that the two never come into
direct contact. Waste products (uremic toxins) diffuse out of the
blood, across the semipermeable membranes, and into the dialysis
solution owing to the concentration gradient. Excess water in the
patent's blood enters the dialysate as a result of a pressure
gradient.
[0229] The venous blood line and venous fistula needle carry the
newly dialyzed blood away from the dialyzer and back into the
patient's circulatory system via a puncture site slightly closer to
the heart than the arterial needle site. The venous set is
comprised of a pressure monitoring chamber with tubing leading to
another pressure transducer in the machine, injection sites, and a
segment of tubing which interfaces to an air detection assembly in
the machine in order to prevent air emboli during treatment.
[0230] A dialysis machine has several systems and components. In an
extracorporeal flow path, which conducts blood from the patient to
the dialyzer and then back to the patient, at least one arterial
blood pump and sometimes a venous blood pump that move the blood
and assist in performing certain types of dialysis treatment such
as ultrafiltration. A hydraulics flow path, which conducts the
dialysate through the dialyzer, includes numerous components to
monitor and control the conditions in that flow path. Flow and
pressure meters may be included, typically at the inlet and outlet
of the dialyzer. A first dialysate pump moves dialysate into the
dialyzer, and a second dialysate pump removes the dialysate from
the dialyzer. A heater may be included to heat the dialysate to
body temperature to avoid undesirable heat transfer to or from the
patient, and/or to heat a disinfecting solution to temperatures
adequate to kill microorganisms. Other components may be included
in dialysis machines designed for specific applications. For
example, in ultrafiltration dialysis treatments, an ultrafiltration
pump is used to control the delivery of desirable components to the
blood.
[0231] Proportioning pumps for one or more dialysis solutions may
be included. Dialysis solution is typically prepared continuously
on-line in present-day machines by combining water which has first
been purified by a separate water treatment system, and liquid
concentrates of electrolytes. Dialysate concentrates have evolved,
from a single formulation which contained acetate as the
physiologic buffering agent for the correction of circulatory
acidosis, to two container systems where bicarbonate replaces
acetate as the buffering agent, and must be kept separate due to
its chemical incompatibility with calcium and magnesium. Two
proportioning pumps are therefore required, the first to mix the
bicarbonate concentrate with water and the second to proportion
this mixture with the concentrated electrolytes to achieve the
final, physiologically compatible dialysis solution.
[0232] The dialysis machine continuously monitors the pressure at
the blood inlet and outlet sides of the dialyzer (by way of the
pressure transducers connected to the blood sets) as well as in the
dialysate circuit. Via microprocessors, the system calculates the
transmembrane pressure (TMP) which determines the amount of water
transmission through the membranes. Dialysis machines may also
comprise a device for measuring the amount of dialysis solution
entering and dialysate leaving the dialyzer, which allows the
calculation of net water removal from the patient. By
electronically comparing the amount of water entering or leaving
the blood with the TMP, the system is able to control actively the
water removed from the patient to a desired target previously
programmed into the system. When low-water-transmission cellulosic
membranes are employed, negative pressure is generated on the
dialysate side of the membrane by the machine in order to
accomplish sufficient water removal. Because suction may be applied
to the dialysate as it transits the dialyzer, it is first be placed
under a greater vacuum in a degassing chamber so that air bubbles
are not generated within the dialyzer that would cause errors in
the calculation of ultrafiltration by the flow sensors and also
reduce the efficiency of the dialyzer. In contrast, when
high-water-transmission, synthetic membranes are used, it is
frequently necessary to apply positive pressure on the dialysate
side to control the rate of ultrafiltration.
[0233] Another non-limiting example of ex vivo therapeutic
applications of the invention is the use of the compositions of the
invention in devices for the intraoperative and post-surgical
salvaging of blood. In this embodiment, a patient's own blood lost
during intraoperative and/or post-surgical procedures, is washed
and filtered and then reinfused into the patient. For a
non-limiting examplary device of this type, see U.S. Pat. No.
5,876,611.
[0234] III. Molecular Scaffolds and Targetable Constructs
[0235] II.A. Structure of Molecular Scaffolds and Targetable
Constructs
[0236] The targetable construct(s) present in a targetable complex
comprises a molecular scaffold which comprises or bears at least
two pairs of carrier epitopes recognized by the arm of an antibody
or antibody fragment in the complex.
[0237] The targetable construct can be of diverse structure, but is
selected not only to elicit sufficient immune responses, but also
for rapid in vivo clearance when used within the bsAb targeting
method. Exemplary targetable constructs for use in the present
application are described in U.S. application Ser. No. 09/337,756
filed Jun. 22, 1999, and in U.S. application Ser. No. 09/823,746,
filed Apr. 3, 2001, the entire contents of which are incorporated
herein by reference.
[0238] Hydrophobic agents are best at eliciting strong immune
responses, whereas hydrophilic agents are preferred for rapid in
vivo clearance, thus, a balance between hydrophobic and hydrophilic
needs to be established. This may be accomplished in a preferred
approach, in part, by relying on the use of hydrophilic chelating
agents to offset the inherent hydrophobicity of many organic
moieties. Also, sub-units of the targetable construct may be chosen
which have opposite solution properties, for example, peptides,
which contain amino acids, some of which are hydrophobic and some
of which are hydrophilic. Aside from peptides, carbohydrates may be
used. Additionally, the targetable construct can comprise PEG
(poly[ethylene] glycol) derivatives to increase its circulation
time in a patient.
[0239] Peptides having as few as one amine residue may be used,
preferably two to ten amino acid residues, if also coupled to other
moieties such as chelating agents. Examples include modified amino
acids, such as bis-DTPA-lysine, and bis-DTPA-diamine. 1
[0240] These agents can be linked covalently to molecules which are
to be targeted. The hapten moiety of the carrier portion should be
a low molecular weight conjugate, preferably having a molecular
weight of 100,000 daltons or less, and advantageously less than
about 20,000 daltons, 10,000 daltons or 5,000 daltons, including
the metal ions in the chelates. For instance, the known peptide
di-indium-DTPA-Tyr-Lys(DTPA)-OH has been used to generate
antibodies against the indium-DTPA portion of the molecule.
However, by use of the non-indium-containing molecule, and
appropriate screening steps, new Abs against the tyrosyl-lysine
dipeptide can be made. More usually, the antigenic peptide will
have four or more residues, such as the peptide
Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH.sub.2. Again, the
non-metal-containing peptide is used as an immunogen, with
resultant Abs screened for reactivity against the Phe-Lys-Tyr-Lys
backbone. Another non-limiting example of an antigenic peptide
having four or more residues is the peptide
DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH.su- b.2, wherein DOTA is
1,4,7,10-tetraazacyclododecanetetraacetic acid and HSG is the
histamine succinyl glycyl group of the formula:
[0241] The non-metal-containing peptide may be used as an
immunogen, with resultant Abs screened for reactivity against the
Phe-Lys-Tyr-Lys backbone.
[0242] In one embodiment, unnatural amino acids, e.g., D-amino
acids, are incorporated into the backbone structure to ensure that,
when used with the final bsAb/linker system, the scFv component
which recognizes the linker moiety is completely specific. The
invention further contemplates other backbone structures such as
those constructed from non-natural amino acids, peptoids,
peptidomimetics, aptamers, peptide nucleic acids (PNAs), and the
like.
[0243] According to one embodiment of the invention, the targetable
construct can encompass a carbohydrate. Suitable such carbohydrates
include carbohydrate chains of two to six sugar units long. The
targetable construct also can comprise a polymeric carbohydrate,
such as dextran.
[0244] In another embodiment of the invention, the haptens of the
targetable construct comprise a known immunogenic recognition
moiety, for example, a known hapten. Using a known hapten, for
example, fluorescein isothiocyanate (FITC), higher specificity of
the targetable construct for the antibody is exhibited. This occurs
because antibodies raised to the hapten are known and can be
incorporated into the inventive antibody. Thus, binding of the
targetable construct with the attached chelator or metal-chelate
complex would be highly specific for the inventive antibody or
antibody fragment. Another example of a hapten to be substituted
onto the targetable construct includes vitamin B12. The use of
vitamin B12 is advantageous since anti-B12 Mabs are known and no
free serum B12 exists, therefore, great specificity for the
antibody may be exhibited. The chelator or its chelate with a metal
cation also can function as the hapten to which an antibody is
raised. Another example of a hapten to be conjugated to a
targetable construct includes biotin.
[0245] III.B. Preparation of Molecular Scaffolds and Targetable
Constructs
[0246] Peptides, including but not limited to, peptides to be used
as molecular scaffolds or immunogens are synthesized conveniently
on an automated peptide synthesizer using a solid-phase support and
standard techniques of repetitive orthogonal deprotection and
coupling.
[0247] Free amino groups in the peptide that are to be used later
for chelate conjugation are advantageously blocked with standard
protecting groups such as an Aloc group. Such protecting groups
will be known to the skilled artisan. See Greene and Wuts,
Protective Groups in Organic Synthesis, John Wiley and Sons, New
York (1999) and Kates S A, Albericio F. Solid-Phase Synthesis: A
Practical Guide. Marcel Dekker, New York (2000). Methods of
synthesizing amino acid-based polymers and glycosylated peptides
are described in, respectively, Sanda and Endo, Syntheses and
Functions of Polymers Based on Amino Acids, Macromol. Chem. Phys.
200:2651-2661, 1999; and Sears and Wong, Toward automated synthesis
of oligosaccharides and glycoproteins, Science 291:2344-2350,
2001.
[0248] III.C. Chemical Conjugation
[0249] Molecular scaffolds may be prepared as a single molecule, or
may be generated by first preparing subunits that are then
covalently attached to each other. The term "conjugation" is used
to indicate the covalent attachment of two or more molecules.
[0250] Peptides are conjugated (i.e., linked, or covalently
attached), to one another using various methods. By way of
non-limiting example, amino acid residues present in the natural
sequence of a first peptide can be directly covalently linked to
amino acid residues in the natural amino acid sequence of a second
peptide as in, e.g., a disulfide bridge; or a cross-linking reagent
(also known as "cross-linker"), typically a bifunctional
(two-armed) chemical linker that forms covalent linkages between
two or more peptides, can be used to covalently link peptides to
each other. Such bifunctional linkers can be homobifunctional
(wherein both "arms" of the linker are the same chemical moiety) or
heterobifunctional (wherein each of the two "arms" is a different
chemical moiety than the other).
[0251] Hermanson (Bioconjugate Techniques, Academic Press, 1996),
herein incorporated by reference, summarizes many of the chemical
methods used to link proteins and other molecules together using
various reactive functional groups present on various cross-linking
or derivatizing reagents. Cross-linking agents are based on
reactive functional groups that modify and couple to amino acid
side chains of proteins and peptides, as well as to other
macromolecules. Cross-linking reagents incorporate two or more
functional reactive groups. The functional reactive groups in a
cross-linking reagent may be the same or different. Many different
cross-linkers are available to cross-link various proteins,
peptides, and macromolecules. Table I lists some of the
cross-linkers that are easily available through commercial sources
according to their class of chemical reactivity. Table 2 lists some
of the properties of chemical cross-linkers and the types of
functional groups with which they react.
1TABLE 1 CLASSES OF CHEMICAL REACTIVITY OF CROSS-LINKERS AND
EXAMPLES OF CROSS-LINKERS Chemical reactivity Abbreviation Compound
homobifunctional DMA Dimethyl adipimidate.2 HCl imidoesters DMP
Dimethyl pimelimidate.2 HCl DMS Dimethyl suberimidate.2 HCl DTBP
Dimethyl 3,3'-dithiobispropionimidate.2HCl homobifunctional N- DSG
Disuccinimidyl glutarate hydroxysuccinimide esters (NHS-esters)
DMSC Dimethyl succinimidate.2 HCl DSS Disuccinimidyl suberate BS
DSP Dithiobis(succinimidylprop- ionate) DTSSP
Dithiobis(sulfosuccinimidylpropionate) DTME
Dithio-bis-maleimidoethane EGS Ethylene glycolbis(succinimidylsuc-
cinate) Sulfo-EGS Ethylene glycolbis(sulfosuccinimidylsuccinate)
DST Disuccinimidyl tartrate Sulfo-DST Disulfosuccinimidyl tartrate
BSOCOES Bis[2- (succinimidooxycarbonyloxy)ethyl- ]sulfone Sulfo-
Bis[2- BSCOCOES (sulfosuccinimidooxycarbon- yloxy)ethyl]sulfone
heterobifunctional BS3 BIS-(sulfosuccinimidyl) suberate NHS-esters
DMM dimethyl malonimidate.2 HCl EMCS
N-[.epsilon.-maleimidocaproyloxy]succinimide ester Sulfo-EMCS
N-[.epsilon.-maleimidocaproyloxy]sulfosuccinimide ester SMCC
succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxyla- te
LC-SMCC succiminidyl-4-(N- maleimidomethyl)cyclohexane-
-1-carboxy-(6- amido-caproate) Sulfo-MBS m-maleimidobenzoyl-N-
hydoxysulfosuccinimide ester Sulfo-SMCC sulfosuccinimidyl 4-(N-
maleimidomethyl)cyclohexane-1- -carboxylate MBS
m-maleimidobenzoyl-N-hydoxysuccinimide ester SMPB succinimidyl
4-[P-Maleimidophenyl] butyrate Sulfo-SMPB sulfosuccinimidyl 4-[p-
maleimidophenyl]butyrate BMH bismaleimidohexane GMBS
N-[.gamma.-Maleimidobutyryloxy] succinimide ester Sulfo-GMBS
N-[.gamma.-Maleimidobutyryloxy] sulfosuccinimide ester
heterobifunctional SIAB N-succinimidyl(4-iodoacetyl)aminobenzoate
haloacetyl NHS-esters Sulfo-SIAB Sulfo-SIAB sulfosuccinimidyl(4-
iodoacetyl)aminobenzoate homobifunctional DPDPB 1,4-Di-[3'-(2'-
pyridyldithiols pyridyldithio)propionamido]butane
heterobifunctional SMPT 4-succinimidyloxycarbonyl-methyl-(2-
pyridyldithiols pyridyldithio)-toluene Sulfo-LC- sulfosuccinimidyl
6-[a-methyl-a-(2-pyridyl- SMPT dithio)toluamido]hexanoate SPDP
N-succinimidyl 3-(2-pyridyldithio)propionate LC-SPDP N-succinimidyl
6-[3'-(2- pyridyldithio)propionamido]hexanoate Sulfo-LC-
sulfosuccinimidyl 6-[3'-(2-pyridyldithio)- SPDP propionamido]
hexanoate carboxyl reactive PDPH 3-(2-Pyridyldithio) propionyl
hydrazide carbonyl reactive EDC 1-ethyl-3-(3-dimethylamino-
propyl)- carbodiimide M2C2H 4-(N-Maleimidomethyl)cyclohex- ane-1-
carboxyl hydrazide DCC N,N-dicyclohexylcarbodimide MPBH
4-(4-N-Maleimidophenyl)butyric acid hydrazide hydrochloride
Photoreactive ABH Azidobenzoyl hydrazide ANB-NOS
N-5-azido-2-nitrobenzoyloxysuccinimide APDP
N-[4-(p-azidosalicylamido)butyl]-3'(2'- pyridyldithio)propionami-
de APG p-Azidophenylglyoxal monhydrate ASBA
4-(p-Azidosalicylamido)butylamine ASIB 1-(p-Azidosalicylamido)-4-
(iodoaceamido)butane BASED Bis-[B-4-azidosalicylamido)et-
hyl]disulfide HSAB N-Hydroxysuccinimidyl-4-azidobenzoate Sulfo-HSAB
N-Hydroxysulfo-succinimdyl-4-azidobenzoate NHS-ASA
N-Hydroxysuccinimidyl-4-azidosalicylic acid Sulfo-NHS-
N-Hydroxysulfo-succinimidly-4-azidosalicylic ASA acid Sulfo-NHS-
Sulfosuccinimidly-[4-azidosalicylamido)- LC-ASA hexanoate PNP-DTP
p-Nitropheyno-2-diazo-3,3,3- trifluoropropionate DTP
2-Diazo-3,3,3-trifluoropropionylchloride SADP
N-succinimidyl-(4-azidopheynyl 1,3' dithiopropionate Sulfo-SADP
Sulfosuccinimidyl-(4- azidophynyldithio)propionate SAED
Sulfosuccinimidyl 2(7-azido-4-
methylcoumarin-3-acetamide)ethyl-1,3- dithiopropionate Sulfo-
Sulfosuccinimidyl 7-azido-4-methycoumarin- SAMCA 3-acetate SAND
Sulfosuccinimidyl 2-(m-azido-o-
nitrobenzamdio)-ethyl-1,3-dithiopropionate SANPH
N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate Sulfo-
Sulfosuccinimidyl 6-(4'-azido-2'- SANPH nitrophenylamino)hexanoate
SASD Sulfosuccinimidyl 2-(p-
azdiosalicylamido)ethyl-1,3'-dithiopropionate Sulfo-SAPB
Sulfosuccinimidyl 4-(p-azidophenyl)-butyrate Heterobifunctional
SDBP N-Hydroxysuccinimidyl 2,3- amine reactive dibromopropionate
Bifunctional aryl DFDNB 1,5-Difluoro-2.4-dinitrobenzene halide
heterobifunctional mal-sac- maleimido-6-aminocaproyl-ester of
nitrophenylsulfonic HNSA 1-hydroxy-2-nitrobenzene-4-sulfonic acid
acid ester
[0252]
2TABLE 2 CHEMICAL CROSS-LINKERS AND SOME OF THEIR PROPERTIES Pierce
Spacer Arm Product Length Cleavable Water Membrane Acronym Number
(angstroms) Links By Soluble Permeable Sulfo-LC-SMPT 21568 20.0
Amines To Thiols Yes No Sulfhydryls SMPT 21558 20.0 Amines To
Thiols Yes No Sulfhydryls Sulfo-KMUS 21111 19.0 Amines To non Yes
No Sulfhydryls LC-SMCC 22362 16.1 Amines To non Yes No Sulfhydryls
KMUA 22211 15.7 Amines To non Yes No Sulfhydryls LC-SPDP 21651 15.6
Amines To non No nd Sulfhydryls Sulfo-LC-SPDP 21650 15.6 Amines To
Thiols Yes No Sulfhydryls SMPB 22416 14.5 Amines To non No Yes
Sulfhydryls Sulfo-SMPB 22317 14.5 Amines To non Yes No Sulfhydryls
SMPH 22363 14.3 Amines To non No nd Sulfhydryls SMCC 22360 11.6
Amines to non No Yes Sulfhydryls Sulfo-SMCC 22322 11.6 Amines to
non Yes No Sulfhydryls SIAB 22329 10.6 Amines to non No Yes
Sulfhydryls Sulfo-SIAB 22327 10.6 Amines To non Yes No Sulfhydryls
Sulfo-GMBS 22324 10.2 Amines To non Yes No Sulfhydryls GMBS 22309
10.2 Amines To non No Yes Sulfhydryls MBS 22311 9.9 Amines To non
No Yes Sulfhydryls Sulfo-MBS 22312 9.9 Amines To non Yes No
Sulfhydryls Sulfo-EMCS 22307 9.4 Amines To non Yes No Sulfhydryls
EMCA 22306 9.4 Amines To non Yes No Sulfhydryls EMCS 22308 9.4
Amines To non No Yes Sulfhydryls SVSB 22358 8.3 Amines To non No
Yes Sulfhydryls BMPS 22298 6.9 Amines To non No nd Sulfhydryls SPDP
21857 6.8 Amines To Thiols No Yes Sulfhydryls SBAP 22339 6.2 Amines
To non No Yes Sulfhydryls BMPA 22296 5.9 Amines To non Yes No
Sulfhydryls AMAS 22295 4.4 Amines To non No nd Sulfhydryls SATP
26100 4.1 Amines To non No Yes Sulfhydryls SIA 22349 1.5 Amines To
non No nd Sulfhydryls Sulfo-LC-SMPT 21568 20.0 Sulfhydryls Thiols
Yes No to Amines SMPT 21558 20.0 Sulfhydryls Thiols No Yes to
Amines AEDP 22101 9.5 Carboxyls Thiols Yes No to Amines EDC 22980
0.0 Carboxyls non Yes No to Amines
[0253] Bifunctional cross-linking reagents may be classified
according to their functional groups, chemical specificity, length
of the cross bridge that they establish, the presence of similar
functional groups or dissimilar functional groups, chemical or
photochemical reactivity, ability to be cleaved internally by
reduction or other means, and the ability of the reagent to be
further modified by radiolabelling (i.e. radioiodination) or
addition of detectable tags or labels. The selective groups on the
cross-linking reagent can be present in a homobifunctional
arrangement in which the selective groups are identical, or can be
present in a heterobifunctional arrangement in which the selective
groups are dissimilar.
[0254] The chemical modification may be done using cross-linking
reagents containing selective groups that react with primary
amines, sulfhydryl (thiol) groups, carbonyl, carboxyl groups,
hydroxyl, or carbohydrates and other groups placed on a protein or
peptide, especially by posttranslational modifications within the
cell. The selective groups include, but are not limited to,
imidoester, N-hydroxysuccinimide ester or sulfosuccinimidyl ester,
ester of 1-hydroxy-2-nitrobenzene-4-sulfonic, maleimide, pyridyl
disulfide, carbodiimide, and haloacetyl groups.
[0255] Sulfhydryl reactive functional groups include maleimides,
alkyl and aryl halides, haloacyls, haloacetyls and pyridyl
disulfides. Maleimides, alkyl and aryl halides, haloacetyls and
haloacyls react with thiols to form stable thioether bonds that are
not reduced by reagents such as 2-mercaptoethanol and
dithiothreitol. Pyridyl disulfides form mixed disulfides with thiol
groups, mixed disulfides may be used as an intermediate for
cross-linking two or more macromolecules. Cross-linkers that first
react with a carboxyl group to form an activated intermediate and
then reacts with an amino group, such as an amino group of lysine
or an amino group of an amino terminal amino acid, may be used.
[0256] A spacer arm or "cross-bridge" region, consisting of a
spacer group or a functional group, such as a disulfide bond or
hindered disulfide bond, connects the two selective or functional
groups. The length of the spacer arm may be varied. The distance
between the functional groups establishes the length of the spacer
arm. Longer spacer arms may be required to diminish or eliminate
steric hindrance between two molecules that are cross-linked
together. Intermolecular cross-linking is more efficient with
longer spacer arms. Short spacer arms favor intramolecular
cross-linking, which is to be avoided in the present invention.
[0257] Spacer arms may have reactive bonds within them that enable
further modifications. For example, internal cleavable bonds may be
placed within the spacer, such as disulfides or hindered
disulfides, one or more ester bonds, or vicinal hydroxyl groups.
Cleavage of internal disulfide bonds may be achieved using
reduction with thiol containing reagents such as 2-mercaptoethanol
and dithiothreitol. One or more metabolizable bonds may be inserted
internally in the cross-linking reagent to provide the ability for
the coupled entities to separate after the bond(s) is broken after
the conjugate is transported into the cell and into the body.
[0258] Homobifunctional cross-linkers contain at least two
identical functional groups. Heterobifunctional cross-linkers
contain two or more functional reactive groups that react with
different specificity. Because heterobifunctional cross-linkers
contain different reactive groups, each end can be individually
directed towards different functional groups on proteins, peptides,
and macromolecules. This feature results in linking, for example,
amino groups on one molecular entity to carboxyl groups on another
entity, or amino groups on one entity to sulfhydryl groups on
another entity.
[0259] Functional groups include reactive portions on proteins,
peptides, and macromolecules that are capable of undergoing
chemical reaction. Functional groups include amino and carboxyl
groups, hydroxyl groups, phenolate hydroxyl groups, carbonyl
groups, guanidinyl groups, and carbon-carbon double bonds. In
addition, photoactive reagents that become reactive when exposed to
light may be used. For example, arylazides may be activated to form
activated intermediates, such as an aryl nitrene or a
dehydroazepine intermediate, that non-selectively inserts into
carbon-hydrogen bonds (i.e. by aryl nitrenes) or reacts with amines
(dehydroazepines). Other examples include fluorinated aryl azides,
benzophenones, certain diazo compounds, and diazrine
derivatives.
[0260] If the desired sulfhydryl groups are not present on the
protein, peptide or macromolecule, a sulfhydryl may be introduced
by chemical modification. As a nonlimiting example, the sFv or a
therapeutic macromolecule can be modified so as to introduce a
thiol by chemical modification. A cysteine amino acid can be placed
in a peptide during peptide synthesis. Sulfhydryl groups can be
added by chemical modification using 2-iminothiolane (IT), also
known as Traut's reagent.
[0261] Sulfhydryl groups can also be added by using a modification
reagent that contains a disulfide bond in addition to a group that
selectively reacts with primary amines. For example, the
heterobifunctional cross-linker sulfosuccinimidyl
6-[3'-(2-pyridyldithio)-propionamido] hexanoate (sulfo-LC-SPDP,
Pierce Chemical Co.) will thiolate peptides when used according to
the manufacturer's directions. Other, non-soluble, forms such as
N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pierce Chemical
Co.) or N-succinimidyl 6-[3'-(2-pyridyldithio)propionamido]hexan-
oate (LC-SPDP, Pierce Chemical Co.) can be used in these reactions
by dissolving in a suitable organic solvent to a concentration of
20 mM, and adding 25-1 to 1 ml of 10 mg/ml peptide. Reducing the
SPDP-derivatized peptide under mild conditions will release
pyridine-2-thione, leaving an aliphatic thiol. An example of a mild
reducing condition is to add {fraction (1/100)}th volume of 1M
dithiothreitol (DTT) to the above SPDP-derivatized target peptide
and incubating for 30 minutes at room temperature, or incubate the
SPDP-derivatized target peptide with 50 mM 2-meraptoethylamine in
PBS-EDTA for 90 minutes at 37.degree. C. The excess SPDP, LC-SPDP
or sulfo-LC-SPDP, and the pyridine-2-thione can then be removed by
HPLC purification.
[0262] These modification reagents may also contain groups near the
added thiol such that they form a hindered disulfide when oxidized.
These reagents, such as
4-succinimidyloxycarbonyl-methyl-(2-pyridyldithio)-tolu- ene
(SMPT), may result in a conjugate that exhibits increased stability
in vivo (Thorpe et al. Cancer Res. 47:5924-5931, 1987). Other
cross-linking reagents can be used for protein thiolation and are
known to those well versed in the art. Many of these reagents are
described in the Pierce Chemical Co. catalog, or by Ji, Meth.
Enzymol. 91:580-609, 1983; and Hermanson, Bioconjugate Techniques,
Academic Press, Inc., San Diego, 1-785, 1996.
[0263] Most commonly, carrier molecule scaffold portions will have
either sulfhydryl or primary amines as the targets of the
cross-linking reagents, and both sulfhydryl and primary amines can
either exist naturally or be the result of chemical modification as
described above. When both carrier molecular scaffold portions have
a reduced sulfhydryl, a homobifunctional cross-linker that contains
maleimide, pyridyl disulfide, or haloacetyl groups can be used for
cross-linking. Examples of such cross-linking reagents include, but
are not limited to, bismaleimidohexane (BMH) or
1,4-Di-[3'-(2'-pyridyldithio)propionamido]but- ane (DPDPB).
Alternatively, a heterobifunctional cross-linker that contains a
combination of maleimide, pyridyl disulfide, or haloacetyl groups
can be used for cross-linking. Less preferably, the cross-linking
reagent can contain thiophthalimide derivatives or disulfide
dioxide derivatives. Also, extrinsic sulfhydryl groups can be
introduced into the carrier molecular scaffold portions and
oxidized to cross-link by disulfide formation.
[0264] When primary amines are selected as the target both on sFv
and therapeutic macromolecule, then a homobifunctional cross-linker
that contains succinimide ester, imidoester, acylazide, or
isocyanate groups can be used for cross-linking. Examples of such
cross-linking reagents include, but are not limited to,
Disuccinimidyl glutarate (DSG), Dithiobis(succinimidylpropionate)
(DSP), Bis[2-(succinimidooxycarbonyloxy- )ethyl]sulfone (BSOCOES),
Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulf- one
(sulfo-BSCOCOES), Disuccinimidyl suberate (DSS),
Bis-(Sulfosuccinimidyl) Suberate (BS3), Disuccinimidyl tartrate
(DST), Disulfosuccinimidyl tartrate (sulfo-DST),
Dithio-bis-maleimidoethane (DTME), Ethylene
glycolbis(succinimidylsuccinate) (EGS),
Dithiobis(sulfosuccinimidylpropionate) (DTSSP), Ethylene
glycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS), Dimethyl
malonimidate.cndot.2 HCl (DMM), Dimethyl succinimidate.cndot.2 HCl
(DMSC), Dimethyl adipimidate.cndot.2 HCl (DMA), Dimethyl
pimelimidate.cndot.2 HCl (DMP), Dimethyl suberimidate.cndot.2 HCl
(DMS), and Dimethyl 3,3'-dithiobispropionimidate.cndot.2HCl (DTBP).
Heterobifunctional cross-linkers that contains a combination of
imidoester or succinimide ester groups can also be used for
cross-linking.
[0265] Heterobifunctional cross-linking reagents that combine
selective groups against different targets are generally preferred
because these allow reactions to be performed selectively and
sequentially, minimizing self-association or polymerization. Also,
heterobifunctional reagents allow selection of chemistry
appropriate for the individual molecules to be joined. Examples of
such cross-linking reagents include, but are not limited to,
N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl
6-[3'-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP),
sulfosuccinimidyl 6-[3'-(2-pyridyldithio)-propionamido] hexanoate
(sulfo-LC-SPDP), m-maleimidobenzoyl-N-hydoxysuccinimide ester
(MBS), m-maleimidobenzoyl-N-hydoxysulfosuccinimide ester
(sulfo-MBS), succinimidyl 4-[P-maleimidophenyl] butyrate (SMPB),
sulfosuccinimidyl 4-[p-maleimidophenyl] butyrate (sulfo-SMPB),
N-[Maleimidobutyryloxy] succinimide ester (GMBS),
N-[maleimidobutyryloxy] sulfosuccinimide ester (sulfo-GMBS),
N-[maleimidocaproyloxy] succinimide ester (EMCS),
N-[maleimidocaproyloxy] sulfosuccinimide ester (sulfo-EMCS),
N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB),
sulfosuccinimidyl(4-iod- oacetyl)aminobenzoate (sulfo-SIAB),
succinimidyl 4-(N-maleimidomethyl)cycl- ohexane-1-carboxylate
(SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclo-
hexane-1-carboxylate (sulfo-SMCC),
succiminidyl-4-(N-maleimidomethyl)cyclo-
hexane-1-carboxy-(6-amido-caproate) (LC-SMCC),
4-succinimidyloxycarbonyl-m- ethyl-(2-pyridyldithio) toluene
(SMPT), and sulfo-LC-SMPT.
[0266] IV. Chelate Moieties
[0267] The presence of hydrophilic chelate moieties on the
targetable construct helps to ensure rapid iii vivo clearance. In
addition to hydrophilicity, chelates are chosen for their
metal-binding properties, and are changed at will since, at least
for those targetable constructs whose bsAb epitope is part of the
peptide or is a non-chelated hapten, recognition of the
metal-chelate complex is no longer an issue.
[0268] The nature of the invention is such that several chelate
moities may be used in a targetable construct or complex. For
example, if two types of bsAbs are to be used in a complex, each of
which has a different binding specificity for a carrier epitope
(i.e., each of which binds a different type of chelate moiety),
then the targetable construct comprises both types of chelate
moieties. Those skilled in the art will be able to choose
appropriate chelate moieties depending on the nature and structure
of the targetable construct and bsAbs to be used, and the intended
application of the targetable constructs and complexes. If need be,
scFvs having specificities for novel carrier epitopes can be
generated and incorporated into a bsAb.
[0269] Particularly useful metal-chelate combinations include
2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with
.sup.47Sc, .sup.52Fe, .sup.55Co, .sup.67Ga, .sup.68Ga, .sup.111In,
.sup.89Zr, .sup.90Y, .sup.161Tb, .sup.177Lu, .sup.212Bi,
.sup.213Bi, and .sup.225Ac for radio-imaging and RAIT. The same
chelators, when complexed with non-radioactive metals, such as
manganese, iron and gadolinium can be used for MRI, when used along
with the bsAbs of the invention. Macrocyclic chelators such as
NOTA, DOTA, and TETA are of use with a variety of metals and
radiometals, most particularly with radionuclides of gallium,
ytrrium and copper, respectively.
[0270] DTPA and DOTA-type chelators, where the ligand includes hard
base chelating functions such as carboxylate or amine groups, are
most effective for chelating hard acid cations, especially Group
IIa and Group IIIa metal cations. Such metal-chelate complexes can
be made very stable by tailoring the ring size to the metal of
interest. Other ring-type chelators such as macrocyclic polyethers,
which are of interest for stably binding nuclides such as
.sup.223Ra for RAIT are encompassed by the invention. Porphyrin
chelators may be used with numerous radiometals, and are also
useful as certain non-radioactive metal complexes for bsAb-directed
immuno-phototherapy. More than one type of chelator may be
conjugated to a carrier to bind multiple metal ions, e.g.,
non-radioactive ions and/or radionuclides. One example is a
bis-.sup.111In-DTPA conjugate that also bears a DOTA-.sup.90Y
chelate. Particularly useful therapeutic radionuclides include, but
are not limited to .sup.32P, .sup.33P, .sup.47Sc, .sup.64Cu,
.sup.67Cu, Ga, .sup.90Y, .sup.111Ag, .sup.111In, .sup.125I,
.sup.131I, .sup.142Pr, .sup.153Sm, .sup.161Tb, .sup.166Dy,
.sup.166Ho, .sup.177Lu, .sup.186R, .sup.188Re, .sup.189Re,
.sup.212Pb, .sup.213Bi .sup.211At, .sup.223Ra and .sup.225Ac.
[0271] Examplary radioactive metal chelate complexes have been
described that use radionuclides such as cobalt-57 (Goodwin et al.,
U.S. Pat. No. 4,863,713), .sup.111In (Barbet et al., U.S. Pat. No.
5,256,395 and U.S. Pat. No. 5,274,076; Goodwin et al., J. Nucl.
Med., 33:1366-1372, 1992; Kranenborg et al., Cancer Res. (suppl.),
55:5864s-5867s, 1995, and Cancer (suppl.) 80:2390-2397, 1997) and
.sup.68Ga (Boden et al., Bioconjugate Chem. 6:373-379, 1995; and
Schuhmacher et al., Cancer Res. 55:115-123, 1995) for
radioimmuno-imaging.
[0272] Because the Abs were raised against the chelators and metal
chelate complexes, they have remarkable specificity for the complex
against which they were originally raised. Indeed, the bsAbs of
Boden et al. have specificity for single enantiomers of
enantiomeric mixtures of chelators and metal-chelate complexes.
[0273] Chelators such as those disclosed in U.S. Pat. No.
5,753,206, especially thiosemi-carbazonylglyoxylcysteine(TscG-Cys)
and thiosemicarbazinyl-acetylcysteine (TscA-Cys) chelators are
advantageously used to bind soft acid cations of Tc, Re, Bi and
other transition metals, lanthanides and actinides that are tightly
bound to soft base ligands, especially sulfur- or
phosphorus-containing ligands. It can be useful to link more than
one type of chelator to a peptide, e.g., a DTPA or similar chelator
for, say In(III) cations, and a thiol-containing chelator, e.g.,
TscG-Cys, for Tc cations. Because antibodies to a di-DTPA hapten
are known (Barbet, U.S. Pat. No. 5,256,395) and are readily coupled
to a targeting antibody to form a bsAb, it is possible to use a
peptide hapten with non-radioactive diDTPA chelates and another
chelate for binding a radioisotope, in a pretargeting protocol, for
targeting the radioisotope. One example of such a peptide is
Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(TscG-Cys-- )-NH.sub.2. This peptide
can be preloaded with In(III) and then labeled with 99-m-Tc
cations, the In(III) ions being preferentially chelated by the DTPA
and the Tc cations binding preferentially to the thiol-containing
TscG-CysC. Other hard acid chelators such as NOTA, DOTA, TETA and
the like can be substituted for the DTPA groups, and Mabs specific
to them can be produced using analogous techniques to those used to
generate the anti-di-DTPA Mab.
[0274] It will be appreciated that two different hard acid or soft
acid chelators can be incorporated into the targetable construct,
e.g., with different chelate ring sizes, to bind preferentially to
two different hard acid or soft acid cations, due to the differing
sizes of the cations, the geometries of the chelate rings and the
preferred complex ion structures of the cations. This will permit
two different metals, one or both of which may be radioactive or
useful for MRI enhancement, to be incorporated into a targetable
construct for eventual capture by a pre-targeted bsAb.
[0275] Chelators are coupled to the carrier portion of a targetable
construct using standard chemistries. For instance, excess
2-(p-isothiocyanato)benzyl-DTPA is reacted with peptide NH.sub.2
groups to form thiourea bonds between the p-isothiocyanate of the
chelator and the free 1-.alpha. and 6-.epsilon.-amino groups of the
peptide, when a peptide is the targetable construct. Alternatively,
the bis-anhydride of DTPA can be coupled directly to a free amine
group on the peptide. The desired chelator-peptide is purified
chromatographically and is ready for use as a metal binding agent.
Similarly, DOTA is mono-activated at one carboxyl group using a
carbodiimide, and two DOTA units are coupled to the peptide's free
amino-groups or DOTA tri-t-butyl ester is activated with a
carbodiimide and the DOTA units are coupled to the free amines on
the peptide. (The protecting groups are removed on cleavage from
the resin.) Chelators bearing groups specifically reactive with
thiols are used for reaction with peptides such as
Ac-Cys-D-Tyr-D-Trp-Gly-D-Cys-Gly-- D-Tyr-D-Trp-NH.sub.2. Such a
chelator is exemplified by 2-(p-bromoacetamido)benzyl-DTPA, which
may be used to alkylate the peptide's free thiol groups under mild,
neutral conditions.
[0276] Chelator-peptide conjugates may be stored for long periods
as solids. They may be metered into unit doses for metal-binding
reactions, and stored as unit doses either as solids, aqueous or
semi-aqueous solutions, frozen solutions or lyophilized
preparations. They may be labeled by well-known procedures.
Typically, a hard acid cation is introduced as a solution of a
convenient salt, and is taken up by the hard acid chelator and
possibly by the soft acid chelator. However, later addition of soft
acid cations leads to binding thereof by the soft acid chelator,
displacing any hard acid cations which may be chelated therein. For
example, even in the presence of an excess of non-radioactive
InCl.sub.3, labeling with .sup.99mTc(V) glucoheptonate or with Tc
cations generated in situ with stannous chloride and
Na99m-TcO.sub.4 proceeds quantitatively on the soft acid chelator.
Other soft acid cations such as .sup.186Re, .sup.188Re, .sup.213Bi
and divalent or trivalent cations of Mn, Co, Ni, Pb, Cu, Cd, Au,
Fe, Ag (monovalent), Zn and Hg, especially .sup.64Cu and .sup.67Cu,
and the like, some of which are useful for radioimmunodiagnosis or
radloimmunotherapy, can be loaded onto the carrier peptide by
analogous methods. Re cations also can be generated in situ from
perrhenate and stannous ions or a prereduced rhenium glucoheptonate
or other transchelator can be used. Because reduction of perrhenate
requires more stannous ion (typically above 200 ug/mL final
concentration) than is needed for the reduction of technetium,
extra care needs to be taken to ensure that the higher levels of
stannous ion do not reduce sensitive disulfide bonds such as those
present in disulfide-cyclized peptides. A convenient way to prepare
ReO metal complexes of the TscG-Cys-ligands is by reacting the
peptide with ReOCI.sub.3(P(Ph.sub.3).sub.2 but it is also possible
to use other reduced species such as
ReO(ethylenediamine).sub.2.
[0277] Preferred chelators include NOTA, DOTA and Tscg and
combinations thereof. These chelators have been incorporated into a
chelator-peptide conjugate motif as exemplified in the following
constructs:
[0278] (a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH.sub.2
[0279] (b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH.sub.2
[0280] (c) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH.sub.2 2
[0281] The chelator-peptide conjugates (d) and (e), above, have
been shown to bind .sup.68Ga and is thus useful in positron
emission tomography (PET) applications.
[0282] Chelators are coupled to the linker moieties using standard
chemistries which are discussed more fully in the working Examples
below. Briefly, the synthesis of the peptide
Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-- Cys-)-NH.sub.2 was
accomplished by first attaching Aloc-Lys(Fmoc)-OH to a Rink amide
resin on the peptide synthesizer. The protecting group
abbreviations "Aloc" and "Fmoc" used herein refer to the groups
allyloxycarbonyl and fluorenylmethyloxy carbonyl. The
Fmoc-Cys(Trt)-OH and TscG were then added to the side chain of the
lysine using standard Fmoc automated synthesis protocols to form
the following peptide: Aloc-Lys(Tscg-Cys(Trt)-rink resin. The Aloc
group was then removed. The peptide synthesis was then continued on
the synthesizer to make the following peptide:
(Lys(Aloc)-D-Tyr-Lys(Aloc)-Lys(Tscg-Cys(Trt)-)-rink resin.
Following N-terminus acylation, and removal of the side chain Aloc
protecting groups. The resulting peptide was then treated with
activated N-trityl-HSG-OH until the resin gave a negative test for
amines using the Kaiser test (Karacay et al., Bioconjugate Chem.
11:842-854, 2000). The synthesis of
Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)-NH.sub.2, as well as the
syntheses of DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH.sub.2; and
DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH.sub.2 are described in greater
detail below.
[0283] Chelator-peptide conjugates may be stored for long periods
as solids. They may be metered into unit doses for metal-binding
reactions, and stored as unit doses either as solids, aqueous or
semi-aqueous solutions, frozen solutions or lyophilized
preparations. They may be labeled by well-known procedures.
Typically, a hard acid cation is introduced as a solution of a
convenient salt, and is taken up by the hard acid chelator and
possibly by the soft acid chelator. However, later addition of soft
acid cations leads to binding thereof by the soft acid chelator,
displacing any hard acid cations which may be chelated therein. For
example, even in the presence of an excess of cold
.sup.111InCl.sub.3, labeling with 99m-Tc(V) glucoheptonate or with
Tc cations generated in situ with stannous chloride and
Na99m-TcO.sub.4 proceeds quantitatively on the soft acid chelator.
Other soft acid cations such as .sup.186Re, .sup.188Re, .sup.213Bi
and divalent or trivalent cations of Mn, Co, Ni, Pb, Cu, Cd, Au,
Fe, Ag (monovalent), Zn and Hg, especially .sup.64Cu and .sup.67Cu,
and the like, some of which are useful for radioimmunodiagnosis or
radioimmunotherapy, can be loaded onto the linker peptide by
analogous methods. Re cations also can be generated in situ from
perrhenate and stannous ions or a prereduced rhenium glucoheptonate
or other transchelator can be used. Because reduction of perrhenate
requires more stannous ion (typically above 200 .mu.g/mL final
concentration) than is needed for the reduction of Tc, extra care
needs to be taken to ensure that the higher levels of stannous ion
do not reduce sensitive disulfide bonds such as those present in
disulfide-cyclized peptides. During radiolabeling with rhenium,
similar procedures are used as are used with the Tc-99m. A
preferred method for the preparation of ReO metal complexes of the
Tscg-Cys-ligands is by reacting the peptide with
ReOCl.sub.3(P(Ph.sub.3).sub.2 but it is also possible to use other
reduced species such as ReO(ethylenediamine).sub.2.
[0284] V. Biologically Active Moieties
[0285] The targetable construct can be conjugated to or complexed
with one or more biologically active agents or moieties. The
following are non-limiting examples of biologically active moieties
and agents.
[0286] One type of biologically active agent or moiety is an enzyme
capable of activating a prodrug at the target site or improving the
efficacy of a normal therapeutic by controlling the body's
detoxification pathways. Suitable enzyme includes malate
dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol dehydrogenase, .alpha.-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
.alpha.-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase.
[0287] Following administration of the bsAb, an enzyme conjugated
to the carrier is administered. After the enzyme is pre-targeted to
the target site, a cytotoxic drug is injected, which is known to
act at the target site, or a prodrug form thereof which is
converted to the drug in situ by the pre-targeted enzyme. The drug
is one which is detoxified to form an intermediate of lower
toxicity, most commonly a glucuronide, using the mammal's ordinary
detoxification processes. The detoxified intermediate, e.g., the
glucuronide, is reconverted to its more toxic form by the
pre-targeted enzyme and thus has enhanced cytotoxicity at the
target site. This results in a recycling of the drug. Similarly, an
administered prodrug can be converted to an active drug through
normal biological processes. The pre-targeted enzyme improves the
efficacy of the treatment by recycling the detoxified drug. This
approach can be adopted for use with any enzyme-drug pair. Similar
pre-targeting strategies have been described in U.S. Application
Ser. No. 60/101,039. Those methodologies are easily adaptable to
the present invention and are hereby incorporated in their entirety
by reference.
[0288] The enzyme-carrier conjugate can be mixed with the targeting
bsAb prior to administration to the patient. After a sufficient
time has passed for the enzyme-carrier-bsAb conjugate to localize
to the target site and for unbound conjugate to clear from
circulation, a prodrug is administered. As discussed above, the
prodrug is then converted to the drug in situ by the pre-targeted
enzyme.
[0289] Certain cytotoxic drugs that are useful for anticancer
therapy are relatively insoluble in serum. Some are also quite
toxic in an unconjugated form, and their toxicity is considerably
reduced by conversion to prodrugs. Conversion of a poorly soluble
drug to a more soluble conjugate, e.g., a glucuronide, an ester of
a hydrophilic acid or an amide of a hydrophilic amine, will improve
its solubility in the aqueous phase of serum and its ability to
pass through venous, arterial or capillary cell walls and to reach
the interstitial fluid bathing the tumor. Cleavage of the prodrug
deposits the less soluble drug at the target site. Many examples of
such prodrug-to-drug conversions are disclosed in U.S. application
Ser. No. 08/445,110.
[0290] Conversion of certain toxic substances such as aromatic or
alicyclic alcohols, thiols, phenols and amines to glucuronides in
the liver is the body's method of detoxifying them and making them
more easily excreted in the urine. One type of anti-tumor drug that
can be converted to such a substrate is epirubicin, a 4-epimer of
doxorubicin (Adriamycin), which is an anthracycline glycoside and
has been shown to be a substrate for human beta-D-glucuronidase.
See, e.g., Arcamone, Cancer Res. 45:5995, 1985. Other analogues
with fewer polar groups are expected to be more lipophilic and show
greater promise for such an approach. Other drugs or toxins with
aromatic or alicyclic alcohol, thiol or amine groups are candidates
for such conjugate formation. These drugs, or other prodrug forms
thereof, are suitable candidates for the site-specific enhancement
methods of the present invention.
[0291] The prodrug CPT-11 (irinotecan) is converted in vivo by
carboxylesterase to the active metabolite SN-38. SN-38 is a highly
effective anti-tumor agent; however, therapeutic doses can not be
administered to patients due to its toxicity. One application of
the invention, therefore, is to target such therapies to the tumor
site using a bsAb specific for a tumor-associated antigen and a
hapten (e.g. di-DTPA) followed by injection of a
di-DTPA-carboxylesterase conjugate. Once a suitable
tumor-to-background localization ratio has been achieved, the
CPT-11 is given and the tumor-localized carboxylesterase serves to
convert CPT-11 to SN-38 at the tumor. Due to its poor solubility,
the active SN-38 will remain in the vicinity of the tumor and,
consequently, will exert an effect on adjacent tumor cells that are
negative for the antigen being targeted. This is a further
advantage of the method. Modified forms of carboxylesterases have
been described and are within the scope of the invention. See,
e.g., Potter et al., Cancer Res. 58:2646-2651 and 3627-3632,
1998.
[0292] Etoposide is a widely used cancer drug that is detoxified to
a major extent by formation of its glucuronide and is within the
scope of the invention. See, e.g., Hande et al., Cancer Res.
48:1829-1834, 1988. Glucuronide conjugates can be prepared from
cytotoxic drugs and can be injected as therapeutics for tumors
pre-targeted with mAb-glucuronidase conjugates. See, e.g., Wang et
al., Cancer Res. 52:4484-4491, 1992. Accordingly, such conjugates
also can be used with the pre-targeting approach described here.
Similarly, designed prodrugs based on derivatives of daunomycin and
doxorubicin have been described for use with carboxylesterases and
glucuronidases. See, e.g., Bakina et al., J. Med Chem.
40:4013-4018, 1997. Other examples of prodrug/enzyme pairs that can
be used within the present invention include, but are not limited
to, glucuronide prodrugs of hydroxy derivatives of phenol mustards
and beta-glucuronidase; phenol mustards or CPT-II and
carboxypeptidase; methotrexate-substituted alpha-amino acids and
carboxypeptidase A; penicillin or cephalosporin conjugates of drugs
such as 6-mercaptopurine and doxorubicin and beta-lactamase;
etoposide phosphate and alkaline phosphatase.
[0293] The enzyme capable of activating a prodrug at the target
site or improving the efficacy of a normal therapeutic by
controlling the body's detoxification pathways may be conjugated to
the hapten. The enzyme-hapten conjugate is administered to the
patient following administration of the pre-targeting bsAb and is
directed to the target site. After the enzyme is localized at the
target site, a cytotoxic drug is injected, which is known to act at
the target site, or a prodrug form thereof which is converted to
the drug in situ by the pre-targeted enzyme. As discussed above,
the drug is one which is detoxified to form an intermediate of
lower toxicity, most commonly a glucuronide, using the mammal's
ordinary detoxification processes. The detoxified intermediate,
e.g., the glucuronide, is reconverted to its more toxic form by the
pre-targeted enzyme and thus has enhanced cytotoxicity at the
target site. This results in a recycling of the drug. Similarly, an
administered prodrug can be converted to an active drug through
normal biological processes. The pre-targeted enzyme improves the
efficacy of the treatment by recycling the detoxified drug. This
approach can be adopted for use with any enzyme-drug pair. In an
alternative embodiment, the enzyme-hapten conjugate can be mixed
with the targeting bsAb prior to administration to the patient.
After a sufficient time has passed for the enzyme-hapten-bsAb
conjugate to localize to the target site and for unbound conjugate
to clear from circulation, a prodrug is administered. As discussed
above, the prodrug is then converted to the drug in situ by the
pre-targeted enzyme.
[0294] One type of biologically active agent or moiety is a
prodrug. The pre-targeting bsAb is administered to the patient and
allowed to localize to the target and substantially clear
circulation. At an appropriate later time, a targetable construct
comprising a prodrug, for example poly-glutamic acid
(SN-38-ester).sub.10, is given, thereby localizing the prodrug
specifically at the tumor target. It is known that tumors have
increased amounts of enzymes released from intracellular sources
due to the high rate of lysis of cells within and around tumors. A
practitioner can capitalize on this fact by appropriately selecting
prodrugs capable of being activated by these enzymes. For example,
carboxylesterase activates the prodrug poly-glutamic acid
(SN-38-ester)loby cleaving the ester bond of the poly-glutamic acid
(SN-38-ester).sub.10 releasing large concentrations of free SN-38
at the tumor. Alternatively, the appropriate enzyme also can be
targeted to the tumor site.
[0295] After cleavage from the targetable construct, the drug is
internalized by the tumor cells. Alternatively, the drug can be
internalized as part of an intact complex by virtue of
cross-linking at the target. The targetable construct can induce
internalization of tumor-bound bsAb and thereby improve the
efficacy of the treatment by causing higher levels of the drug to
be internalized.
[0296] A variety of carriers are well-suited for conjugation to
prodrugs, including polyamino acids, such as polylysine,
polyglutamic (E) and aspartic acids (D), including D-amino acid
analogs of the same, co-polymers, such as poly(Lys-Glu) {poly[KE]},
advantageously from 1:10 to 10:1. Copolymers based on amino acid
mixtures such as poly(Lys-Ala-Glu-Tyr) (KAEY; 5:6:2:1) can also be
employed. Smaller polymeric carriers of defined molecular weight
can be produced by solid-phase peptide synthesis techniques,
readily producing polypeptides of from 2-50 residues in chain
length. A second advantage of this type of reagent, other than
precise structural definition, is the ability to place single or
any desired number of chemical handles at certain points in the
chain. These can be used later for attachment of recognition and
therapeutic haptens at chosen levels of each moiety.
[0297] Poly(ethylene) glycol [PEG] has desirable in vivo properties
for a bi-specific antibody prodrug approach. Ester linkages between
the hydroxyl group of SN-38 and both ends of a standard di-hydroxyl
PEG can be introduced by insertion of diacids such as succinic acid
between the SN-38 and PEG hydroxyl groups, to generate species such
as
SN-38-O--CO(CH.sub.2).sub.2CO--O-PEG-O--CO(CH.sub.2).sub.2CO--OSN-38.
The di-SN-38-PEG produced can be considered as the shortest member
of the class of SN-38-polymer prodrugs. The desirable in vivo
properties of PEG derivatives and the limited loading capacity due
to their dimeric functionality led to the preparation of PEG
co-polymers having greater hapten-bearing capacity such as those
described by Poiani et al. See, e.g., Poiani et al., Bioconjugate
Chem. 5:621-630, 1994. PEG derivatives are activated at both ends
as their bis(succinimidyl)carbonate derivatives and co-polymerized
with multi-functional diamines such as lysine. The product of such
co-polymerization, containing
(-Lys(COOH)--PEG-Lys(COOH)--PEG-).sub.n repeat units wherein the
lysyl carboxyl group is not involved in the polymerization process,
can be used for attachment of SN-38 residues. The SN-38 residues
are reacted with the free carboxyl groups to produce SN-38 esters
of the (-Lys-(COOH)-PEG-Lys(COOH)-PEG-).sub.n chain.
[0298] Other synthetic polymers that can be used to carry
recognition haptens and prodrugs include
N-(2-hydroxypropyl)methacrylamide (HMPA) copolymers,
poly(styrene-co-maleic acid/anhydride (SMA), poly(divinylether
maleic anhydride) (DIVEMA), polyethyleneimine, ethoxylated
polyethylene-imine, starburst dendrimers and
poly(N-vinylpyrrolidone) (PVP). As an example, DIVEMA polymer
comprised of multiple anhydride units is reacted with a limited
amount of SN-38 to produce a desired substitution ratio of drug on
the polymer backbone. Remaining anhydride groups are opened under
aqueous conditions to produce free carboxylate groups. A limited
number of the free carboxylate groups are activated using standard
water-soluble peptide coupling agents, e.g.
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC),
and coupled to a recognition moiety bearing a free amino group. An
example of the latter is histamine, to which antibodies have been
raised in the past.
[0299] A variety of prodrugs can be conjugated to the carrier
portion of the targetable construct. The above exemplifications of
polymer use are concerned with SN-38, the active metabolite of the
prodrug CPT-11 (irinotecan). SN-38 has an aromatic hydroxyl group
that was used in the above descriptions to produce aryl esters
susceptible to esterase-type enzymes. Similarly the camptothecin
analog topotecan, widely used in chemotherapy, has an available
aromatic hydroxyl residue that can be used in a similar manner as
described for SN-38, producing esterase-susceptible
polymer-prodrugs.
[0300] Doxorubicin also contains aromatic hydroxyl groups that can
be coupled to carboxylate-containing polymeric carriers using
acid-catalyzed reactions similar to those described for the
camptothecin family. Similarly, doxorubicin analogs like
daunomycin, epirubicin and idarubicin can be coupled in the same
manner. Doxorubicin and other drugs with amino `chemical handles`
active enough for chemical coupling to polymeric carriers can be
effectively coupled to carrier molecules via these free amino
groups in a number of ways. Polymers bearing free carboxylate
groups can be activated in situ (EDC) and the activated polymers
mixed with doxorubicin to directly attach the drug to the
side-chains of the polymer via amide bonds. Amino-containing drugs
can also be coupled to amino-pendant polymers by mixing
commercially available and cleavable cross-linking agents, such as
ethylene glycobis(succinimidylsuccinate) (EGS, Pierce Chemical Co.,
Rockford, Ill.) or bis-[2-(succinimido-oxycarb-
onyloxy)ethyl]sulfone (BSOCOES, Molecular Biosciences, Huntsville,
Ala.), to cross-link the two amines as two amides after reaction
with the bis(succinimidyl) ester groups. This is advantageous as
these groups remain susceptible to enzymatic cleavage. For example,
(doxorubicin-EGS).sub.n-poly-lysine remains susceptible to
enzymatic cleavage of the diester groups in the EGS linking chain
by enzymes such as esterases. Doxorubicin also can be conjugated to
a variety of peptides, for example, HyBnK(DTPA)YK(DTPA)-NH.sub.2,
using established procedures
(HyBn=p-H.sub.2NNHC.sub.6H.sub.4CO.sub.2H). See Kaneko et al., J.
Bioconjug. Chem. 2:133-141, 1991.
[0301] The therapeutic conjugate may comprise doxonibicin coupled
to a carrier comprising amine residues and a chelating agent, such
as DTPA, to form a DTPA-peptide-doxorubicin conjugate, wherein the
DTPA forms the recognition moiety for a pretargeted bsMAb.
Preferably, the carrier comprises a tyrosyl-lysine dipeptide, e.g.,
Tyr-Lys(DTPA)-NH.sub.2, and more preferably still it comprises
Lys(DTPA)-Tyr-Lys(DTPA)-NH.sub.2. Doxorubicin phenyl hydrazone
conjugates to bis-DPTA containing peptides are particularly
desirable in a therapeutic context.
[0302] Methotrexate also has an available amino group for coupling
to activated carboxylate-containing polymers, in a similar manner
to that described for doxorubicin. It also has two glutamyl
carboxyl groups (alpha and gamma) that can be activated for
coupling to amino-group containing polymers. The free carboxylate
groups of methotrexate can be activated in situ (EDC) and the
activated drug mixed with an amino-containing polymer to directly
attach the drug to the side-chains of the polymer via amide bonds.
Excess unreacted or cross-reacted drug is separated readily from
the polymer-drug conjugate using size-exclusion or ion-exchange
chromatography.
[0303] Maytansinoids and calicheamicins (such as esperamycin)
contain mixed di- and tri-sulfide bonds that can be cleaved to
generate species with a single thiol useful for chemical
manipulation. The thiomaytensinoid or thioespera-mycin is first
reacted with a cross-linking agent such as a maleimido-peptide that
is susceptible to cleavage by peptidases. The C-terminus of the
peptide is then activated and coupled to an amino-containing
polymer such as polylysine.
[0304] An immunomodulator, such as a cytokine, may also be
conjugated to, or form an alternative or additional biologically
active moiety. As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, such as tumor
necrosis factor (TNF), and hematopoietic factors, such as
interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10,
IL-12, IL-18 and IL-21), colony stimulating factors (e.g.,
granulocyte-colony stimulating factor (G-CSF) and granulocyte
macrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,
interferons-.alpha., -.beta. and -.gamma.), the stem cell growth
factor designated "S1 factor", and erythropoietin and
thrombopoietin. Examples of suitable immunomodulator moieties
include IL-2, IL-6, IL-10, IL-12, IL-18, interferon-.gamma.,
TNF-.alpha., and the like. Alternatively, subjects can receive
invention compositions and a separately administered cytokine,
which can be administered before, concurrently or after
administration of the invention compositions. The invention
compositions may also be conjugated to the immunomodulator.
[0305] VI. Combination Therapy
[0306] The bi-specific antibody-directed delivery of therapeutics
or prodrug polymers to in vivo targets can be combined with
bi-specific antibody delivery of radionuclides, such that
combination chemotherapy and radioimmunotherapy is achieved. Each
therapy can be conjugated to the targetable construct and
administered simultaneously, or the nuclide can be given as part of
a first targetable construct and the drug given in a later step as
part of a second targetable construct. In one simple embodiment, a
peptide containing a single prodrug and a single nuclide is
constructed. For example, the tripeptide Ac-Glu-Gly-Lys-NH.sub.2
can be used as a carrier portion of a targetable construct, whereby
SN-38 is attached to the gamma glutamyl carboxyl group as an aryl
ester, while the chelate DOTA is attached to the epsilon amino
group as an amide, to produce the complex
Ac-Glu(SN-38)-Gly-Lys(DOTA)-NH.sub.2. The DOTA chelate can then be
radiolabeled with various metals for imaging and therapy purposes
including In-111, Y-90, Sm-153, Lu-177 and Zr-89. As the metal-DOTA
complex may represent the recognizable hapten on the targetable
construct, the only requirement for the metal used as part of the
DOTA complex is that the secondary recognition antibody also used
recognizes that particular metal-DOTA complex at a sufficiently
high affinity. Generally, this affinity (log K.sub.a) is between
6-11. Polymeric peptides such as
poly[Glu(SN-38).sub.10-Lys(Y-90-DOTA).sub.2] can be given as
readily as the more chemically defined lower MW reagent above, and
are indeed preferred. Also, triply substituted polymers can be
used, such as
poly[Glu(Sn-38).sub.10-Lys(Y-90-DOTA).sub.n(histamine-succi-
nate).sub.m, where n and m are integers, such that the recognition
agent is independent of the radioimmunotherapy agent. The prodrug
is activated by carboxylesterases present at the tumor site or by
carboxylesterases targeted to the site using a second targetable
construct.
[0307] Alternatively, a combination therapy can be achieved by
administering the chemotherapy and radioimmunotherapy agents in
separate steps. For example, a patient expressing CEA-tumors is
first administered bsAb with at least one arm which specifically
binds CEA and at least one other arm which specifically binds the
targetable construct whose hapten is a conjugate of yttrium-DOTA.
Later the patient is treated with a targetable construct comprising
a conjugate of yttrium-DOTA-beta-glucuron- idase. After sufficient
time for bsAb and enzyme localization and clearance, a second
targetable construct, comprising
Ac-Glu(SN-38)-Gly-Lys(Y-90-DOTA)-NH.sub.2, is given. The second
targetable construct localizes to the tumor by virtue of bsAb at
the tumor that are not already bound to a first targetable
construct. First targetable constructs which are localized to the
target site act on the Ac-Glu(SN-38)-Gly-Lys(Y-90-DOTA)-NH.sub.2 to
liberate the free SN-38 drug. Localization of both the prodrug and
its respective enzyme to the target site enhances the production of
active drug by ensuring that the enzyme is not substrate limited.
This embodiment constitutes a marked improvement of current prodrug
methodologies currently practiced in the art.
[0308] Another advantage of administering the prodrug-polymer in a
later step, after the nuclide has been delivered as part of a
previously given targetable construct, is that the synergistic
effects of radiation and drug therapy can be manipulated and,
therefore, maximized. It is hypothesized that tumors become more
`leaky` after RAIT due to radiation damage. This can allow a
polymer-prodrug to enter a tumor more completely and deeply. This
results in improved chemotherapy.
[0309] Alternatively, the RAIT therapy agent can be attached to
bsAb rather the targetable construct. For example, an anti-CEA x
anti-DTPA bsAb conjugated to Y-90-DOTA is administered first to a
patient with CEA-expressing tumors. In this instance, advantage is
taken of the selectivity of certain anti-chelate mabs in that an
anti-indium-DTPA antibody do not bind to a yttrium-DOTA chelate.
After the Y-90-DOTA-anti-CEA x anti-indium-DTPA has maximized at
the tumor and substantially cleared non-target tissue, a conjugate
of indium-DTPA-glucuronidase is injected and localized specifically
to the CEA tumor sites. The patient is then injected with a
polymer-prodrug such as poly(Glu)(SN-38).sub.10. The latter is
cleaved selectively at the tumor to active monomeric SN-38,
successfully combining chemotherapy with the previously
administered RAIT.
[0310] It should also be noted that a bi-specific antibody or
antibody fragment can be used in the present method, with at least
one binding site specific to an antigen at a target site and at
least one other binding site specific to an enzyme. Such an
antibody can bind the enzyme prior to injection, thereby obviating
the need to covalently conjugate the enzyme to the antibody, or it
can be injected and localized at the target site and, after
non-targeted antibody has substantially cleared from the
circulatory system of the mammal, enzyme can be injected in an
amount and by a route which enables a sufficient amount of the
enzyme to reach the pre-targeted bsAb and bind to it to form an
antibody-enzyme conjugate in situ.
[0311] VII. Kits
[0312] In accordance with yet another aspect of the present
invention, the present invention provides a kit suitable for
treating or identifying diseased tissues in a patient, comprising a
bi-specific antibody or antibody fragment having at least one arm
that specifically binds a targeted tissue and at least one other
arm that specifically binds a targetable construct, a first
targetable construct which comprises a carrier portion which
comprises or bears at least one epitope recognizable by the at
least one other arm of the bi-specific antibody or antibody
fragment, and one or more conjugated therapeutic agents, or
enzymes, and, optionally, a clearing composition useful for
clearing non-localized antibodies and antibody fragments.
[0313] A "clearing agent" is an agent that clears unbound
targetable construct from circulation, thereby facilitating
circulating moiety from a patient's body, removal from blood
circulation, or inactivation thereof in circulation. Preferably,
the clearing agent has physical properties, such as size, charge,
configuration or combinations thereof, that limit clearing agent
access to the population of target cells recognized by a targetable
construct used in the same treatment protocol as the clearing
agent. This enhancement may be further improved by the
administration of an anti-idiotypic clearing agent, such as an
anti-idiotypic monoclonal antibody specific for the determinant of
the targeting conjugate, which binds to the tumor site. The
clearance effect may be further enhanced by using a galactosylated
clearing agent, because a galactosylated clearing agent is rapidly
cleared through the liver.
[0314] When the first targetable construct comprises an enzyme, the
kit may optionally contain a prodrug, when the enzyme is capable of
converting the prodrug to a drug at the target site, a drug which
is capable of being detoxified in the patient to form an
intermediate of lower toxicity, when the enzyme is capable of
reconverting the detoxified intermediate to a toxic form, and,
therefore, of increasing the toxicity of the drug at the target
site, or a prodrug which is activated in the patient through
natural processes and is subject to detoxification by conversion to
an intermediate of lower toxicity, when the enzyme is capable of
reconverting the detoxified intermediate to a toxic form, and,
therefore, of increasing the toxicity of the drug at the target
site, or a second targetable construct which comprises a carrier
portion which comprises or bears at least one epitope recognizable
by the at least one other arm of the bi-specific antibody or
antibody fragment, and a prodrug, when the enzyme is capable of
converting the prodrug to a drug at the target site. Instruments
which facilitate identifying or treating diseased tissue also can
be included in the kit. Examples include, but are not limited to
application devices, such as syringes.
[0315] A therapeutic kit of the invention comprises any of the
following reagents and/or components in any combination.
[0316] 1. One or more therapeutic agents.
[0317] 2. If the therapeutic agent(s) are not formulated for
delivery via the alimentary canal, which includes but is not
limited to sublingual delivery, a device capable of delivering the
therapeutic agent through some other routes. One type of device for
parenteral delivery is a syringe that is used to inject the
therapeutic agent into the body of an animal in need of the
therapeutic agent. Inhalation devices may also be used.
[0318] b3. Separate containers, each of which comprises one or more
reagents of the kit. In a preferred embodiment, the containers are
vials contain sterile, lyophilized formulations of a therapeutic
composition that are suitable for reconstitution. Other containers
include, but are not limited to, a pouch, tray, box, tube, or the
like. Kit components may be packaged and maintained sterilely
within the containers.
[0319] 4. Instructions to a person using a kit for its use. The
instructions can be present on one or more of the kit components,
the kit packaging and/or a kit package insert. Such instructions
include, by way of non-limiting example, instructions for use of
the kit and its reagents, for reconstituting lyophilized reagents
or otherwise preparing reagents.
[0320] A preferred kit of the present invention comprises the
elements useful for performing an immunoassay. A kit of the present
invention can comprise one or more experimental samples (i.e.,
formulations of the present invention) and one or more control
samples bound to at least one pre-packed dipstick or ELISA plate,
and the necessary means for detecting immunocomplex formation
(e.g., labelled secondary antibodies or other binding compounds and
any necessary solutions needed to resolve such labels, as described
in detail above) between antibodies contained in the bodily fluid
of the animal being tested and the proteins bound to the dipstick
or ELISA plate. It is within the scope of the invention that the
kit can comprise simply a formulation of the present invention and
that the detecting means can be provided in another way.
[0321] VIII. Characterization of Targetable Constructs and
Complexes
[0322] Any of the following methodologies, as well as others known
in the art and/or described in the Examples herein, can be used to
examine one or more attributes of a targetable construct or
complex.
[0323] VIII.A. Affinity for Epitopes
[0324] Affinity can be either absolute or relative. By absolute
affinity, it is meant that the assay for affinity gives defined
numerical determinations of the affinity of one compound for
another. Comparison of the affinity of the complex being tested to
that of a reference compound whose binding affinity is known allows
for the determination of relative binding affinity of the test
ligand.
[0325] Whether absolute or relative, affinity of one molecule for
another can be measured by any method known in the art. By way of
non-limiting example, such methods include competition assays,
surface plasmon resonance, half-maximal binding assays, competition
assays, Scatchard analysis, direct force techniques (Wong et al.,
Direct force measurements of the streptavidin-biotin interaction,
Biomol. Eng. 16:45-55, 1999), and mass spectrometry (Downard,
Contributions of mass spectrometry to structural immunology, J.
Mass Spectrom. 35:493-503, 2000).
[0326] VIII.A.1. Absolute Affinity
[0327] As regards absolute affinity, "low affinity" refers to
binding wherein the association constant (Ka) between two molecules
is about 10.sup.5 M to 10.sup.7 M. "Moderate affinity" refers to
binding wherein the association constant (Ka) between two molecules
is at least about 10.sup.7 M to 10.sup.8 M. "High affinity" refers
to a binding wherein the association constant between the two
molecules is at least about 10.sup.8 M to about 10.sup.14 M, and
preferably about 10.sup.9 M to about 10.sup.14 M, more preferably
about 10.sup.10 M to about 10.sup.14 M, and most preferably greater
than about 1014 M.
[0328] The dissociation constant, Kd, is an equilibrium constant
for the dissociation of one species into two, e.g., the
dissociation of a complex of two or more molecules into its
components, for example, dissociation of a substrate from an
enzyme. Exemplary Kd values for compositions of the present
invention are from about 10.sup.-7 M (100 nM) to about 10.sup.-12 M
(0.001 nM). The stability constant is an equilibrium constant that
expresses the propensity of a species to form from its component
parts. The larger the stability constant, the more stable is the
species. The stability constant (formation constant) is the
reciprocal of the instability constant (dissociation constant).
[0329] The affinity of the complexes of the invention for a target
epitope, or the affinity of a bi-specific antibody for a carrier
epitope, is driven by non-covalent interactions. There are four
main non-covalent attractive forces between molecules: (i)
electrostatic forces, which occur between between oppositely
charged molecules such as amino groups and carboxylic groups; (ii)
hydrogen bonds, which are formed when hydrogen atoms are shared
between electronegative atoms such as nitrogen and oxygen; (iii)
Van der Waals forces, which are generated between electron clouds
around molecules oppositely polarized by neighboring atoms; and
(iv) hydrophobic interactions, which are formed when water is
excluded from the interface allowing hydrophobic molecules to
interact in a waterless environment.
[0330] Non-covalent interactions can, but rarely do, have the
strength of a covalent linkage (i.e., a chemical bond). In some
instances, the affinity of the complexes of the invention for a
target epitope, although driven by non-covalent interactions, is so
high as to approach the strength of a covalent bond. This provides
for complexes that are very stable relative to other complexes.
[0331] Preferably, the affinity of a targetable complex for its
cognate target epitope, and/or the affinity of a bsAb for the
carrier epitopes of a targetable construct, is a Kd of about 100 nM
to about 0.01 nM; more preferably, greater than about 100 nM, or
greater than about 10 nM; most preferably, greater than about 1 nM,
or greater than about 0.1 nM. Typical Kd for target epitopes are
from about 0.1 nM to 100 nM, preferably from about 0.1 nM to 10 nM,
more preferably from about 0.5 nM to 5 nM, or about 1 nM.
[0332] In the invention, when multiple copies of a carrier epitope
are present on the targetable construct, the affinity of an
antibody for its cognate carrier epitope may be greater than the
affinity of an antibody for a free carrier epitope or for a
monovalent tragetable construct comprising the carrier epitope.
Additionally or alternatively, a multivalent targetable construct
having x carrier epitopes has a greater affinity for its target
epitope than would x number of constructs. Put another way, the
compositions of the invention provide for synergistic, rather than
merely additive, binding effects.
[0333] VIII.A.2. Surface Plasmon Resonance
[0334] Binding parameters such as Kd may be measured using surface
plasmon resonance on a chip, for example, with a BIAcore.RTM. chip
coated with immobilized binding components. Surface plasmon
resonance is used to characterize the microscopic association and
dissociation constants of reaction between an antibody or antibody
fragment and its ligand. Such methods are generally described in
the following references which are incorporated herein by
reference. (Vely et al., BIAcore analysis to test
phosphopeptide-SH2 domain interactions, Meth. Mol. Biol.
121:313-21, 2000; Liparoto et al., Biosensor analysis of the
interleukin-2 receptor complex, J. Mol. Recog. 12:316-21, 1999;
Lipschultz et al., Experimental design for analysis of complex
kinetics using surface plasmon resonance, Methods 20:310-8, 2000;
Malmqvist., BIACORE: an affinity biosensor system for
characterization of biomolecular interactions, Biochem. Soc.
Transactions 27:33540, 1999; Alfthan, Surface plasmon resonance
biosensors as a tool in antibody engineering, Biosensors &
Bioelectronics 13:653-63, 1998; Fivash et al., BIAcore for
macromolecular interaction, Curr. Opin. Biotech. 9:97-101, 1998;
Price et al., Summary report on the ISOBM TD-4 Workshop: analysis
of 56 monoclonal antibodies against the MUC1 mucin, Tumour Biol. 19
Suppl 1:1-20, 1998; Malmqvist et al., Biomolecular interaction
analysis: affinity biosensor technologies for functional analysis
of proteins, Curr. Opin. Chem. Biol. 1:378-83, 1997; O'Shannessy et
al., Interpretation of deviations from pseudo-first-order kinetic
behavior in the characterization of ligand binding by biosensor
technology, Anal. Biochem. 236:275-83, 1996; Malmborg et al.,
BIAcore as a tool in antibody engineering, J. Immunol. Meth.
183:7-13, 1995; Van Regenmortel, Use of biosensors to characterize
recombinant proteins, Dev. Biol. Standardization 83:143-51, 1994;
O'Shannessy, Determination of kinetic rate and equilibrium binding
constants for macromolecular interactions: a critique of the
surface plasmon resonance literature, Curr. Opin. Biotechnol.
5:65-71, 1994). Models using BIAcore to examine the binding of
fixed ligands to multivalent compounds have been described (Muller
et al., Model and simulation of multivalent binding to fixed
ligands, Anal. Biochem. 261:149-158, 1998).
[0335] BIAcore.RTM. uses the optical properties of surface plasmon
resonance (SPR) to detect alterations in protein concentration
bound within to a dextran matrix lying on the surface of a
gold/glass sensor chip interface, a dextran biosensor matrix. In
brief, proteins are covalently bound to the dextran matrix at a
known concentration and a ligand for the protein (e.g., antibody)
is injected through the dextran matrix. Near infrared light,
directed onto the opposite side of the sensor chip surface is
reflected and also induces an evanescent wave in the gold film,
which in turn, causes an intensity dip in the reflected light at a
particular angle known as the resonance angle. If the refractive
index of the sensor chip surface is altered (e.g., by ligand
binding to the bound protein) a shift occurs in the resonance
angle. This angle shift can be measured and is expressed as
resonance units (RUs) such that 1000 RUs is equivalent to a change
in surface protein concentration of 1 ng/mm 2. These changes are
displayed with respect to time along the y-axis of a sensorgram,
which depicts the association and dissociation of any biological
reaction.
[0336] Additional details may be found in Jonsson et al.,
Introducing a biosensor based technology for real-time biospecific
interaction analysis, Ann. Biol. Clin. 51:19-26, 1993; Jonsson et
al., Real-time biospecific interaction analysis using surface
plasmon resonance and a sensor chip technology, Biotechniques
11:620-627, 1991; Johnsson et al., Comparison of methods for
immobilization to carboxymethyl dextran sensor surfaces by analysis
of the specific activity of monoclonal antibodies, J. Mol. Recog.
8:125-131, 1995; and Johnsson, Immobilization of proteins to a
carboxymethyldextran-modified gold surface for biospecific
interaction analysis in surface plasmon resonance sensors, Anal.
Biochem. 198:268-277, 1991; Karlsson et al., Kinetic analysis of
monoclonal antibody-antigen interactions with a new biosensor based
analytical system, J. Immunol. Meth. 145:229, 1991; Weinberger et
al., Recent trends in protein biochip technology, Pharmacogenomics
1:395-416, 2000; Lipschultz et al., Experimental design for
analysis of complex kinetics using surface plasmon resonance,
Methods 20:310-8, 2000.
[0337] VIII.A.3. Relative Affinity
[0338] Affinity may also be defined in relative terms, e.g., by
IC.sub.50. In the context of affinity, the IC.sub.50 of a compound
is the concentration of that compound at which 50% of a reference
ligand is displaced from a target epitope in vitro or targeted
tissue in vivo. When the target epitope is CEA, the reference
ligand can be a complex comprising the [hMN].sub.2-[734scFv].sub.2
or [hMN.sup.(1253A)].sub.2-[73- 4scFv].sub.2 bi-specific antibody.
Typically, IC.sub.50 is determined by competitive ELISA.
[0339] VIII.B. Biodistribution and Clearing Characteristics
[0340] Methods of evaluating biodistribution patterns of targetable
complexes are described in U.S. provisional Application Ser. No.
60/361,037 (Atty. Docket No. 018733-1037), which was filed Mar. 1,
2002 and is entitled "Bispecific antibody point mutations for
enhancing rate of clearance." This application is hereby
incorporated in its entirety by reference.
[0341] Methods of evaluating clearing characteristics of targetable
complexes are described in U.S. Provisional Application Ser. No.
60/361,037 (Atty Docket No. 018733-1037), which was filed Mar. 1,
2002 and is entitled "Bispecific antibody point mutations for
enhancing rate of clearance." This application is hereby
incorporated in its entirety by reference.
[0342] VIII.C. Formation of Defined Species of Multimers
[0343] It is often the case that mixing several compounds that are
capable of binding to each other results in a variety of multimers.
For example, mixing binding compounds A and B can result in species
of complexes such as AB, (AB).sub.2, (AB).sub.3, (AB).sub.4, etc. A
desirable attribute of some of the complexes of the invention is
that the components thereof, when mixed together, predominately
form a single type of multimer. In the case of some of the
complexes of the invention, for example, dimeric complexes are
predominately formed with little or no other species of multimers
being present.
[0344] For example, mixing a targetable construct and one type
bi-specific antibodies at relative concentrations ranging anywhere
from about 10.sup.-9, 10.sup.-8, 10.sup.-7, 10.sup.-6, 10.sup.-5,
10.sup.-4, 10.sup.-3, 10.sup.-2, 10.sup.-1, 1, 10.sup.1, 10.sup.2,
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9 to about 10.sup.10 preferably results in a mixture in
which greater than about 50% of the multimeric complexes have a
defined stoichiometry of two molecules of the bi-specific antibody,
and one molecule of the targetable construct. Preferably
.gtoreq.75% to about .gtoreq.85%, more preferably .gtoreq.95%, and
most preferably .gtoreq.99% of the multimeric complexes so formed
have a defined stoichiometry of two molecules of the bi-specific
antibody, and one molecule of the targetable construct.
[0345] VIII.D. Stability
[0346] VIII.D.1. Types of Stability
[0347] In general, two types of stability are of interest: chemical
stability and conformational stability. Both types contribute to
functional stability: an agent may be chemically stable (i.e.,
resistant to degradation) but may not be biologically active if it
does not have the proper conformation. The term "functional
stability" refers to the amount of functional (biologically active)
agent that is retained over time.
[0348] Chemical stability is measured as is known in the art, e.g.,
by preparing a mixture of labeled agent, incubating the mixture
under a given set of conditions (temperature, pH, etc.), and
determining the amount of labeled agent remaining in samples taken
at one or more time points. In addition to physiological conditions
(see below), conditions of interest may be those that influence the
shelf-life of an agent and/or ease of manipulation thereof. The
stability of an agent as regards a specific degradative molecule,
e.g., in the case of proteins, proteases, can be determined in
vitro using similar methodologies.
[0349] Conformational stability can be measured using a variety of
techniques known in the art including, by way of non-limiting
example, circular dichroism (CD), fluorescence, fluorescent energy
transfer (FRET), fluorescent energy transfer confocal microscopy,
nuclear magnetic resonance (NMR) spectroscopy, neutron scattering,
synchrotron radiolysis, mass spectrometry, and electrospray
ionization mass spectrometry. See, for example, van Mierlo and
Steensma, Protein folding and stability investigated by
fluorescence, circular dichroism (CD), and nuclear magnetic
resonance (NMR) spectroscopy: the flavodoxin story, J. Biotechnol.
79:281-98, 2000; Tehei et al., Fast dynamics of halophilic malate
dehydrogenase and BSA measured by neutron scattering under various
solvent conditions influencing protein stability, Proc. Natl. Acad.
Sci. USA. 98:14356-61, 2001; Maleknia and Downard, Unfolding of
apomyoglobin helices by synchrotron radiolysis and mass
spectrometry, Eur. J. Biochem. 268:5578-88, 2001; Kim et al.,
Site-specific amide hydrogen/deuterium exchange in E. coli
thioredoxins measured by electrospray ionization mass spectrometry,
J. Am. Chem. Soc. 123:9860-6, 2001; Doig et al., Structure,
stability and folding of the alpha-helix, Biochem. Soc. Symp.
68:95-110, 2001; Kolakowski and Konermann, From small-molecule
reactions to protein folding: studying biochemical kinetics by
stopped-flow electrospray mass spectrometry, Anal. Biochem.
292:107-14, 2001; Helfrich and Jones, High-throughput
flow-injection technique for stability sensing characterization of
biomolecules in solution, Am. Laboratory 33:24-29, 2001;
Hammarstrom et al., Protein compactness measured by fluorescence
resonance energy transfer: Human carbonic anhydrase ii is
considerably expanded by the interaction of GroEL, J. Biol. Chem.
276:21765-75, 2001; Talaga et al., Dynamics and folding of single
two-stranded coiled-coil peptides studied by fluorescent energy
transfer confocal microscopy, Proc. Natl. Acad. Sci. USA
97:13021-6, 2000; and Kumar and Nussinov, Review: How do
thermophilic proteins deal with heat? Cell. Mol. Life Sci.
58:1216-1233, 2001.
[0350] In addition to the conformational stability of individual
components of the targetable complexes (e.g., targetable constructs
and bsAbs), the stability of the targetable complexes per se is
also a factor. Preferably, the targetable complexes of the
invention are stable in vitro and in vivo. That is, once the
targetable constructs and bsAbs are combined and form targetable
complexes, the constructs and bsAbs have little tendency to
dissociate from the complexes.
[0351] VIII.D.2. Stability Under Physiological Conditions
[0352] Another desirable attribute of a compound intended for in
vivo use is stability particularly under physiological conditions.
As those in the art will appreciate, what constitutes
"physiological conditions" will vary, for example, depending on
whether an in vivo or ex vivo state is under consideration, the
type of organism and its age, weight, health, sex, level of
activity, metabolic state, etc. Parameters that vary in various
physiological conditions include, but are not limited to, the type
of solvent, pH, buffering capacity, the concentrations and types of
salts and ions, temperature, and the like. In any event, it is well
within the skill of the ordinary artisan to define and determine
what particular conditions exist for a given physiological
state.
[0353] The stability of a compound can be expressed as the
compound's half-life in a body fluid such as, by way of
non-limiting example, serum, blood, urine, lymph, plasma,
interstitial fluid, bile, gastric juices and the like. By way of
non-limiting example, stability can be measured and expressed as
the in vivo or in vitro half-life of a compound in serum or
blood.
[0354] For example, serum half-life is a time point at which half
of the administered amount of targeting protein or conjugate
thereof remains in the serum. Serum determinations over a series of
time points can generate a curve which is useful for determining
whole body exposure to an agent.
[0355] IX. Biosensors
[0356] IX.A. Biosensors in General
[0357] The present invention is directed to a device that comprises
a sensor adapted to detect one or more specific health and/or
nutrition markers in a subject or in the environment. The device
may also signal the caretaker, the subject, or an actuator of the
occurrence. The sensor comprises a biosensor. As used herein, the
term "biosensor" is defined as a component comprising one or more
binding moities being adapted to detect a ligand found in one or
more target pathogenic microorganisms or related biomolecules.
[0358] Generally, biosensors function by providing a means of
specifically binding, and therefore detecting, a target
biologically active analyte. In this way, the biosensor is highly
selective, even when presented with a mixture of many chemical and
biological entities. Often the target biological analyte is a minor
component of a complex mixture comprising a multiplicity of
biological and other components. Thus, in many biosensor
applications, detection of target analytes occurs in the
parts-per-billion, parts-per-trillion, or even lower ranges
levels.
[0359] IX.B. Biosensor Design
[0360] The biosensor of the present invention may comprise a
bio-recognition element, or molecular recognition element, that
provides the highly specific binding or detection selectivity for a
particular analyte. The bio-recognition element or system is often
an antibody. In a biosensor of the invention, the bio-recognition
element, or system, is a targetable complex comprising bsAbs. The
bio-recognition element is responsible for the selective
recognition of the analyte and the physico-chemical signal that
provides the basis for the output signal.
[0361] Biocatalytic and bioaffinity biosensor systems are described
in more detail in J. Chromatography 510:347-354, 1990, and in the
Kirk-Othmer Encyclopedia of Chemical Technology, 4.sup.th ed.
(1992), John Wiley & Sons, NY, each of which is incorporated by
reference herein.
[0362] The biosensors of the present invention may detect
biologically active analytes related to impending (i.e., future
presentation of symptoms is likely) or current human systemic
disease states, including, but not limited to, pathogenic bacteria,
parasites (e.g., any stage of the life cycle, including eggs or
portions thereof, cysts, or mature organisms), viruses, fungi,
antibodies to pathogens, and/or microbially produced toxins.
Additionally, the biosensor may target biologically active analytes
related to impending or current localized health issues, such as
stress proteins (e.g., cytokines) and interleukin 1-alpha that may
precede the clinical presentation of skin irritation or
inflammation. In some embodiments, the biosensor functions as a
proactive sensor, detecting and signaling the subject, a caretaker
or medical personnel of the impending condition prior to the
presentation of clinical symptoms. This allows time to administer
prophylactic or remedial treatments to the subject which can
significantly reduce, if not prevent, the severity and duration of
the symptoms. Further, the sensor, by detecting the presence of a
target biological analyte in a sample from the subject, may detect
residual contamination on a surface, such as skin or environmental
surface, in contact with the biosensor, and provide and appropriate
signal.
[0363] The physico-chemical signal generated by the bio-recognition
element or elements may be communicated visually to the caretaker
or medical personnel (i.e., via a color change visible to the human
eye). Other embodiments may produce optical signals, which may
require other instrumentation to enhance the signal. These include
flourescence, bioluminesence, total internal reflectance resonance,
surface plasmon resonance, Raman methods and other laser-based
methods, such as LED or laser diode sensors. Exemplary surface
plasmon resonance biosensors are available as IBIS I and IBIS II
from XanTec Analysensysteme of Muenster, Germany, which may
comprise bioconjugate surfaces as bio-recognition elements.
Alternatively, the signal may be processed via an associated
transducer which, for example, may produce an electrical signal
(e.g., current, potential, inductance, or impedance) that may be
displayed (e.g., on a readout such as an LED or LCD display) or
which triggers an audible or tactile (e.g., vibration) signal or
which may trigger an actuator, as described herein. The signal may
be qualitative (e.g., indicating the presence of the target
biological analyte) or quantitative (i.e., a measurement of the
amount or concentration of the target biological analyte). In such
embodiments, the transducer may optionally produce an optical,
thermal or acoustic signal. In any event, the signal may also be
durable (i.e., stable and readable over a length of time typically
at least of the same magnitude as the usage life of the device) or
transient (i.e., registering a real-time measurement).
Additionally, the signal may be transmitted to a remote indicator
site (e.g., via a wire, or transmitter, such as an infrared or rf
transmitter) including other locations within or on the device or
remote devices. Further, the sensor, or any of its components, may
be adapted to detect and/or signal only concentrations of the
target biological analyte above a predefined threshold level (e.g.,
in cases wherein the target biological analyte is normally present
in the bodily waste or when the concentration of the analyte is
below a known "danger" level).
[0364] The target analytes that the biosensors of the present
invention are adapted to detect may also be viruses. An exemplary
biosensor adapted to detect HIV is described in U.S. Pat. Nos.
5,830,341 and 5,795,453, referenced above. The disclosure of each
of these patents is incorporated by reference herein. Biosensors
are adopted to use in different tissues; see, e.g., U.S. Pat. No.
6,342,037; and using different binding molecules, see, e.g., U.S.
Pat. No. 6,329,160.
[0365] When the targetable complexes of the invention are
incorporated into a biosensor, they may be immobilized in the
biosensor by techniques known in the art such as entrapment,
adsorption, crosslinking, encapsulation, covalent attachment, any
combination thereof, or the like. Further, the immobilization can
be carried out on many different substrates such as known the art.
In certain preferred embodiments, the immobilization substrate may
be selected from the group of polymer-based materials, hydrogels,
tissues, nonwoven materials or woven materials.
[0366] In certain embodiments, biosensor embodiments, may comprise,
be disposed on, or be operatively associated with a microchip, such
as a silicon chip, MEMs (i.e., micro electromechanical system)
device, or an integrated circuit. Microchip-based biosensors may be
known as "biochips." Regardless of the type of sensor, the
microchip may comprise a multiplicity of sensor components having
similar or different sensitivities, kinetics, and/or target
analytes (i.e., markers) in an array adapted to detect differing
levels or combinations of the analyte(s). Further, each sensor in
such an array may provide a different type of signal, including
those types disclosed herein, and may be associated with different
actuators and/or controllers. Also, each sensor in an array may
operate independently or in association with (e.g., in parallel,
combination, or series) any number of other sensors in the
array.
[0367] A biosensor of the invention may comprise a detectable
compound that produces a signal once analytes are bound. See, by
way of non-limiting example, Billinton et al., Development of a
green fluorescent protein reporter for a yeast genotoxicity
biosensor, Biosensors & Bioelectronics 13:831-838, 1998. A
biosensor according to the invention may use microbalance sensor
systems (Hengerer et al., Determination of phage antibody
affinities to antigen by a microbalance sensor system,
BioTechniques 26:956-964, 1999).
[0368] X. Target Antigens and Epitopes
[0369] A target epitope is comprised within, displayed by and/or
released from targeted tissues of a subject, samples or cell
cultures thereof. A sample may be a bodily tissue or fluid tissue
and may be within a subject, or biopsied or removed from a subject,
or a whole or any portion of a bodily organ. Additionally, the
tissue may be "sample" in that the tissue is recently removed from
a subject without any preservation steps between the excision and
the methods of the current invention. The tissue may also have been
preserved by such standard tissue preparation techniques including,
but not limited to, freezing, quick freezing, paraffin embedding
and tissue fixation, prior to application of the methods of the
current invention.
[0370] As used herein, the term "subject" refers to any animal
(i.e., vertebrates and invertebrates) including, but not limited to
humans and other primates, rodents (e.g., mice, rats, and guinea
pigs), lagamorphs (e.g., rabbits), bovines (e.g., cattle), ovines
(e.g., sheep), caprines (e.g., goats), porcines (e.g., swine),
equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats),
domestic fowl (e.g., chickens, turkeys, ducks, geese, other
gallinaceous birds, etc.), as well as feral or wild animals,
including, but not limited to, such animals as ungulates (e.g.,
deer), bear, fish, lagamorphs, rodents, birds, etc. It is not
intended that the term be limited to a particular age or sex. Thus,
adult and newborn subjects, as well as fetuses, whether male or
female, are encompassed by the term. However, the preferred species
for use of this technology is Homo sapiens, and the next preferred
use is in domestic pets, such as horses, dogs, and cats.
[0371] By "displayed" it is meant that a portion of the membrane
protein is present on the surface of a cell, tissue and/or organ,
and is thus in contact with the external environment of the cell,
tissue or organ. A target epitope may be associated with a disease
including but not limited to cancers and pathogenic infections.
[0372] X.A. Antigens and Epitopes Associated with
Hyperproliferative Diseases
[0373] The mutant bispecific antibodies used in the present
invention are specific to a variety of cell surface or
intracellular antigens associated with hyperproliferative diseases.
Normal tissue homeostasis is achieved by an intricate balance
between the rate of cell proliferation and cell death. Disruption
of this balance either by increasing the rate of cell proliferation
or decreasing the rate of cell death can result in the abnormal
growth of cells and is thought to be a major event in the
development of cancer and other hyperproliferative diseases. A
"hyperproliferative disease" is one in which cells have an
abnormally high rate of cell division and/or an abnormally low rate
of necrosis and/or apoptosis. Non-limiting examples include
tumorigenesis; tumor progression; cancers, such as leukemia, solid
tumors and metastases; psoriasis; benign hyperproliferative
diseases, such as benign prostatic hypertrophy, benign hyperplasia
of the skin, and hemangiomas; chronic inflammatory proliferative
diseases, such as psoriasis and rheumatoid arthritis; proliferative
ocular disorders, such as diabetic retinopathy and macular
degeneration; and proliferative cardiovascular diseases, such as
restenosis. Restenosis, characterized by the regrowth of smooth
muscle cells into the lumen of blood vessels following angioplasty
or other arterial damage, is a frequent and recurring problem in
the long term success of angioplasty, and also occurs after
arterial reconstructions, atherectomy, stent implantation, and
laser angioplasty.
[0374] These antigens may be substances produced by, e.g., the
tumor or may be substances which accumulate at a tumor site, on
tumor cell surfaces or within tumor cells, whether in the
cytoplasm, the nucleus or in various organelles or subcellular
structures, including cell-surface or intracellular receptors.
Among such tumor-associated markers are those disclosed, but not
intended to be limiting, by Herberman, Immunodiagnosis of Cancer,
in Fleisher ed., The Clinical Biochemistry of Cancer, page 347
(American Association of Clinical Chemists, 1979) and in U.S. Pat.
Nos. 4,150,149; 4,361,544; and 4,444,744.
[0375] Tumor-associated markers have been categorized by Herberman,
supra, in a number of categories including oncofetal antigens,
placental antigens, oncogenic or tumor virus associated antigens,
tissue associated antigens, organ associated antigens, ectopic
hormones and normal antigens or variants thereof. Occasionally, a
sub-unit of a tumor-associated marker is advantageously used to
raise antibodies having higher tumor-specificity, e.g., the
beta-subunit of human chorionic gonadotropin (HCG) or the gamma
region of carcino embryonic antigen (CEA), which stimulate the
production of antibodies having a greatly reduced cross-reactivity
to non-tumor substances as disclosed in U.S. Pat. Nos. 4,361,644
and 4,444,744.
[0376] Examples, which are non-limiting, of suitable
tumor-associated markers or receptors, include the B-cell complex
structures (e.g., CD19, CD20, CD21, CD22, CD23, CD80), other
receptors expressed on hematopoietic and certain solid tumors
(e.g., CD15, CD33, CD45, NCA90, NCA95, CD74, HLA-DR), and
tumor-associated markers expressed on diverse cancers (e.g.,
carcinoembryonic antigen, Le(y), MUC-1, MUC-2, MUC-3, MUC-4, Tag-72
[B72.3 and CC49 constituting the antibodies against Tag-72], EGP-1,
EGP-2, the antigen specific for A33 antibody, PSA, PSMA, EGFR,
HER2/neu, PAM-4, AFP, HCG and its subunits, melanoma-associated
antigens (e.g., S100), glioma-associated antigens, ovarian
cancer-associated antigens, etc.), as well as target molecules
expressed by the vasculature of the tumors (tumor angiogenesis
markers, usually produced by the vascular endothelium), such as
VEGF and tenascin (the latter in brain tumors, for example), and
also to oncogene-associated markers, such as p53. Other
tumor-associated antigens include, but are not limited to A3, BrE3,
CD1, CD1a, CD3, CD5, CD15, CD25, CD30, CD33, CD45, CD79a, CSAp,
EGP-1, EGP-2, Ep-CAM, Ba 733, KC4, KS-1, KS1-4, MAGE, RS5, IL-6,
insulin growth factor-1 (IGF-1), Tn antigen, Thomson-Friedenreich
antigens, tumor necrosis antigens, 17-1A, an angiogenesis marker, a
cytokine, an immunomodulator, an oncogene marker (e.g., p53), and
an oncogene product. In addition to the exemplary antibodies to
such antigens disclosed herein, antibodies to these antigens are
known in the art (see, for example, Kim et al., Expression and
Characterization of a Recombinant Fab Fragment Derived from an
Anti-Human alpha-Fetoprotein Monoclonal Antibody, Mol. Cells 11:
158-163, 2001; and Haisma et al., Construction and characterization
of a fusion protein of single-chain anti-CD40 antibody and human
-glucuronidase for antibody-directed enzyme prodrug therapy, Blood
92:184-190, 1998.
[0377] Another marker of interest is transmembrane activator and
CAML-interactor (TACI). See Yu et al., Nat. Immunol. 1:252-256,
2000. Briefly, TACI is a marker for B-cell malignancies (e.g.,
lymphoma). Further it is known that TACI and B cell maturation
antigen (BCMA) are bound by the tumor necrosis factor homolog a
proliferation-inducing ligand (APRIL). APRIL stimulates in vivo
proliferation of primary B and T cells and increases spleen weight
due to accumulation of B cells in vivo. APRIL also competes with
TALL-I (also called BLyS or BAFF) for receptor binding. Soluble
BCMA and TACI specifically prevent binding of APRIL and block
APRIL-stimulated proliferation of primary B cells. BCMA-Fc also
inhibits production of antibodies against keyhole limpet hemocyanin
and Pneumovax in mice, indicating that APRIL and/or TALL-I
signaling via BCMA and/or TACI are required for generation of
humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two
ligand-two receptor pathway involved in stimulation of B and T cell
function.
[0378] Tumor-specific antigens (TSAs), tumor-associated
differentiation antigens (TADAs) and other antigens associated with
cancers and other hyperproliferative diseases also include, but are
not limited to, C1 IAC, a human cancer associated protein (U.S.
Pat. No. 4,132,769); the CA125 antigen, an antigen associated with
cystadenocarcinoma of the ovary, (Hanisch et al., Carbohydr. Res.
178:29-47, 1988; U.S. Pat. No. 4,921,790); CEA (carcinembryonic
antigen), an antigen present on many adenocarcinomas (Horig et al.,
Strategies for cancer therapy using carcinembryonic antigen
vaccines, Expert Reviews in Molecular Medicine,
http://www-ermm.cbcu.cam.ac.uk: 1, 2000); CORA (carcinoma or
orosomucoid-related antigen) described by Toth et al. (U.S. Pat.
No. 4,914,021); DF3 antigen from human breast carcinoma (U.S. Pat.
Nos. 4,963,484 and 5,053,489); DU-PAN-2, a pancreatic carcinoma
antigen (Lan et al., Cancer Res. 45:305-310, 1985); HCA, a human
carcinoma antigen (U.S. Pat. No. 5,693,763); Her2, a breast cancer
antigen (Fendly et al., The Extracellular Domain of HER2/neu Is a
Potential Immunogen for Active Specific Immunotherapy of Breast
Cancer, J. Biol. Resp. Modifiers 9:449-455, 1990); MSA, a breast
carcinoma glycoprotein (Tjandra et al., Br. J. Surg. 75:811-817,
1988); MFGM, a breast carcinoma antigen (Ishida et al., Tumor Biol.
10: 12-22, 1989); PSA, prostrate specific antigen (Nadji et al.,
Prostatic-specific-antigen, Cancer 48:1229-1232, 1981); STEAP (six
transmembrane epithelial antigens of the prostate) proteins (U.S.
Pat. No. 6,329,503); TAG-72, a breast carcinoma glycoprotein
(Kjeldsen et al., Cancer Res. 48:2214-2220, 1988); YH206, a lung
carcinoma antigen (Hinoda et al., Cancer J. 42:653-658, 1988); the
p97 antigen of human melanoma (Estin et al., Recombinant Vaccinia
Virus Vaccine Against the Human Melanoma Antigen p97 for Use in
Immunotherapy, Proc. Natl. Acad. Sci. USA 85:1052-1056, 1988); and
the melanoma specific antigen described in U.S. Pat. No.
6,025,191).
[0379] X.B. Antigens from Pathogens
[0380] X.B.1. Viruses
[0381] By way of non-limiting example, pathogens include viruses
including, but not limited to, hepatitis type A, hepatitis type B,
hepatitis type C, influenza, varicella, adenovirus, herpes simplex
type I (HSV-I), herpes simplex type II (HSV-11), rinderpest,
rhinovirous, echovirus, rabies virus, Ebola virus, rotavirus,
respiratory syncytial virus, papilloma virus, papova virus,
cytomegalovirus (CMV), echinovirus, arbovirus, huntavirus,
coxsackie virus, mumps virus, measles virus, rubella virus, polio
virus, human immunodeficiency virus type I (HIV-I) and human
immunodeficiency virus type II (HIV-I), Sendai virus, feline
leukemia virus, Reovirus, poliovirus, human serum parvo-like virus,
simian virus 40 (SV40), respiratory syncytial virus (RSV), mouse
mammary tumor virus (MMTV), Varicella-Zoster virus, Dengue virus,
rubella virus, measles virus, adenovirus, human T-cell leukemia
viruses, Epstein-Barr virus, murine leukemia virus, vesicular
stomatitis virus (VSV), smallpox (Variola virus), Sindbis virus,
lymphocytic choriomeningitis virus, Rinderpest virus, wart virus
and blue tongue virus.
[0382] X.B.2. Intracellular Pathogens
[0383] By way of non-limiting example, pathogens include
intracellular obligates, including but not limited to Chlamydia
sp., Rickettsia sp., intracellular protozoa, including but not
limited to, species of Leishmania, Kokzidioa, and Trypanosoma,
including without limitation intracellular spirochetes, including
but not limited to, Borrelia burgdorfei, the causative agent of
Lyme disease; and species of Plasmodia, sporozoan obligate
intracellular parasites of liver and red blood cells, including but
not limited to P. falciparum, the causative agent of malaria,
Trypanosoma brucei, a hemoflagellate causing sleeping sickness, and
Trypanosoma cruzi, the cause of Chagas disease. For reviews of the
immunology of such pathogens, see Blackman, Proteases involved in
erythrocyte invasion by the malaria parasite: function and
potential as chemotherapeutic targets, Curr Drug Targets 1:59-83,
2000; Kosma, Chlamydial lipopolysaccharide, Biochim. Biophys. Acta.
1455:387-402, 1999; Casadevall, Antibody-mediated protection
against intracellular pathogens, Trends Microbiol. 6:102-7, 1998;
Hoffman and Franke, Inducing protective immune responses against
the sporozoite and liver stages of Plasmodium, Immunol. Lett.
41:89-94, 1994; Keusch, Immune responses in parasitic diseases.
Part A: general concepts, Rev. Infect. Dis. 4:751-5, 1982; and
Colli and Alves, Relevant glycoproteins on the surface of
Trypanosoma cruzi.
[0384] X.B.3. Bacteria
[0385] Bacterial pathogens include, but are not limited to,
Streptococcus agalactiae, Legionella pneumophilia, Streptococcus
pyogenes, Escherichia coli, Salmonella typhimurium, Neisseria
gonorrhoeae, Neisseria meningitidis, Pneumococcus sp., Hemophilis
influenzae B, Yersina pestis, Mycobacteria sp. including by way of
non-limiting example Mycobacterium leprae and Mycobacterium
tuberculosis, Treponema pallidum, Pseudomonas aeruginosa,
Francisella tularensis, Brucella sp. including Brucella abortus,
Bacillus anthracis including Anthrax spores, Clostridium botulinum
including Botulism toxin, and Clostridium tetani including Tetanus
toxin). See U.S. Pat. No. 5,332,567.
[0386] X.B.4. Pathogenic Fungi
[0387] Fungal pathogens include, but are not limited to, Candida
sp., Aspergillus sp., Mucor sp., Rhizopus sp., Fusarium sp.,
Penicillium marneffei and Microsporum. Trichophyton mentagrophytes,
Candida albicans, Histoplasma capsulatum, Blastomyces dermatitidis,
and Coccidioides immitis are fungal pathogens of particular
interest.
[0388] XI. Antibodies
[0389] The Fvs of the invention constructs are derived from an
antibody and specifically bind a targeted tissue. Exemplary Fvs are
derived from anti-CD20 antibodies, such as those described in U.S.
Provisional Application Ser. No. 60/356,132, entitled "Anti-CD20
Antibodies And Fusion Proteins Thereof And Methods Of Use",
Attorney Docket No. 18733-1073, filed Feb. 14, 2002 (the contents
of which are incorporated by reference herein in their entirety)
and hMN-14 antibodies, such as those disclosed in U.S. Pat. No.
5,874,540 (the contents of which are incorporated by reference
herein in their entirety), which is a Class III
anti-carcinoembryonic antigen antibody (anti-CEA antibody).
[0390] The Fvs can be from murine antibodies, cdr-grafted
(humanized) antibodies, or human antibodies. The Fvs can be derived
from human monoclonal antibodies, transgenic mice with human
Fv-libraries, or phage/ribosome human IgG libraries.
[0391] When the Fvs are derived from CDR-grafted antibodies,
appropriate variable region framework sequences may be used having
regard to the class or type of the donor antibody from which the
antigen binding regions are derived. Preferably, the type of human
framework used is of the same or similar class or type as the donor
antibody. Advantageously, the framework is chosen to maximize or
optimize homology with the donor antibody sequence, particularly at
positions spatially close to or adjacent the CDRs. Examples of
human frameworks which may be used to construct CDR-grafted
antibodies are LAY, POM, TUR, TEI, KOL, NEWM, REI and EU. KOL and
NEWM and are suitable for heavy chain construction. REI is suitable
for light chain construction and EU is suitable for both heavy
chain and light chain construction.
[0392] The light or heavy chain variable regions of the CDR-grafted
antibodies may be fused to human light or heavy chain constant
domains as appropriate, (the term "heavy chain constant domains" as
used herein is to be understood to include hinge regions unless
specified otherwise). The human constant domains of the CDR-grafted
antibodies, where present, may be selected having regard to the
proposed function of the antibody, in particular, the effector
functions which may be required. For example, IgG1 and IgG3 isotype
domains may be used when the CDR-grafted antibody is intended for
therapeutic purposes and antibody effector functions are required.
Alternatively, IgG2 and IgG4 isotype domains may be used when the
CDR-grafted antibody is intended for purposes for which antibody
effector functions are not required, e.g. for imaging, diagnostic
or cytotoxic targeting purposes. Light chain human constant domains
which may be fused to the light chain variable region include human
Lambda or, especially, human Kappa chains.
[0393] Antibodies may further contain desirable mutations, e.g.,
mutations facilitating clearance of antibody constructs. A mutation
may encompass, for example, a "conservative" change, wherein a
substituted amino has similar structural or chemical properties,
such as charge or size (e.g., replacement of leucine with
isoleucine). A mutation also encompasses, for example, a
"non-conservative" change (e.g., replacement of a glycine with a
tryptophan).
[0394] The scFv component of the bi-specific mutant antibody
specifically binds a targetable construct. The use of any scFv
component is contemplated by the present invention. Preferred scFv
components are 679 scFv (derived from a murine anti-HSG) and
734scFv (derived from a murine anti-diDTPA). The scFv can be
murine, cdr-grafted (humanized) or human.
[0395] The light or heavy chain variable regions of the CDR-grafted
antibodies may be fused to human light or heavy chain constant
domains as appropriate, (the term "heavy chain constant domains" as
used herein are to be understood to include hinge regions unless
specified otherwise). The human constant domains of the CDR-grafted
antibodies, where present, may be selected having regard to the
proposed function of the antibody, in particular the effector
functions which may be required. For example, IgG1 and IgG3 isotype
domains may be used when the CDR-grafted antibody is intended for
therapeutic purposes and antibody effector functions are required.
Alternatively, IgG2 and IgG4 isotype domains may be used when the
CDR-grafted antibody is intended for purposes for which antibody
effector functions are not required, e.g. for imaging, diagnostic
or cytotoxic targeting purposes. Light chain human constant domains
which may be fused to the light chain variable region include human
Lambda or, especially, human Kappa chains.
[0396] The murine monoclonal antibody designated 679 (an IgG1, K)
binds with high affinity to molecules containing the tri-peptide
moiety histamine succinyl glycyl (HSG) (Morel et al., Mol. Immunol.
27:995-1000, 1990). The nucleotide sequence pertaining to the
variable domains (V.sub.H and V.sub.K) of 679 has been determined
(Qu et al, unpublished results). V.sub.K is one of two isotypes of
the antibody light chains, V.sub.L. The function of the two
isotypes is identical. 679 can be humanized or fully human to help
avoid an adverse response to the murine antibody.
[0397] hMN14 is a humanized monoclonal antibody that binds
specifically to CEA (Shevitz et al., J. Nucl. Med. 34, 217P, 1993;
U.S. Pat. No. 6,254,868). While the original Mabs were murine,
humanized antibody reagents are now utilized to reduce the human
anti-mouse antibody response. The variable regions of this antibody
were engineered into an expression construct (hMN14-scFv-L5). A
preferred mutant hMN14 is hMN-14IgG.sup.1253A wherein amino acid
residue 253 is changed from isoleucine to alanine.
[0398] 734 is a murine monoclonal antibody designated that binds
with high affinity to the metal-chelate complex indium-DTPA
(diethylenetriamine-pen- taacetic acid).
[0399] Single light chain and two heavy chain variable region
sequences encoding the humanized anti-hCD20 (hA20) antibody were
designed and constructed, as in U.S. Provisional Application Ser.
No. 60/356,132, entitled "Anti-CD20 Antibodies And Fusion Proteins
Thereof And Methods Of Use", Attorney Docket No. 18733-1073, filed
Feb. 14, 2002, and U.S. application Ser. No. 10/366,709, filed Feb.
14, 2003 (the contents of each of which are incorporated by
reference herein in their entirety). ha20 contains the V.sub.H and
V.sub.K genes of A20, an anti-CD20 antibody, obtained by RT-PCR
using the primer pairs VH1BACK/VH1FOR and VK1BACK/VK1FOR,
respectively Orlandi et al., Proc. Natl. Acad. Sci. USA 86: 3833,
1989. Human REI framework sequences were used for V.sub.K, and a
combination of EU and NEWM framework sequences were used for
V.sub.H. There are a number of amino acid changes in each chain
outside of the CDR regions when compared to the starting human
antibody frameworks. The heavy chain of hA20, hA20V.sub.H1,
contains nine changes, while hA20V.sub.H2 contains three changes
from the human EU frameworks. hA20V.sub.H2 is preferred because it
contains more amino acids from the human antibody framework region
than hA20V.sub.H1. The light chain of hA20, hA20V.sub.K, contains
seven amino acid changes from the REI framework.
[0400] The hLL-2 antibody is a humanized antibody prepared by
combining the CDR regions of murine LL-2 antibody (mLL-2) with
variable region framework sequences obtained from human antibodies.
The sequence of the heavy and light chain variable regions of hLL-2
are shown in FIG. 1 of U.S. Pat. No. 5,789,554. As shown in that
figure, the kappa light chain of hLL-2 contains the four light
chain CDR regions from mLL-2 and the four framework regions of
human antibody REI. The heavy chain of hLL-2 contains the three
heavy chain CDRs from mLL-2 combined with three framework regions
from human antibody EU, together with a fourth framework region
from human antibody NEWM.
[0401] XI.A. Definitions
[0402] The term "antibody" is meant to encompass an immunoglobulin
molecule obtained by in vitro or in vivo generation of an
immunogenic response, and includes both polyclonal, antipeptide and
monoclonal antibodies. The term "antibody" also includes
genetically engineered antibodies and/or antibodies produced by
recombinant DNA techniques and "humanized" antibodies. As described
below, humanized and even fully human antibodies can be produced by
phage display, gene and chromosome transfection methods, as well as
by other means.
[0403] An "immunogenic response" or "antigenic response" is one
that results in the production of antibodies directed to a compound
after the appropriate cells have been contacted therewith. The
compound that is used to elicit an immunogenic response is referred
to as an immunogen or antigen. The antibodies produced in the
immunogenic response specifically bind the immunogen used to elicit
the response.
[0404] The compound that is used to elicit an immunogenic response
is referred to as an immunogen or antigen. An "epitope" or
"antigenic determinant" is an area on the surface of an immunogen
that stimulates a specific immune response directed to the epitope.
In proteins, particularly denatured proteins, an epitope is
typically defined and represented by a contiguous amino acid
sequence. However, in the case of nondenatured proteins, epitopes
also include structures, such as active sites, that are formed by
the three-dimensional folding of a protein in a manner such that
amino acids from separate portions of the amino acid sequence of
the protein are brought into close physical contact with each
other.
[0405] A "hapten" is a small molecule that cannot provoke an immune
response unless first bound to an immunogenic carrier molecule.
Although a hapten cannot itself provoke an immune response, it is
specifically bound by antibodies generated during an immunogenic
response to the hapten-carrier conjugate.
[0406] The term "antibody fragment" refers to functional fragments
of antibodies, i.e., polypeptides that are smaller than an antibody
which have sequences from the antibody, but nevertheless have the
ability to specifically bind to an antigenic determinant. Antibody
fragments can be prepared by in vitro manipulation of antibodies
(e.g., by limited proteolysis of an antibody), or via recombinant
DNA technology (e.g., the preparation of single-chain antibodies
from phage display libraries).
[0407] XI.B. Antibody Structure
[0408] Naturally occurring (wildtype) antibody molecules are
Y-shaped molecules consisting of four polypeptide chains, two
identical heavy chains and two identical light chains, which are
covalently linked together by disulfide bonds. Both types of
polypeptide chains have constant regions, which do not vary or vary
minimally among antibodies of the same class (i.e., IgA, IgM,
etc.), and variable regions. The variable regions are unique to a
particular antibody and comprise a recognition element for an
epitope. The carboxy-terminal regions of both heavy and light
chains are conserved in sequence and are called the constant
regions (also known as C-domains). The amino-terminal regions (also
known as V-domains) are variable in sequence and are responsible
for antibody specificity. The antibody specifically recognizes and
binds to an antigen mainly through six short
complementarity-determining regions (CDRs) located in their
V-domains.
[0409] Each light chain of an antibody is associated with one heavy
chain, and the two chains are linked by a disulfide bridge formed
between cysteine residues in the carboxy-terminal region of each
chain, which is distal from the amino terminal region of each chain
that constitutes its portion of the antigen binding domain.
Antibody molecules are further stabilized by disulfide bridges
between the two heavy chains in an area known as the hinge region,
at locations nearer the carboxy terminus of the heavy chains than
the locations where the disulfide bridges between the heavy and
light chains are made. The hinge region also provides flexibility
for the antigen-binding portions of an antibody.
[0410] An antibody's specificity is determined by the variable
regions located in the amino terminal regions of the light and
heavy chains. The variable regions of a light chain and associated
heavy chain form an "antigen binding domain" that recognizes a
specific epitope; an antibody thus has two antigen binding domains.
The antigen binding domains in a wildtype antibody are directed to
the same epitope of an immunogenic protein, and a single wildtype
antibody is thus capable of binding two molecules of the
immunogenic protein at the same time. Thus, a wildtype antibody is
monospecific (i.e., directed to a unique antigen) and divalent
(i.e., capable of binding two molecules of antigen).
[0411] XI.C. Types of Antibodies
[0412] "Polyclonal antibodies" are generated in an immunogenic
response to a protein having many epitopes. A composition (e.g.,
serum) of polyclonal antibodies thus includes a variety of
different antibodies directed to the same and to different epitopes
within the protein. Methods for producing polyclonal antibodies are
known in the art (see, e.g., Cooper et al., Section III of Chapter
11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et
al., eds., John Wiley and Sons, New York, 1992, pages 11-37 to
11-41).
[0413] "Antipeptide antibodies" (also known as "monospecific
antibodies") are generated in a humoral response to a short
(typically, 5 to 20 amino acids) immunogenic polypeptide that
corresponds to a few (preferably one) isolated epitopes of the
protein from which it is derived. A plurality of antipeptide
antibodies includes a variety of different antibodies directed to a
specific portion of the protein, i.e, to an amino acid sequence
that contains at least one, preferably only one, epitope. Methods
for producing antipeptide antibodies are known in the art (see,
e.g., Cooper et al., Section III of Chapter 11 in: Short Protocols
in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and
Sons, New York, 1992, pages 11-42 to 11-46).
[0414] A "monoclonal antibody" is a specific antibody that
recognizes a single specific epitope of an immunogenic protein. In
a plurality of a monoclonal antibody, each antibody molecule is
identical to the others in the plurality. In order to isolate a
monoclonal antibody, a clonal cell line that expresses, displays
and/or secretes a particular monoclonal antibody is first
identified; this clonal cell line can be used in one method of
producing the antibodies of the invention. Methods for the
preparation of clonal cell lines and of monoclonal antibodies
expressed thereby are known in the art (see, for example, Fuller et
al., Section II of Chapter 11 in: Short Protocols in Molecular
Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New
York, 1992, pages 11-22 to 11-11-36).
[0415] A "naked antibody" is an antibody that lacks the Fc portion
of a wildtype antibody molecule. The Fc portion of the antibody
molecule provides effector functions, such as complement fixation
and ADCC (antibody dependent cell cytotoxicity), which set
mechanisms into action that may result in cell lysis. See, e.g.,
Markrides, Therapeutic inhibition of the complement system,
Pharmacol. Rev. 50:59-87, 1998. In some systems, it appears that
the therapeutic action of an antibody depends upon the effector
functions of the Fc region (see, e.g., Golay et al., Biologic
response of B lymphoma cells to anti-CD20 monoclonal antibody
rituximab in vitro: CD55 and CD59 regulate complement-mediated cell
lysis, Blood 95:3900-3908, 2000).
[0416] However, it is possible that the Fc portion is not required
for therapeutic function in every instance, as other mechanisms,
such as apoptosis, can come into play. Moreover, the Fc region may
be deleterious in some applications as antibodies comprising an Fc
region are taken up by Fc receptor-bearing cells, thereby reducing
the amount of thereapeutic antibody taken up targeted cells.
Vaswani and Hamilton, Humanized antibodies as potential therapeutic
drugs. Ann. Allergy Asthma Immunol. 81:105-119, 1998. Components of
the immune system may recognize and react to antibodies that are
clumped together on the surface of tumor cells. It is thus
envisioned that the resulting immune response will target and
destroy, or at least limit the proliferation of, the tumor
cells.
[0417] One way to get naked antibodies deilivered to surfaces where
they will clump together is to use a targetable construct or
complex to bring different naked antibodies together on a targeted
cellular surface. By way of non-limiting example, an anti-C20
antibody (e.g., Rituxan) and an anti-C22 antibody might be
administered separately or together, allowed to clear so that
unbound antibodies are removed from the system. The addition of a
targetable construct that binds and connects both types antibodies,
thereby forming a targetable construct in situ, which is expected
to mimic a group of anti-C20 and anti-C22 antibodies clumped on the
surface of a tumor cell.
[0418] Naked antibodies are also of interest for therapy of
diseases caused by parasites, such as malaria. Vukovic et al.,
Immunoglobulin G3 antibodies specific for the 19-kilodalton
carboxyl-terminal fragment of Plasmodium yoelii merozoite surface
protein 1 transfer protection to mice deficient in Fc-RI receptors,
Infect. Immun. 68:3019-22, 2000.
[0419] Single chain antibodies (scFv) generally do not include
portions of the Fc region of antibodies that are involved in
effector functions and are thus naked antibodies, although methods
are known for adding such regions to known scFv molecules if
desired. See Helfrich et al., A rapid and versitile method for
harnessing scFv antibody fragments with various biological
functions, J. Immunol. Meth. 237:131-145,2000; and de Haard et al.,
Creating and engineering human antibodies for immunotherapy, Adv.
Drug Delivery Rev. 31:5-31, 1998.
[0420] XI.D. Antibody Fragments
[0421] XI.D. 1. Proteolytic Antibody Fragments
[0422] Antibody fragments produced by limited proteolysis of
wildtype antibodies are called proteolytic antibody fragments.
These include, but are not limited to, the following.
[0423] "F(ab').sub.2 fragments" are released from an antibody by
limited exposure of the antibody to a proteolytic enzyme, e.g.,
pepsin or ficin. An F(ab').sub.2 fragment comprises two "arms,"
each of which comprises a variable region that is directed to and
specifically binds a common antigen. The two Fab' molecules are
joined by interchain disulfide bonds in the hinge regions of the
heavy chains; the Fab' molecules may be directed toward the same
(bivalent) or different (bispecific) epitopes.
[0424] "Fab' fragments" contain a single anti-binding domain
comprising an Fab and an additional portion of the heavy chain
through the hinge region.
[0425] "Fab'-SH fragments" are typically produced from F(ab').sub.2
fragments, which are held together by disulfide bond(s) between the
H chains in an F(ab').sub.2 fragment. Treatment with a mild
reducing agent such as, by way of non-limiting example,
beta-mercaptoethylamine, breaks the disulfide bond(s), and two Fab'
fragments are released from one F(ab').sub.2 fragment. Fab'-SH
fragments are monovalent and monospecific.
[0426] "Fab fragments" (i.e., an antibody fragment that contains
the antigen-binding domain and comprises a light chain and part of
a heavy chain bridged by a disulfide bond) are produced by papain
digestion of intact antibodies. A convenient method is to use
papain immobilized on a resin so that the enzyme can be easily
removed and the digestion terminated. Fab fragments do not have the
disulfide bond(s) between the H chains present in an F(ab').sub.2
fragment.
[0427] XI.D.2. Recombinant Antibody Fragments
[0428] "Single-chain antibodies" are one type of antibody fragment.
The term single chain antibody is often abbreviated as "scFv" or
"sFv." These antibody fragments are produced using molecular
genetics and recombinant DNA technology. A single-chain antibody
consists of a polypeptide chain that comprises both a V.sub.H and a
V.sub.L portion. Unlike wildtype antibodies, wherein two separate
heavy and light polypeptide chains are conjoined to form a single
antigen-binding variable region, a single-chain antibody is a
single polypeptide that comprises an antigen-binding variable
region. That is, a single-chain antibody comprises the variable,
antigen-binding determinative region of a single light and heavy
chain of an antibody linked together by a chain of 10-25 amino
acids.
[0429] The term "single-chain antibody" includes but is not limited
to a disulfide-linked Fv (dsFv) in which two single-chain
antibodies linked together by a disulfide bond; a bispecific sFv (a
sFv or a dsFv molecule having two antigen-binding domains, each of
which may be directed to a different epitope); a diabody (a
dimerized sFv formed when the V.sub.H domain of a first sFv
assembles with the V.sub.L domain of a second sFv and the V.sub.L
domain of the first sFv assembles with the V.sub.H domain of the
second sFv; the two antigen-binding regions of the diabody may be
directed towards the same or different epitopes); and a triabody (a
trimerized sFv, formed in a manner similar to a diabody, but in
which three antigen-binding domains are created in a single
complex; the three antigen binding domains may be directed towards
the same or different epitopes).
[0430] "Camelid antibodies" are unlike mammalian antibodies in that
they need only V-domain, namely V.sub.H, to specifically and
effectively bind an antigen. Camelid antibodies or fragments
thereof have the advantages of being water soluble and showing good
expression in yeast and Aspergillus moulds. For reviews, see
Muyldermans, Single domain camel antibodies: current status, J.
Biotechnol. 74:277-302, 2001; and Wemery, Camelid immunoglobulins
and their importance for the new-born--a review, J. Vet. Med. B.
Infect. Dis. Vet. Public Health 48:561-8, 2001. See also Spinelli
et al., Camelid heavy-chain variable domains provide efficient
combining sites to haptens, Biochemistry 39:1217-22, 2000;
Muyldermans et al., Unique single-domain antigen binding fragments
derived from naturally occurring camel heavy-chain antibodies, J.
Mol. Recog. 12:131-40, 1999; and Davies et al., Single antibody
domains as small recognition units: design and in vitro antigen
selection of camelized, human V.sub.H domains with improved protein
stability, Protein Eng. 9:531-7, 1996. Other immunoglobulin-like
molecules from other species may also be used. See, e.g., Roux et
al., Structural analysis of the nurse shark (new) antigen receptor
(NAR): molecular convergence of NAR and unusual mammalian
immunoglobulins, Proc. Natl. Acad. Sci. USA. 95:11804-9, 1998.
Methods of producing camelid antibodies are known in the art. See,
for example, U.S. Pat. Nos. 6,015,695; 6,005,079; 5,874,541;
5,840,526; 5,800,988; and 5,759,808, each of which is entitled
Immunoglobulins Devoid of Light Chains.
[0431] "Humanized antibodies" have been modified, by genetic
manipulation and/or in vitro treatment to be more human, in terms
of amino acid sequence, glycosylation pattern, etc., in order to
reduce the antigenicity of the antibody or antibody fragment in an
animal to which the antibody is intended to be administered. See
Gussow and Seemann, Humanization of monoclonal antibodies, Meth.
Enz. 203:99-121, 1991 and Vaswani and Hamilton, Humanized
antibodies as potential therapeutic drugs, Ann. Allergy Asthma
Immunol. 81:105-119, 1998.
[0432] "Fully human antibodies" are human antibodies produced in
transgenic animals such as Xenomice. XenoMouse strains are
genetically engineered mice in which the murine IgH and Igk loci
have been functionally replaced by their Ig counterparts on yeast
artificial YAC transgenes. These human Ig transgenes can carry the
majority of the human variable repertoire and can undergo class
switching from IgM to IgG isotypes. The immune system of the
xenomouse recognizes administered human antigens as foreign and
produces a strong humoral response. The use of XenoMouse in
conjunction with well-established hybridomas techniques, results in
fully human IgG mAbs with sub-nanomolar affinities for human
antigens (see U.S. Pat. No. 5,770,429, entitled "Transgenic
non-human animals capable of producing heterologous antibodies",
U.S. Pat. No. 6,162,963, entitled "Generation of xenogenetic
antibodies"; U.S. Pat. No. 6,150,584, entitled "Human antibodies
derived from immunized xenomice", U.S. Pat. No. 6,114,598, entitled
"Generation of xenogeneic antibodies"; and U.S. Pat. No. 6,075,181,
entitled "Human antibodies derived from immunized xenomice"; for
reviews, see Green, Antibody engineering via genetic engineering of
the mouse: XenoMouse strains are a vehicle for the facile
generation of therapeutic human monoclonal antibodies, J. Immunol.
Meth. 231:11-23, 1999; Wells, Eek, a XenoMouse: Abgenix, Inc.,
Chem. Biol. 7:R185-6, 2000; and Davis et al., Transgenic mice as a
source of filly human antibodies for the treatment of cancer,
Cancer Metastasis Rev. 18:421-5, 1999).
[0433] "Complementary determining region peptides" or "CDR
peptides" are another form of an antibody fragment. A CDR peptide
(also known as "minimal recognition unit") is a peptide
corresponding to a single complementarity-determining region (CDR),
and can be prepared by constructing genes encoding the CDR of an
antibody of interest. Such genes are prepared, for example, by
using the polymerase chain reaction to synthesize the variable
region from RNA of antibody-producing cells. See, for example,
Larrick et al., Methods: A Companion to Methods in Enzymology
2:106, 1991.
[0434] "T-cell receptor (TCR) fragments" are soluble peptides
having amino acid sequences corresponding to the variable and
constant regions of a T-cell receptor. Soluble TCR fragments can be
prepared as a single chain (scTCR) or as separate components with
dimerization domain that allow the separate peptides to stably
associated with each other. (Willcox et al., Production of soluble
alpha:beta T-cell receptor heterodimers suitable for biophysical
analysis of ligand binding, Protein Science 8:2418-2423, 1999).
Soluble TCR molecules made using chinese hamster ovary (CHO) cells
are described by Lin et al., Expression of T Cell Antigen Receptor
Heterodimers in a Lipid-Linked Form, Science 249:677-679, 1990; and
Davis et al., TCR Recognition and Selection In Vivo, Cold Spring
Harbor Symposia on Quantitative Biology, LIV, 119-128, 1989). Both
of these articles describe the use of a GPI linkage approach to
produce soluble TCR molecules. For other examply methods of
producing soluble TCR fragments, see, for example, U.S. Pat. No.
6,165,745, Recombinant production of immunoglobulin-like domains in
prokaryotic cells; U.S. Pat. No. 6,080,840, Soluble T cell
receptors; U.S. Pat. No. 5,723,309, Production of subunits of
soluble T cell receptors by co-transfection; U.S. Pat. No.
5,552,300, T cell antigen receptor V region proteins and methods of
preparation thereof; Novotny et al., Proc. Natl. Acad Sci USA.
88:8646-8650, 1991; Ward, Scand. J. Immunol. 34:215-220, 1991; and
Pecorari et al., Folding, Heterodimeric Association and Specific
Peptide Recognition of a Murine .alpha..beta. T-cell Receptor
Expressed in Escherichia coli, J. Mol. Biol. 285:1831-1843,
1999.
[0435] "Chimeric antibody derivatives" such as chimeric TCR:Ab
molecules have been produced by shuffling the variable and constant
domains of murine T-cell receptors with the constant region of an
immunoglobulin kappa light chain. For example, Gregoire et al.
(Proc. Natl. Acad. Sci. USA 88:8077-8081, 1991) show a murine
chimera consisting of the C-alpha and V-alpha genes of the KB5-C2
joined to the C region of the kappa light chain of the S105
monoclonal antibody, and a V-beta-C-beta-C-kappa. chimera. Both are
transfected into a mammalian B cell myeloma that does not express
native immunoglobulin heavy or light chains. See also, Weber et
al., Nature 356:793-795, 1992.
[0436] In "cysteine-modified antibodies," a cysteine amino acid is
inserted or substituted on the surface of antibody by genetic
manipulation and used to conjugate the antibody to another molecule
via, e.g., a disulfide bridge. Cysteine substitutions or insertions
for antibodies have been described (see U.S. Pat. No. 5,219,996).
Methods for introducing Cys residues into the constant region of
the IgG antibodies for use in site-specific conjugation of
antibodies are described by Stimmel et al. (J. Biol. Chem
275:330445-30450, 2000).
[0437] XII. Pharmaceutical Compositions
[0438] XII.A. Definitions
[0439] A "pharmaceutical composition" refers to a composition
comprising a drug wherein the carrier is a pharmaceutically
acceptable carrier, while a "veterinary composition" is one wherein
the carrier is a veterinarily acceptable carrier. The term
"pharmaceutically acceptable carrier" or "veterinarily acceptable
carrier" includes any medium or material that is not biologically
or otherwise undesirable, i.e, the carrier may be administered to
an organism along with a composition or compound of the invention
without causing any undesirable biological effects or interacting
in a deleterious manner with the complex or any of its components
or the organism. Examples of pharmaceutically acceptable reagents
are provided in The United States Pharmacopeia, The National
Formulary, United States Pharmacopeial Convention, Inc., Rockville,
Md. 1990, hereby incorporated in its entirety by reference herein
into the present application, as is Phamaceutical Dosage Forms
& Drug Delivery Systems, 7th Edition, Ansel et al., editors,
Lippincott Williams & Wilkins, 1999.
[0440] The drug (i.e., targetable construct or complex) is included
in the pharmaceutical composition in an amount sufficient to
produce the desired effect upon the patient. The pharmaceutical
compositions of the invention can further comprise other chemical
components, such as diluents and excipients. A "diluent" is a
chemical compound diluted in a solvent, preferably an aqueous
solvent, that facilitates dissolution of the drug in the solvent,
and it may also serve to stabilize the biologically active form of
the drug or one or more of its components. Salts dissolved in
buffered solutions are utilized as diluents in the art. For
example, preferred diluents are buffered solutions containing one
or more different salts. A preferred buffered solution is phosphate
buffered saline (particularly in conjunction with compositions
intended for pharmaceutical administration), as it mimics the salt
conditions of human blood. Since buffer salts can control the pH of
a solution at low concentrations, a buffered diluent rarely
modifies the biological activity of a biologically active
peptide.
[0441] An "excipient" is any more or less inert substance that can
be added to a composition in order to confer a suitable property,
for example, a suitable consistency or to form a drug. Suitable
excipients and carriers include, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, agar, pectin, xanthan gum, guar gum, locust bean gum,
hyaluronic acid, casein potato starch, gelatin, gum tragacanth,
polyacrylate, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
If desired, disintegrating agents can also be included, such as
cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Other suitable excipients and
carriers include hydrogels, gellable hydrocolloids, and chitosan.
Chitosan microspheres and microcapsules can be used as carriers.
See WO 98/52547 (which describes microsphere formulations for
targeting compounds to the stomach, the formulations comprising an
inner core (optionally including a gelled hydrocolloid) containing
one or more active ingredients, a membrane comprised of a water
insoluble polymer (e.g., ethylcellulose) to control the release
rate of the active ingredient(s), and an outer layer comprised of a
bioadhesive cationic polymer, for example, a cationic
polysaccharide, a cationic protein, and/or a synthetic cationic
polymer; U.S. Pat. No. 4,895,724. Typically, chitosan is
cross-linked using a suitable agent, for example, glutaraldehyde,
glyoxal, epichlorohydrin, and succinaldehyde. Compositions
employing chitosan as a carrier can be formulated into a variety of
dosage forms, including pills, tablets, microparticles, and
microspheres, including those providing for controlled release of
the active ingredient(s). Other suitable bioadhesive cationic
polymers include acidic gelatin, polygalactosamine, polyamino acids
such as polylysine, polyhistidine, polyornithine, polyquaternary
compounds, prolamine, polyimine, diethylaminoethyldextran (DEAE),
DEAE-imine, DEAE-methacrylate, DEAE-acrylamide, DEAE-dextran,
DEAE-cellulose, poly-p-aminostyrene, polyoxethane,
copolymethacrylates, polyamidoamines, cationic starches,
polythiodiethylaminomethylethylene and polyvinylpyridine.
[0442] XII.B. Formulation of Pharmaceutical Compositions
[0443] The targetable constructs and complexes of the invention can
be formulated in any suitable manner. The targetable constructs and
complexes may be uniformly (homogeneously) or non-uniformly
(heterogenously) dispersed in the carrier. Suitable formulations
include dry and liquid formulations. Dry formulations include
freeze dried and lyophilized powders, which are particularly well
suited for aerosol delivery to the sinuses or lung, or for long
term storage followed by reconstitution in a suitable diluent prior
to administration. Other preferred dry formulations include those
wherein a pharmaceutical composition according to the invention is
compressed into tablet or pill form suitable for oral
administration or compounded into a sustained release formulation.
When the pharmaceutical composition is intended for oral
administration but the targetable construct or complex is to be
delivered to epithelium in the intestines, it is preferred that the
formulation be encapsulated with an enteric coating to protect the
formulation and prevent premature release of the targetable
constructs and complexes included therein. As those in the art will
appreciate, the pharmaceutical compositions of the invention can be
placed into any suitable dosage form. Pills and tablets represent
some of such dosage forms. The pharmaceutical compositions can also
be encapsulated into any suitable capsule or other coating
material, for example, by compression, dipping, pan coating, spray
drying, etc. Suitable capsules include those made from gelatin and
starch. In turn, such capsules can be coated with one or more
additional materials, for example, and enteric coating, if desired.
Liquid formulations include aqueous formulations, gels, and
emulsions.
[0444] Some preferred embodiments concern compositions that
comprise a bioadhesive, preferably a mucoadhesive, coating. A
"bioadhesive coating" is a coating that allows a drug to adhere to
a biological surface or substance better than occurs absent the
coating. A "mucoadhesive coating" is a preferred bioadhesive
coating that allows a substance, for example, a composition
according to the invention, to adhere better to mucosa occurs
absent the coating. For example, micronized particles (e.g.,
particles having a mean diameter of about 5, 10, 25, 50, or 100 m)
can be coated with a mucoadhesive. The coated particles can then be
assembled into a dosage form suitable for delivery to an organism.
Preferably, and depending upon the location where the cell surface
transport moiety to be targeted is expressed, the dosage form is
then coated with another coating to protect the formulation until
it reaches the desired location, where the mucoadhesive enables the
formulation to be retained while the compositions or compounds of
the invention interact with the target cell surface transport
moiety.
[0445] XII.C. Administration of Pharmaceutical Compositions
[0446] The pharmaceutical compositions of the invention facilitate
administration of monoclonal antibodies to an organism, preferably
an animal, preferably a mammal, bird, fish, insect, or arachnid.
Preferred mammals include bovine, canine, equine, feline, ovine,
and porcine animals, and non-human primates. Humans are
particularly preferred. Multiple techniques of administering or
delivering a compound exist in the art including, but not limited
to, oral, rectal (e.g., an enema or suppository) aerosol (e.g., for
nasal or pulmonary delivery), parenteral, and topical
administration. Preferably, sufficient quantities of the
composition or compound of the invention are delivered to achieve
the intended effect. The particular amount of composition or
compound to be delivered will depend on many factors, including the
effect to be achieved, the type of organism to which the
composition is delivered, delivery route, dosage regimen, and the
age, health, and sex of the organism. As such, the particular
dosage of a composition or compound of the invention included in a
given formulation is left to the ordinarily skilled artisan's
discretion.
[0447] Those skilled in the art will appreciate that when the
pharmaceutical compositions of the present invention are
administered as agents to achieve a particular desired biological
result, which may include a therapeutic or protective effect(s)
(including vaccination), it may be necessary to combine the
composition or compound of the invention with a suitable
pharmaceutical carrier. The choice of pharmaceutical carrier and
the preparation of the composition or compound as a therapeutic or
protective agent will depend on the intended use and mode of
administration. Suitable formulations and methods of administration
of therapeutic agents include, but are not limited to, those for
oral, pulmonary, nasal, buccal, ocular, dermal, rectal, or vaginal
delivery.
[0448] Depending on the mode of delivery employed, the
context-dependent functional entity can be delivered in a variety
of pharmaceutically acceptable forms. For example, the
context-dependent functional entity can be delivered in the form of
a solid, solution, emulsion, dispersion, micelle, liposome, and the
like, incorporated into a pill, capsule, tablet, suppository,
areosol, droplet, or spray. Pills, tablets, suppositories,
areosols, powders, droplets, and sprays may have complex,
multilayer structures and have a large range of sizes. Aerosols,
powders, droplets, and sprays may range from small (1 micron) to
large (200 micron) in size.
[0449] Pharmaceutical compositions of the present invention can be
used in the form of a solid, a lyophilized powder, a solution, an
emulsion, a dispersion, a micelle, a liposome, and the like,
wherein the resulting composition contains one or more of the
targetable constructs or complexes of the present invention, as an
active ingredient, in admixture with an organic or inorganic
carrier or excipient suitable for enteral or parenteral
applications. The active ingredient may be compounded, for example,
with the usual non-toxic, pharmaceutically acceptable carriers for
tablets, pellets, capsules, suppositories, solutions, emulsions,
suspensions, and any other form suitable for use. The carriers
which can be used include glucose, lactose, mannose, gum acacia,
gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn
starch, keratin, colloidal silica, potato starch, urea, medium
chain length triglycerides, dextrans, and other carriers suitable
for use in manufacturing preparations, in solid, semisolid, or
liquid form. In addition auxiliary, stabilizing, thickening and
coloring agents and perfumes may be used. Examples of a stabilizing
dry agent includes triulose, preferably at concentrations of 0.1%
or greater (See, e.g., U.S. Pat. No. 5,314,695).
[0450] XII.D. Dosages
[0451] Although individual needs may vary, determination of optimal
ranges for effective amounts of pharmaceutical compositions is
within the skill of the alt. Human doses can be extrapolated from
animal studies (Katocs et al., Chapter 27 In: Remington's
Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing
Co., Easton, Pa., 1990). Generally, the dosage required to provide
an effective amount of a pharmaceutical composition, which can be
adjusted by one skilled in the art, will vary depending on the age,
health, physical condition, weight, type and extent of the disease
or disorder of the recipient, frequency of treatment, the nature of
concurrent therapy (if any) and the nature and scope of the desired
effect(s). See, for example, Nies et al., Chapter 3 In: Goodman
& Gilman's The Pharmacological Basis of Therapeutics, 9th Ed.,
Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996)
[0452] Dosing of therapeutic compositions is dependent on severity
and responsiveness of the disease state to be treated, with the
course of treatment lasting from several days to several months, or
until a cure is effected or a diminution of the disease state is
achieved. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient. The
term "patient" is intended to encompass animals (e.g., cats, dogs
and horses) as well as humans. Persons of ordinary skill can easily
determine optimum dosages, dosing methodologies and repetition
rates. Optimum dosages may vary depending on the relative potency
of individual therapeutic agents, and can generally be estimated
based on EC.sub.50 found to be effective in in vitro and in vivo
animal models.
[0453] The range of doses (the amount of targetable construct or
complex administered) is broad, since in general the efficacy of a
therapeutic effect for different mammals varies widely with doses
typically being 20, 30 or even 40 times smaller (per unit body
weight) in man than in the rat. In general, dosage is from 0.01 g
to 100 g per kg of body weight, preferably 0.01 g to 10 g/kg of
body weight, 0.01 g to 1000 mg/kg of body weight, 0.01 g to 100
mg/kg of body weight, 0.01 g to 10 mg/kg of body weight, 0.01 g to
1 mg/kg of body weight, 0.01 g to to 100 g/kg of body weight, 0.01
g to to 10 g/kg of body weight, 0.01 g to 1 g/kg of body weight,
0.01 g to 10 g/kg of body weight, 0.01 g to 1 g/kg of body weight,
0.01 g to 0.1 g/kg of body weight, and ranges based on the
boundaries of the preceding ranges of concentrations. Thus, for
example, the preceding description of dosages encompasses dosages
within the range of 100 to 10 g per kg of body weight, 10 g to 1000
mg/kg of body weight, 1000 mg to 100 mg, etc.
[0454] Doses may be given once or more daily, weekly, monthly or
yearly, or even once every 2 to 20 years. Persons of ordinary skill
in the art can easily estimate repetition rates for dosing based on
measured residence times and concentrations of the targetable
construct or complex in bodily fluids or tissues. Following
successful treatment, it may be desirable to have the patient
undergo maintenance therapy to prevent the recurrence of the
disease state, wherein the therapeutic agent is administered in
maintenance doses, ranging from 0.01 ug to 100 g per kg of body
weight, once or more daily, to once every 20 years.
[0455] The specific dose is calculated according to the approximate
body weight or surface area of the patient. Other factors in
determining the appropriate dosage can include the disease or
condition to be treated or prevented, the severity of the disease,
the route of administration, and the age, sex and medical condition
of the patient. Further refinement of the calculations necessary to
determine the appropriate dosage for treatment is routinely made by
those skilled in the art, especially in light of the dosage
information and assays disclosed herein. The dosage can also be
determined through the use of known assays for determining dosages
used in conjunction with appropriate dose-response data.
[0456] An individual patient's dosage can be adjusted as the
progress of the disease is monitored. Blood levels of the
targetable construct or complex in a patient can be measured to see
if the dosage needs to be adjusted to reach or maintain an
effective concentration. Pharmacogenomics may be used to determine
which targetable constructs and/or complexes, and dosages thereof,
are most likely to be effective for a given individual (Schmitz et
al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica
Chimica Acta 308:33-41, 2001).
[0457] XIII. References, Patents and Published Patent
Applications
[0458] XIII.A. Scientific References
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[0469] Schuhmacher, J., Klivenyi,G., Matys, R., Stadler, M.,
Regiert, T., Hauser, H., Doll, J., Maier-Borst, W., Zoller, M.
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[0470] Sharkey, R M., Karacay, H. Griffiths, G L., Behr, T M.,
Blumenthal, R D., Mattes, M J., Hansen, H J., Goldenberg, D M.
Development of a streptavidin-anti-carcinoembryonic antigen
antibody, radiolabeled biotin pretargeting method for
radioimmunotherapy of colorectal cancer. Studies in a human colon
cancer xenograft model. Bioconjugate Chem 1997; 8:595-604.
[0471] Stickney, D R., Anderson, L D., Slater, J B., Ahlem, C N.,
Kirk, G A., Schweighardt, S A and Frincke, J M. Bifunctional
antibody: a binary radiopharmaceutical delivery system for imaging
colorectal carcinoma. Cancer Res. 1991;51: 6650-6655.
[0472] XIII.B. U.S. Patents
[0473] U.S. Pat. No. 6,358,489, Fluorination of proteins and
peptides for F-18 positron emission tomography
[0474] U.S. Pat. No. 6,331,175, Method and kit for imaging and
treating organs and tissues.
[0475] U.S. Pat. No. 6,319,500, Detection and treatment of
infections with immunoconjugates.
[0476] U.S. Pat. No. 6,306,393, Immunotherapy of B-cell
malignancies using anti-CD22 antibodies.
[0477] U.S. Pat. No. 6,254,868, Glycosylated humanized B-cell
specific antibodies.
[0478] U.S. Pat. No. 6,228,362, Boron neutron capture therapy using
pre-targeting methods.
[0479] U.S. Pat. No. 6,187,287, Immunoconjugates and humanized
antibodies specific for B-cell lymphoma and leukemia cells.
[0480] U.S. Pat. No. 6,187,284, Fluorination of proteins and
peptides for F-18 positron emission tomography.
[0481] U.S. Pat. No. 6,183,744, Immunotherapy of B-cell
malignancies using anti-CD22 antibodies.
[0482] U.S. Pat. No. 6,132,718, Multi-stage cascade boosting
vaccine.
[0483] U.S. Pat. No. 6,126,916, Radiometal-binding peptide
analogues.
[0484] U.S. Pat. No. 6,120,768, Dota-biotin derivatives.
[0485] U.S. Pat. No. 6,096,289, Intraoperative, intravascular, and
endoscopic tumor and lesion detection, biopsy and therapy.
[0486] U.S. Pat. No. 6,090,381 Stimulation of an immune response
with antibodies labeled with the .alpha.-galactosyl epitope.
[0487] U.S. Pat. No. 6,083,477 Non-antigenic toxin-conjugate and
fusion protein of internalizing receptor system.
[0488] U.S. Pat. No. 6,077,499 Targeted combination immunotherapy
of cancer
[0489] U.S. Pat. No. 6,071,490 Position emission tomography using
gallium-68 chelates
[0490] U.S. Pat. No. 6,010,680 Thiolation of proteins for
radionuclide-based radioimmunodetection and radioimmunotherapy
[0491] U.S. Pat. No. 5,976,492 Radioactive phosphorus labeled
proteins for targeted radiotherapy
[0492] U.S. Pat. No. 5,965,131 Delivery of diagnostic and
therapeutic agents to a target site
[0493] U.S. Pat. No. 5,958,408 Delivery of diagnostic and
therapeutic agents to a target site
[0494] U.S. Pat. No. 5,922,302 Detection and therapy of lesions
with biotin/avidin-metal chelating protein conjugates
[0495] U.S. Pat. No. 5,874,540 CDR-grafted type III anti-CEA
humanized mouse monoclonal antibodies
[0496] U.S. Pat. No. 5,851,527 Method for antibody targeting of
therapeutic agents
[0497] U.S. Pat. No. 5,846,741 Boron neutron capture therapy using
pre-targeting methods
[0498] U.S. Pat. No. 5,843,397 Cytotoxic therapy for graft
rejection
[0499] U.S. Pat. No. 5,798,100 Multi-stage cascade boosting
vaccine
[0500] U.S. Pat. No. 5,789,554 Immunoconjugates and humanized
antibodies specific for B-cell lymphoma and leukemia cells
[0501] U.S. Pat. No. 5,776,095 Method and kit for imaging and
treating organs and tissues
[0502] U.S. Pat. No. 5,776,094 Method and kit for imaging and
treating organs and tissues
[0503] U.S. Pat. No. 5,776,093 Method for imaging and treating
organs and tissues
[0504] U.S. Pat. No. 5,772,981 Thiolation of proteins for
radionuclide-based radioimmunodetection and radioimmunotherapy
[0505] U.S. Pat. No. 5,753,206 Radiometal-binding analogues of
luteinizing hormone releasing hormone
[0506] U.S. Pat. No. 5,746,996 Thiolation of peptides for
radionuclide-based radiodetection and radiotherapy
[0507] U.S. Pat. No. 5,736,119 Detection and therapy of lesions
with biotin/avidin-metal chelating protein conjugates
[0508] U.S. Pat. No. 5,728,369 Radioactive phosphorus labeling of
proteins for targeted radiotherapy
[0509] U.S. Pat. No. 5,716,595 Intraoperative, intravascular and
endoscopic tumor and lesion detection and therapy with monovalent
antibody fragments
[0510] U.S. Pat. No. 5,705,158 Treatment of infectious and
inflammatory lesions
[0511] U.S. Pat. No. 5,698,405 Method of reducing
immunogenicity
[0512] U.S. Pat. No. 5,698,178 Polyspecific immunoconjugates and
antibody composites for targeting the multidrug resistant
phenotype
[0513] U.S. Pat. No. 5,697,902 Method for imaging and treating
organs and tissues
[0514] U.S. Pat. No. 5,686,578 Polyspecific immunoconjugates and
antibody composites for targeting the multidrug resistant
phenotype
[0515] U.S. Pat. No. 5,677,427 Chimeric antibody for detection and
therapy of infectious and inflammatory lesions
[0516] U.S. Pat. No. 5,670,132 Modified radioantibody fragments for
reduced renal uptake
[0517] U.S. Pat. No. 5,637,288 Chimeric antibody for detection and
therapy of infectious and inflammatory lesions
[0518] U.S. Pat. No. 5,635,603 Preparation and use of
immunoconjugates
[0519] U.S. Pat. No. 5,632,968 Detection of cardiovascular
lesions
[0520] U.S. Pat. No. 5,612,016 Conjugates of antibodies and
bifunctional ligands
[0521] U.S. Pat. No. 5,609,846 Radiolabelled antibody cytotoxic
therapy of infectious or autoimmune disease
[0522] U.S. Pat. No. 5,601,825 Therapeutic conjugates of toxins and
drugs
[0523] U.S. Pat. No. 5,541,297 Therapeutic conjugates of toxins and
drugs
[0524] U.S. Pat. No. 5,525,338 Detection and therapy of lesions
with biotin/avidin conjugates.
[0525] U.S. Pat. No. 5,514,363 Method for radiolabeling antibody
fragments.
[0526] U.S. Pat. No. 5,482,698 Detection and therapy of lesions
with biotin/avidin polymer conjugates.
[0527] U.S. Pat. No. 5,443,953 Preparation and use of
immunoconjugates.
[0528] U.S. Pat. No. 5,439,665 Detection and treatment of
infectious and inflammatory lesions.
[0529] U.S. Pat. No. 5,364,612 Detection of cardiovascular
lesions.
[0530] U.S. Pat. No. 5,334,708 Method for radiolabeling monovalent
antibody fragments.
[0531] U.S. Pat. No. 5,332,567 Detection and treatment of
infections with immunoconjugates.
[0532] U.S. Pat. No. 5,328,679 Methods for technetium/rhenium
labeling of proteins.
[0533] U.S. Pat. No. 5,128,119 Methods for technetium/rhenium
labeling of f(ab').sub.2 fragments.
[0534] U.S. Pat. No. 5,120,525 Radiolabeled antibody cytotoxic
therapy of cancer.
[0535] U.S. Pat. No. 5,101,827 Lymphographic and organ imaging
method and kit.
[0536] U.S. Pat. No. 5,061,641 Method for radiolabeling
proteins.
[0537] U.S. Pat. No. 4,932,412 Intraoperative and endoscopic tumor
detection and therapy.
[0538] U.S. Pat. No. 4,925,648 Detection and treatment of
infectious and inflammatory lesions.
[0539] U.S. Pat. No. 4,900,684 CEA immunoassay free of human
anti-mouse antibody false positives.
[0540] U.S. Pat. No. 4,824,659 Antibody conjugates.
[0541] U.S. Pat. No. 4,792,521 Non-enzymatic immunohistochemical
staining system and reagents.
[0542] U.S. Pat. No. 4,737,453 Sandwich immunoassay utilizing a
separation specific binding substance.
[0543] U.S. Pat. No. 4,735,210 Lymphographic and organ imaging
method and kit.
[0544] U.S. Pat. No. 4,699,880 Method of producing monoclonal
anti-idiotype antibody.
[0545] U.S. Pat. No. 4,680,338 Bifunctional linker.
[0546] U.S. Pat. No. 4,624,846 Method for enhancing target
specificity of antibody localization and clearance of non-target
diagnostic and therapeutic principles.
[0547] U.S. Pat. No. 4,595,654 Method for detecting immune
complexes in serum.
[0548] XIII.C. Published PCT Patent Applications
[0549] WO 02/12347, Immunotherapy for Chronic Myelocytic
Leukemia
[0550] WO 02/08293, Multivalent Target Binding Protein
[0551] WO 02/02150, Stable Radioiodine Conjugates And Methods For
Their Synthesis.
[0552] WO 01/97855, Targeted Combination Immunotherapy Of Cancer
And Infectious Diseases.
[0553] WO 00/74718, Immunotherapy Of Autoimmune Disorders Using
Antibodies Which Target B-Cells.
[0554] WO 00/67795, Immunotherapy Of B-Cell Malignancies Using
Anti-Cd22 Antibodies.
[0555] WO 00/33874, Boron Neutron Capture Therapy Using
Pre-Targeting Methods.
[0556] WO 00/21573, Site-Specific Labeling Of Disulfide-Containing
Targeting Vectors.
[0557] WO 00/16808, Methods And Compositions For Increasing The
Target-Specific Toxicity Of A Chemotherapy Drug.
[0558] WO 00/14537, Diagnosis Of Multidrug Resistance In Cancer And
Infectious Lesions.
[0559] WO 99/66951, Use Of Bi-Specific Antibodies For Pre-Targeting
Diagnosis And Therapy.
[0560] WO 99/59633, Therapeutics Using A Bispecific Anti-HLA Class
Ii Invariant Chain X Anti-Pathogen Antibody.
[0561] WO 99/56792, Positron Emission Tomography Using Gallium-68
Chelates.
[0562] WO 99/46389, Recombinant Onconase, And Chemical Conjugates
And Fusion Proteins Of Recombinant Onconase.
[0563] WO 99/30745, Dota-Biotin Derivatives.
[0564] WO 99/24472, Glycosylated Antibodies And Antibody Fragments
Having Reactive Ketone Groups.
[0565] WO 99/11590, Fluorination Of Proteins And Peptides For F-18
Positron Emission Tomography.
[0566] WO 99/11294, Stable Radioiodine Conjugates And Methods For
Their Synthesis.
[0567] WO 98/50435, Immunotoxins, Comprising An One Protein,
Directed Against Malignant Cells.
[0568] WO 98/42378, Immunotherapy Of B-Cell Malignancies Using
Anti-CD22 Antibodies.
[0569] WO 98/34957, Stimulation Of An Immune Response With
Antibodies Labeled With The .+-.-Galactosyl Epitope.
[0570] WO 98/16254, Non-Antigenic Toxin-Conjugate And Fusion
Protein Of Internalizing Receptor System.
[0571] WO 98/08548, Stable Radioiodine Conjugates And Methods For
Their Synthesis WO 98/04917, Boron Neutron Capture Therapy Using
Pre-Targeting Methods Immunomedics Inc.
[0572] WO 98/04293, Improved Detection And Therapy Of Lesions With
Biotin-Chelate Conjugates.
[0573] WO 98/02192, Radiometal-Binding Peptide Analogues.
[0574] WO 97/41898, Targeted Combination Immunotherapy Of
Cancer.
[0575] WO 97/40384, Mass Spectrometry And X-Ray Crystallization
Analysis Of Biological Material Via Solid Phase Support.
[0576] WO 97/34636, Humanization Of An Anti-Carcinoembryonic
Antigen Anti-Idiotype Antibody And Use As A Tumor Vaccine And For
Targeting Applications.
[0577] WO 97/34632, Glycosylated Humanized B-Cell Specific
Antibodies.
[0578] WO 97/23237, Use Of Immunoconjugates To Enhance The Efficacy
Of Multi-Stage Cascade Boosting Vaccines.
[0579] WO 97/11370, Recombinant Proteins Having Multiple Disulfide
Bonds And Thiol-Substituted Conjugates Thereof.
[0580] The contents of the articles, patents, and patent
applications, and all other documents and electronically available
information mentioned or cited herein, are hereby incorporated by
reference in their entirety to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference. Applicants reserve the right to
physically incorporate into this application any and all materials
and information from any such articles, patents, patent
applications, or other documents.
[0581] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including," containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the inventions embodied therein herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention.
[0582] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0583] In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
EXAMPLES
[0584] Reagents
[0585] Mono-DPTA Peptides
[0586] IMP 233 Ac-Phe-Gln-Tyr-Lys(DTPA)-NH.sub.2 Described
herein
[0587] IMP 240 Ac-Lys(DPTA)-Cys-NH.sub.2 Described herein
[0588] Di-DPTA Peptides
[0589] IMP 156 Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH.sub.2 See Boerman
et al., Pretargeting of renal cell carcinoma: improved tumor
targeting with a bivalent chelate, Cancer Res. 59:4400-5, 1999.
[0590] IMP 192 Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(TSCG-Cys)-NH.sub.2
See Karacay et al., Experimental pretargeting studies of cancer
with a humanized anti-CEA x murine anti-[In-DTPA] bispecific
antibody construct and a (99m)Tc-/(188)Re-labeled peptide,
Bioconjug Chem. 11:842-54, 2000.
[0591] IMP 222 Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH.sub.2 Described
herein
[0592] IMP 240 Ac-Lys(DTPA)-Cys-NH.sub.2 Described herein
[0593] Tetra-DPTA Peptides
[0594] IMP 246 [Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH.sub.2].sub.2 (aka
[IMP 222].sub.2) Described herein
[0595] Bi-Specific Antibodies
[0596] hMN-14IgG-(734scFV).sub.2
[0597] See published PCT Application WO 99/66951 by Hansen et al.,
entitled "Use of Bi-specific Antibodies for Pre-targeting Diagnosis
and Therapy."
[0598] hMN-14IgG.sup.(1253A)-(734scFv).sub.2
[0599] This mutant derivative of hMN-14IgG-(734scFv).sub.2, which
has a substitution of its 253rd amino acid residue from isoleucine
to alanine, is described in U.S. Provisional Application Ser. No.
60/361,037 (Atty Docket No. 018733-1037), which was filed Mar. 1,
2002, and is entitled "Bispecific Antibody Point Mutations for
Enhancing Rate of Clearance."
Example 1
Synthesis of Mono-DTPA peptide IMP 233
[0600] IMP 233 Ac-Phe-Gln-Tyr-Lys(DTPA)-NH.sub.2
[0601] The IMP 233 peptide was synthesized by solid phase peptide
synthesis using the Fmoc strategy and Rink Amide Resin (0.25 g, 0.8
mmol/g substitution). The protected amino acids Fmoc-Lys(Aloc)-OH,
Fmoc-Tyr(But)-OH, Fmoc-Gln(Trt)OH, Fmoc-Phe-OH and Acetic anhydride
were added in that order to the resin.
[0602] The Aloc side chain protecting group was removed and the
DTPA tetra-t-butyl ester was added. The peptide was cleaved and
purified by HPLC to afford 0.009 g of the purified peptide (ESMS
MH.sup.+1002).
Example 2
Synthesis of Di-DTPA Peptide IMP 156
[0603] IMP 156 Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH.sub.2
[0604] The IMP 156 peptide was synthesized by solid phase peptide
synthesis using the Fmoc method and Rink Amide Resin (or Sieber
amide resin). The protected amino acids; Fmoc-Lys(Aloc)-OH,
Fmoc-Tyr(But)-OH, Fmoc-Lys(Aloc)OH, Fmoc-Phe-OH and Acetic
anhydride were added in that order to the resin.
[0605] The Aloc side chain protecting groups were removed and the
DTPA tetra-t-butyl ester was added. The peptide was cleaved and
purified by HPLC (ESMS MH.sup.+1377).
Example 3
Synthesis of Di-DTPA Peptide IMP 222
[0606] IMP 222 Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH.sub.2
[0607] The IMP 222 peptide was synthesized by solid phase peptide
synthesis using the Fmoc and Rink Amide Resin (0.25 g, 0.8 mmol/g
substitution). The protected amino acids; Fmoc-Lys(Aloc)-OH,
Fmoc-Tyr(But)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Cys(Trt)-OH and Acetic
anhydride were added in that order to the resin. The Aloc side
chain protecting groups were removed and the DTPA tetra-t-butyl
ester was added. The peptide was cleaved and purified by HPLC to
afford 0.125 g of the purified peptide (ESMS MH+1333).
Example 4
Synthesis of Tetra-DTPA Peptide IMP 246
[0608] IMP 246 [Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH.sub.2].sub.2
[0609] The peptide, 0.0531 G (IMP 222, 3.98.times.10.sup.-5 MOL,
AC-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH.sub.2) was dissolved in a
solution which contained 1.0 mL DMSO, 0.2 mL
Diisopropylethylaminie, and 0.3 mL water. The solution was
incubated at room temperature for four days and then purified by
reverse phase HPLC to afford 0.0336 g of the disulfide (ESMS
MH.sup.+2663).
Example 5
IMP 246 Kits
[0610] The peptide (0.0022 g) was dissolved in 100 mL of a solution
that contained 0.418 g citric acid and 10.06 g of HPCD buffered at
pH 4.3. The solution was sterile filtered through a 0.22 .mu.M
Millex GV filter in 1 mL aliquots into vials which were immediately
frozen and lyophilized.
Example 6
In-111 Labeling of IMP 246
[0611] The In-111 (0.4 mCi) was diluted with 0.5 mL water and added
to a lyophilized IMP 246 kit. The solution was incubated at room
temperature for 10 min. A 1.5 mL aliquot of a solution containing
2.56.times.10.sup.-5 M Indium in 0.5 M NaOAc Buffer pH 7.17 was
then added to the kit.
Example 7
Evaluation of Affinity of di-DTPA Peptide IMP 192 Complexes
Comprising hMN-141gG.sup.(1253A)-(734scFv).sub.2 by HPLC
[0612] The binding of In-DTPA peptides to the anti-In-DTPA antibody
hMN-141 g.sup.(1253A)-(734scFV).sub.2 was examined by size
exclusion HPLC. The bsMAb was radiodinated using chloramine T
(Greenwood and Hunter). Binding of the radioiodinated bsMAbs to
CEA, W12 (rat anti-MN-14 idiotypic antibody) and radiolabeled
peptidyl DTPA chelate was examined on analytical size exclusion
HPLC. Approximately 90% of the radioidinated bsMAb bound to CEA
upon treatment with 10-20.times.molar excess of CEA. The bsMAb
complexed with radiolabeled indium-DTPA chelates (IMP-156 or
IMP-192).
[0613] An IMP 192 kit was labeled with Tc-99m 20.9 mCi. Aliquots
from the kit were diluted and mixed with
hMN-14IgG.sup.(1253A)-(734scFV).sub.2 in the following molar ratios
(peptide/Ab) 1:5, 1:1, and 20:1. The peptide/antibody mixtures, the
peptide alone and the antibody alone were examined on a Bio-Sil SEC
250 300 mm.times.7.8 mm HPC column eluted at 1 mL/min with 0.2 M
phosphate buffer pH 6.8.
[0614] The HPLC traces (FIGS. 3-7) show that essentially only one
peptide/antibody complex is formed. A known standard of
hMN-14IgG.sup.(1253A)-(734scFV).sub.2 eluted from the column at
about 9.41 minutes (FIG. 3). A known standard of Tc-99m IMP 192
eluted from the column at about 14.85 minutes (FIG. 4). When a 1:1
mixture of hMN-14IgG.sup.(1253A)-(734scFV).sub.2 to Tc-99m IMP 192
was applied to the column, only one peak was observed at about 9.56
minutes (FIG. 5). In contrast, when a 1:5 mixture of
hMN-14IgG.sup.(1253A)-(734scFV).sub.2 to Tc-99m IMP 192 was applied
to the column, two major peaks were observed, one at about 9.56
minutes [hMN-14IgG.sup.(1253A)-(734scFV).sub.2] and the other at
about 14.80 minutes (Tc-99m IMP 192) (FIG. 6). When a 20:1 mixture
of hMN-14 IgG.sup.(1253A)-(734scFv).sub.2 to Tc-99m IMP 192 was
applied to the column, only one peak was observed at 9.56 minutes
(FIG. 7).
Example 8
Stoichiometry of Targetable Complexes
[0615] This Example describes experiments designed to determine the
stoichiometry of different species of targetable complexes that
arise when the targetable divalent construct IMP 246 is mixed with
bsAbs hMN-14IgG.sup.(1253A)-(734scFV).sub.2, and
hMN-14IgG-(734scFV).sub.2.
[0616] The peptide IMP 246 was dissolved in 100 mL of a solution
which contained 0.418 g citric acid and 10.06 g of HPCD buffered at
pH 4.3. The solution was filter sterilized through a 0.22 .mu.m
Millex GV filters in 1 mL aliquots into vials that were immediately
frozen and lyophilized.
[0617] The IMP 246 was labeled with In-111 as follows. The In-111
was diluted with 0.5 mL water and added to a lyophilized IMP 246
kit. The solution was allowed to incubate at room temperature for
10 min. A 1.5 mL aliquot of a solution containing 2.56.times.10-5
Indium in 0.5 M NaOAc buffer pH 7.17 was then added to the kit.
[0618] The In-111-labelled IMP 246 was mixed with bsAb in a 1:10
(IMP 246:bsAb) mole ratio and then examined by size exclusion HPLC.
The results are shown in FIG. 8. When mixed with the mutant bsAb,
i.e., hMN-14IgG.sup.(1253A)-(734scFV).sub.2, about 90% of the
In-111 label was found at a clean sharp peak at 8.5 min (FIG. 8A).
When mixed with hMN-14IgG-(734scFv).sub.2, the peak was somewhat
broader, comprised about 83% of the In-111 label and was found at
7.5 min (FIG. 8B). In contrast, the antibody:peptide complexes
formed when IMP 192 was used were found at 9.5 min (FIG. 7).
Example 9
Synthesis of Mono-DTPA Peptide IMP 240 IMP 240
Ac-Lys(DTPA)-Cys-NH.sub.2
[0619] The Tetra t-butyl ester of DTPA (J Med Chem 1996 Aug.
30;39(18):3451-60, Reassessment of diethylenetriaminepentaacetic
acid (DTPA) as a chelating agent for indium-111 labeling of
polypeptides using a newly synthesized monoreactive DTPA
derivative. Arano Y, Uezono T, Akizawa H, Ono M, Wakisaka K,
Nakayama M, Sakahara H, Konishi J, Yokoyama A.) 1.424 g was
dissolved in 5.5 mL dioxane. N-Hydroxysuccinimide, 0.304 g was
added followed by 0.4 mL of diisopropylcarbodiimide (DIC) and mixed
for 1 hr at room temperature. The remaining reagents 1.049 g
Na.sub.2CO.sub.3 and 0.862 g Ac-Lys-OH were mixed then added 5 mL
water was added. The lysine/carbonate solution was then mixed with
the activated DTPA reagent. The reaction was stirred at room
temperature overnight and then quenched with 20 mL 1 M citric acid.
The citric acid solution was extracted with 2.times.50 mL portions
of chloroform, dried over Na.sub.2SO.sub.4 and concentrated under
reduced pressure to obtain 2.031 g of crude product. The crude
product was dissolved in 6 mL dioxane and mixed with 0.264 g
N-Hydroxysuccinimide and 0.38 mL DIC. The reaction was mixed at
room temperature for 2.5 hr then 0.414 g H-Cys(Trt)-NH.sub.2 was
added along with 0.3 mL diisopropylethylamine. The reaction was
stirred at room temperature overnight and then filtered. The solids
were washed with dioxane and the filtrates were combined. The crude
product was concentrated under reduced pressure. The crude product
was treated with a cleavage solution of 25 mL Trifluoro acetic
acid, 1 mL triisopropylsilane and 1 mL anisole. The cleavage
reaction mixture was poured into 2.times.40 mL ether after 3.5 hr.
The peptide was collected by centrifugation. The precipitated
peptide was washed with 3.times.30 mL ether. The precipitate was
dried then purified by reverse phase HPLC to obtain 0.2152 g of the
desired product MNa+688.
Example 10
Binding Studies Using Surface Plasmon Resonance
[0620] The binding of In-DTPA peptides to the anti-In-DTPA antibody
hMN-14IgG.sup.(1253A)-(734scFv).sub.2 was examined by affinity
blocking studies using the IMP 240 peptide,
Ac-Lys(DPTA)-Cys-NH.sub.2, which comprises a single DPTA group and
a single Cys residue that can be used to form disulfide bridges
with a second molecule having a free sulfhydryl group.
[0621] Preparation of IMP 240-coated BIACore Chip
[0622] The binding studies were performed on a chip coated with IMP
240 using the disulfide connection as recommended by BIAcore. The
chip surface was regenerated after each assay with 100 .mu.L 0.025
M In-DTPA in HBS-In-citrate buffer as described. The binding
studies were done with picomoles of antibody therefore binding was
very sensitive to the slightest trace of In-DTPA left after the
displacement wash.
[0623] IMP 156 Binding to hMN-14IgG.sup.(1253A)-(734scFV).sub.2
[0624] The affinity studies show that when IMP 156 was mixed with
hMN-14IgG.sup.(1253A)-(734scFv).sub.2 in a 1:1 ratio the antibody
binding to the IMP 240 chip was blocked. This indicates that both
734 binding sites were filled with the two DTPA's on a single
peptide.
[0625] IMP 233 Binding to hMN-14
IgG.sub.(1253A)-(734scFV).sub.2
[0626] The affinity studies show the binding of the antibody was
not completely blocked even when four equivalents of the mono-DTPA
peptide were premixed with the antibody. Although not wishing to be
bound by any particular theory, this is probably due to the
dissociation of a portion of the mono-DTPA peptide from the
antibody during the test.
[0627] Binding Studies of IMP 156 Binding to c734 IgG
[0628] Studies were performed with c734-IgG to compare the In-DTPA
peptide binding behavior of this IgG to the
hMN-14IgG.sup.(1253A)-(734scFV).sub.2- . It was necessary to add
two or more equivalents of IMP 156 to block the binding of c734 to
the chip whereas the hMN-14IgG.sup.(1253A)-(734scFV).s- ub.2 was
blocked by one equivalent of IMP 156.
[0629] This indicates that both In/DTPA's of IMP 156 were
simultaneously bound to both scFv's of
hMN-14IgG.sup.(1253A)-(734scFV).sub.2 whereas only one In/DTPA of
IMP 156 was bound to the binding arm of c734-IgG and another
In/DTPA on another molecule of IMP 156 was needed to block the
other binding arm of c734-IgG.
[0630] Binding Studies of IMP 233 Binding to c734 IgG
[0631] The affinity studies showed that even 4:1 IMP 233/c734 IgG
did not completely block the binding of the antibody to the IMP 240
chip.
[0632] This experiment demonstrated that it was difficult to
completely block the binding of the binding of c734 IgG to the IMP
240 chip with a peptide bearing a singe In-DTPA (IMP 233). This
meant that either there was a certain amount of free c734 IgG (or
at least one binding arm available for binding) or the In-DTPA on
the chip was able to displace the In-DTPA of IMP 233. The fact that
IMP 156 completely blocked the binding of
hMN-14IgG.sup.(1253A)-(734scFV).sub.2 (when mixed in a 1:1 ratio
Peptide: Antibody) to the IMP 240 chip demonstrated that both of
the DTPA binding sites were filled and that the affinity of the
hMN-14IgG.sup.(1253A)-(734scFV).sub.2 for in2-IMP 156 was far
higher than the affinity of a single In-DTPA for a c734 IgG binding
arm.
Example 11
Attachment of IMP 222 to a BIAcore Chip
[0633] The CM-5 Chip has a carbohydrate attached to a gold surface
which has been derivitized with carboxylic acids. The carboxylic
acids on the surface were activated with NHS(N-hydroxysuccinimide)
and a water soluble carbodiimide, EDC (N-ethyl-N'-(3-dimethyl
aminopropyl)-carbodiimide hydrochloride) essentially according to
the manufacturer's instructions (see Application Note 9, Biacore
International AB, Uppsala, Sweden, published on-line at
http://www.biacore.com/products/pdf/ANLigandImmobili- zation.
pdf).
[0634] A solution of 2-(2-pyridinylthio)ethaneamine hydrochloride
(PDEA) in 0.1 M pH 8.5 borate buffer was then added essentially
according to the manufacturer's instructions. The IMP 222 peptide,
Ac-Cys-Lys(DTPA)-Tyr-Ly- s(DTPA)-NH.sub.2, was dissolved in 0.01 M
pH 4.3 formate buffer then added to the chip as described.
[0635] The peptide forms a disulfide linkage to the chip when added
in this manner. Finally, the remaining unreacted active ester and
PDEA are quenched by the addition of a solution containing cysteine
and NaCl in 0.1 M formate buffer pH 4.3.
Example 12
Serum Stability
[0636] Stability of bsAb in Serum
[0637] Bi-specific antibody (bsAb) was radioiodinated and tested
for stability in fresh human serum at 37.degree. C. under a
humidified 5% CO.sub.2 atmosphere. Aliquots were examined on
SE-HPLC. In order to detect radioiodine associated with serum
proteins, the aliquots were mixed with WI2 to shift the bsMAb peak
to earlier retention times. The bsMAbs showed about 3-5% loss of
binding capacity to WI2 after 48 h incubation in serum. Slight
aggregate formation (about 4-7%) was observed upon incubation of
the bsMAbs in serum for 72 hours.
[0638] Stability of Targetable Constructs in Serum
[0639] A 40 .mu.L aliquot of the labeled peptide was diluted with
400 .mu.L of fresh mouse serum and incubated at 37.degree. C. for
17.5 hours. A 10 .mu.L aliquot was removed and mixed with 5 .mu.L
of 3.2 mg/mL 14IgG.sup.(1253A)-(734scFv).sub.2 and diluted with 40
.mu.L water. A 10 .mu.L aliquot of the diluted mixture was then
examined by size exclusion HPLC.
[0640] Stability of Targetable Complexes in Serum
[0641] Peptide Plus hMN-14 IgG.sup.(1251A)-(734scFV).sub.2 Serum
Stability:
[0642] A 4 .mu.L aliquot of the labeled peptide solution was mixed
with 10 .mu.L of the 3.2 mg/mL
hMN-14IgG.sup.(1253A)-(734scFv).sub.2 and diluted in 400 .mu.L of
fresh mouse serum. The serum sample was incubated at 37.degree. C.
for 16 hr and then analyzed by size exclusion HPLC.
[0643] The IMP 246 peptide was unstable in mouse serum after 16
hours, as demonstrated by the presence of several overlapping peaks
in the HPLC tracing. Three broad peaks comprised about 20%, 41% and
37% of the area under the curve of the HPLC tracing. In contrast,
the stability of IMP 246 was greatly increased in the presence of
the bi-specific antibody, as .gtoreq.90% of the area under the
curve of the HPLC tracing was found in a distinct peak at the
predicted position.
Example 13
Evaluation of Complexes Comprising hA20-IgG-(734scFv).sub.2 in
vitro
[0644] hA20-IgG-(734scFv).sub.2 is made using methods described in
PCT Application Publication No. WO 99/66951 by Hansen et al., with
the only change being that cDNA coding for the variable chains of
hA20 is used in place of cDNA coding for hMN-14 variable chains.
The cDNA coding for the constant regions of both hMN-14 and hA20
are identical, as is the cDNA coding for the linker and scFv of
monoclonal antibody 734.
[0645] Raji cells in culture are incubated with
hA20IgG-(734scFv).sub.2. To one set of wells is added IMP-246 to
cross-link molecules of hA20IgG-(734scFv).sub.2 bound to CD20 on
the surface of the Raji cells. A second set of wells to which
IMP-246 is not added serve as controls, and both sets of wells
incubated at 37 degrees C. After 3 days, the cells to which IMP-246
was added are determined to have undergone extensive apoptosis.
Minimal apoptosis was observed in the control wells to which
IMP-246 was not added.
[0646] An average of 5 molecules IMP-222 are conjugated to human
serum albumin (hAlb-222). The experiment described in the paragraph
above is repeated, but hAb-222 is used to cross-link molecules of
hA20IgG-(734scFv).sub.2 bound to CD20 on the surface of the Raji
cells, in place of IMP-246. Extensive apoptosis of cells is
observed in wells to which hAlb-222 was added.
[0647] Illustrative Embodiments
[0648] Additional embodiments are within the scope of the
invention. For example, the invention is further illustrated by the
following numbered embodiments:
[0649] 1. A targetable construct comprising (i) a molecular
scaffold and (ii) two pairs of a carrier epitope, wherein said
targetable construct, when combined with a bi-specific antibody
comprising (i) two copies of a first arm comprising a binding site
for said carrier epitope, and (ii) two copies of a second arm
comprising a binding site for a target epitope, forms a targetable
complex, wherein one or more of the following applies:
[0650] (a) said targetable complexes have a Kd for said target
epitope from about 0.1 nM to about 100 nM,
[0651] (b) mixing said targetable construct and said bi-specific
antibody at relative concentrations ranging from about 10.sup.-3 to
about 10.sup.3 results in a mixture in which greater than about 75%
of the complexes therein have a defined stoichiometry of two
molecules of said bi-specific antibody, and one molecule of said
targetable construct, and
[0652] (c) a pair of carrier epitopes is simultaneously bound by
said two copies of a first arm comprising a binding site for said
carrier epitope, wherein said two copies of a first arm comprising
a binding site for said carrier epitope are part of said
bi-specific antibody.
[0653] 2. The targetable construct of embodiment 1, wherein mixing
said targetable construct and said bi-specific antibody at relative
concentrations ranging from about 10.sup.-3 to about 10.sup.3
results in a mixture in which greater than about 85% of the
multimeric complexes have a defined stoichiometry of two molecules
of said bi-specific antibody, and one molecule of said targetable
construct.
[0654] 3. The targetable construct of embodiment 1, wherein mixing
said targetable construct and said bi-specific antibody at relative
concentrations ranging from about 10.sup.-3 to about 103 results in
a mixture in which greater than about 95% of the multimeric
complexes have a defined stoichiometry of two molecules of said
bi-specific antibody, and one molecule of said targetable
construct.
[0655] 4. The targetable construct of embodiment 1, wherein mixing
said targetable construct and said bi-specific antibody at relative
concentrations ranging from about 10.sup.-3 to about 10.sup.3
results in a mixture in which greater than about 99% of the
multimeric complexes have a defined stoichiometry of two molecules
of said bi-specific antibody and one molecule of said targetable
construct.
[0656] 5. The targetable construct of embodiment 1, wherein said Kd
for said target epitope is from about 0.1 nM to 10 nM.
[0657] 6. The targetable construct of embodiment 5, wherein said Kd
for said target epitope is from about 0.5 nM to about 5 nM.
[0658] 7. The targetable construct of embodiment 6, wherein said Kd
for said target epitope is about 1 nM.
[0659] 8. The targetable construct of embodiment 1, wherein said
molecular scaffold is a peptide or peptide derivative.
[0660] 9. The targetable construct of embodiment 1, wherein said
targetable construct is IMP 246.
[0661] 10. The targetable construct of embodiment 1, wherein said
targetable construct comprises two constructs that are conjugated
to each other, wherein each construct comprises a molecular
scaffold and a pair of carrier epitopes.
[0662] 11. The targetable construct of embodiment 10, wherein each
of said constructs is independently selected from the group
consisting of IMP 156, IMP 192 and IMP 222.
[0663] 12. The targetable construct of embodiment 1, wherein said
carrier epitope is a hapten.
[0664] 13. The targetable construct of embodiment 1, wherein said
carrier epitope is a chelator, or a complex between a chelator and
a metal ion.
[0665] 14. The targetable construct of embodiment 13, wherein said
chelator is selected from the group consisting of DTPA, DOTA,
benzyl DTPA, NOTA, and TETA.
[0666] 15. The targetable construct of embodiment 1, wherein said
bi-specific antibody is [IgG]-[scFv]2; wherein IgG is a human,
chimeric or CDR-grafted antibody; further wherein scFv is a human,
chimeric or CDR-grafted single chain antibody specific for a
hapten; and further wherein said scFv is extended from the carboxyl
terminal amino acid of the heavy chains of said IgG by a linker
peptide.
[0667] 16. The targetable construct of embodiment 15, wherein said
bi-specific antibody is selected from the group consisting of
[hMN14-IgG1]-[734scFv].sub.2 and
[hMN14-IgG1.sup.(1253A)]-[734scFV].sub.2- .
[0668] 17. The targetable construct of embodiment 15, wherein said
bi-specific antibody is selected from the group consisting of
[hMN14-IgG1]-[679scFv].sub.2 and
[hMN14-IgG1.sup.(1253A)]-[679scFv].sub.2- .
[0669] 18. The targetable construct of embodiment 15, wherein said
bi-specific antibody is selected from the group consisting of
[hA20-IgG1]-[734scFv].sub.2 and
[hA20-IgG1.sup.(1253A)]-[734scFv].sub.2.
[0670] 19. The targetable construct of embodiment 15, wherein said
bi-specific antibody is selected from the group consisting of
[hA20-IgG1]-[679scFv].sub.2 and
[hA20-IgG1.sup.(1253A)]-[679scFv].sub.2.
[0671] 20. The targetable construct of embodiment 15, wherein said
bi-specific antibody is selected from the group consisting of
[hLL2-IgG1]-[734scFv].sub.2 and
[hLL2-IgG1.sup.(1253A)]-[734scFv].sub.2.
[0672] 21. The targetable construct of embodiment 15, wherein said
bi-specific antibody is selected from the group consisting of
[hLL2-IgG1]-[679scFv].sub.2 and
[hLL2-IgG1.sup.(1253A)]-[670scFv].sub.2.
[0673] 22. The targetable construct of embodiment 1, wherein said
targetable construct further comprises a bioactive moiety.
[0674] 23. The targetable construct of embodiment 22, wherein said
bioactive moiety is selected from the group consisting of a drug, a
prodrug, an enzyme, a hormone, an immunomodulator, an
oligonucleotide; a radionuclide, an image enhancing agent and a
toxin.
[0675] 24. The targetable construct of embodiment 23, wherein said
enzyme is selected from the group consisting of malate
dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol dehydrogenase, .alpha.-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
.alpha.-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase.
[0676] 25. The targetable construct of embodiment 23, wherein said
immunomodulator is selected from the group consisting of a
cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic
growth factor, a colony stimulating factor (CSF), an interferon
(IFN), erythropoietin, thrombopoietin and a combination
thereof.
[0677] 26. The targetable construct of embodiment 23, wherein said
immunomodulator consists essentially of IL-1, IL-2, IL-3, IL-6,
IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF, interferon-.gamma.,
-.alpha., -.beta. or -.gamma., TNF-.alpha., and "S1 factor.
[0678] 27. The targetable construct of embodiment 23, wherein said
oligonucleotide is an anti-sense oligonucleotide.
[0679] 28. The targetable construct of embodiment 27, wherein said
anti-sense oligonucleotide is bcl-2 or p53.
[0680] 29. A solid support to which the targetable construct of
embodiment 1 is bound.
[0681] 30. A biosensor comprising the targetable construct of
embodiment 1.
[0682] 31. A targetable construct comprising (i) a molecular
scaffold and (ii) two pairs of carrier epitopes, wherein the first
of said two pairs of carrier epitopes is specifically bound by a
first bi-specific antibody, and the second of said two pairs of
carrier epitopes is specifically bound by a second bi-specific
antibody, wherein said targetable construct forms a targetable
complex when combined with
[0683] (i) a first bi-specific antibody, said first bi-specific
antibody copmrising (a) two copies of a first arm comprising a
binding site for said carrier epitope, and (b) two copies of a
second arm comprising a binding site for a first target epitope,
and
[0684] (ii) a second bi-specific antibody, said second bi-specific
antibody comprising (a) two copies of a first arm comprising a
binding site for said carrier epitope, and (b) two copies of a
second arm comprising a binding site for a second target
epitope;
[0685] wherein said first bi-specific antibody and said second
bi-specific antibody can be the same or different, said pairs of
carrier epitopes can be the same or different, and said target
epitopes can be the same or different, and wherein one or more of
the following applies:
[0686] (a) said targetable complex has a Kd of from about 0.1 nM to
about 100 nM for either or both of said target epitopes,
[0687] (b) mixing said targetable construct and said bi-specific
antibodies at relative concentrations ranging from about 10.sup.-3
to about 10.sup.3 results in a mixture in which greater than about
75% of the multimeric complexes have a defined stoichiometry of one
molecule of said first bi-specific antibody, one molecule of said
second bi-specific antibody, and one molecule of said targetable
construct; and
[0688] (c) a pair of carrier epitopes is simultaneously bound by
said two copies of a first arm comprising a binding site for said
carrier epitope, wherein said two copies of a first arm comprising
a binding site for said carrier epitope are part of said
bi-specific antibody.
[0689] 32. The targetable construct of embodiment 31, wherein said
pairs of carrier epitopes are different, but said target epitopes
are the same.
[0690] 33. The targetable construct of embodiment 31, wherein said
first antibody and said second antibody are different, but said
target epitopes are the same.
[0691] 34. The targetable constrict of embodiment 31, wherein said
carrier epitopes are the same, and said first arm comprising a
binding site for said carrier epitope of said first bi-specific
antibody and said first arm comprising a binding site for said
carrier epitope of said second bi-specific antibody are the same,
but said first target epitope and said second target epitope are
not the same.
[0692] 35. The targetable construct of embodiment 31, wherein
mixing said targetable construct and said bi-specific antibodies at
relative concentrations ranging from about 10.sup.-3 to about
10.sup.3 results in a mixture in which greater than about 85% of
the multimeric complexes have a defined stoichiometry of two
molecules of said bi-specific antibody or antibody derivative and
one molecule of said targetable construct.
[0693] 36. The targetable construct of embodiment 31, wherein
mixing said targetable construct and said bi-specific antibody at
relative concentrations ranging from about 10.sup.-3 to about
10.sup.3 results in a mixture in which greater than about 95% of
the multimeric complexes have a defined stoichiometry of two
molecules of said bi-specific antibody or antibody derivative and
one molecule of said targetable construct.
[0694] 37. The targetable construct of embodiment 31, wherein
mixing said targetable construct and said bi-specific antibody at
relative concentrations ranging from about 10.sup.-3 to about
10.sup.3 results in a mixture in which greater than about 99% of
the multimeric complexes have a defined stoichiometry of two
molecules of said bi-specific antibody or antibody derivative and
one molecule of said targetable construct.
[0695] 38. The targetable construct of embodiment 31, wherein said
Kd for said target epitope is from about 0.1 nM to 10 nM.
[0696] 39. The targetable construct of embodiment 38, wherein said
Kd for said target epitope is from about 0.5 nM to about 5 nM.
[0697] 40. The targetable construct of embodiment 39, wherein said
Kd for said target epitope is about 1 nM.
[0698] 41. The targetable construct of embodiment 31, wherein said
targetable construct comprises two constructs that are conjugated
to each other, wherein each construct comprises a molecular
scaffold and a pair of carrier epitopes.
[0699] 42. The targetable construct of embodiment 41, wherein each
of said constructs is independently selected from the group
consisting of IMP 156, IMP 192 and IMP 222.
[0700] 43. The targetable construct of embodiment 31, wherein said
molecular scaffold is a peptide or peptide derivative.
[0701] 44. The targetable construct of embodiment 31, wherein said
carrier epitope is a hapten.
[0702] 45. The targetable construct of embodiment 31, wherein said
carrier epitope is a chelator, or a complex between a chelator and
a metal ion.
[0703] 46. The targetable construct of embodiment 45, wherein said
chelator is selected from the group consisting of DTPA, DOTA,
benzyl DTPA, NOTA, and TETA.
[0704] 47. The targetable construct of embodiment 31, wherein said
targetable construct further comprises a bioactive moiety.
[0705] 48. The targetable complex of embodiment 47, wherein said
bioactive moiety is selected from the group consisting of a drug, a
prodrug, an enzyme, a hormone, an immunomodulator, an
oligonucleotide, a radionuclide, an image enhancing agent and a
toxin.
[0706] 49. The targetable construct of embodiment 48, wherein said
enzyme is selected from the group consisting of malate
dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol dehydrogenase, .alpha.-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
.beta.-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase.
[0707] 50. The targetable construct of embodiment 48, wherein said
immunomodulator is selected from the group consisting of a
cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic
growth factor, a colony stimulating factor (CSF), an interferon
(IFN), erythropoietin, thrombopoietin and a combination
thereof.
[0708] 51. The targetable construct of embodiment 48, wherein said
immunomodulator is selected from the group consisting of IL-1,
IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF,
interferon-.gamma., -.alpha., -.beta. or -.gamma., TNF-.alpha., and
"S1 factor.
[0709] 52. The targetable construct of embodiment 48, wherein said
oligonucleotide is an anti-sense oligonucleotide.
[0710] 53. The targetable construct of embodiment 52, wherein said
anti-sense oligonucleotide is bcl-2 or p53.
[0711] 54. The targetable construct of embodiment 34, wherein said
first arm comprising a binding site for said carrier epitope of
said first bi-specific antibody, and said first arm comprising a
binding site for said carrier epitope of said second bi-specific
antibody, are both [734scFv].sub.2.
[0712] 55. A solid support to which the targetable construct of
embodiment 31 is bound.
[0713] 56. A biosensor comprising the targetable construct of
embodiment 31.
[0714] 57. A targetable construct comprising a molecular scaffold
and X pairs of carrier epitopes, wherein each of said pairs of
carrier epitopes is specifically bound by one of Xbi-specific
antibodies, each bi-specific antibody comprising (a) two copies of
a first arm comprising a binding site for a carrier epitope, and
(b) two copies of a second arm comprising a binding site for one of
Y target epitopes, and wherein
[0715] (i) X is a whole integer .gtoreq.3,
[0716] (ii) Y is a whole integer .gtoreq.1,
[0717] (iii) said Xbi-specific antibodies can be the same or a
mixture of different bi-specific antibodies,
[0718] (iv) said Xpairs of carrier epitopes can be the same or a
mixture of different carrier epitopes, and
[0719] (v) when Y.gtoreq.2, said Y target epitopes can be the same
or a mixture of different target epitopes,
[0720] and wherein one or more of the following applies:
[0721] (a) said targetable complex has a Kd of from about 0.1 nM to
about 100 nM for at least one of said target epitopes,
[0722] (b) a pair of carrier epitopes is simultaneously bound by
said two copies of a first arm comprising a binding site for said
carrier epitope, wherein said two copies of a first arm comprising
a binding site for said carrier epitope are part of said
bi-specific antibody.
[0723] 58. The targetable construct of embodiment 57, wherein said
Kd for at least one of said target epitopes is from about 0.1 nM to
10 nM.
[0724] 59. The targetable construct of embodiment 58, wherein said
Kd for at least one of said target epitopes is from about 0.5 nM to
about 5 nM.
[0725] 60. The targetable construct of embodiment 59, wherein said
Kd for for at least one of target epitope is about 1 nM.
[0726] 61. The targetable construct of embodiment 57, wherein said
molecular scaffold is a peptide or peptide derivative.
[0727] 62. The targetable construct of embodiment 57, wherein at
least one of said carrier epitopes is a hapten.
[0728] 63. The targetable construct of embodiment 57, wherein said
targetable construct further comprises a bioactive moiety.
[0729] 64. The targetable construct of embodiment 63, wherein said
bioactive moiety is selected from the group consisting of a drug, a
prodrug, an enzyme, a radionuclide, an image enhancing agent and a
toxin.
[0730] 65. The targetable construct of embodiment 64, wherein said
enzyme is selected from the group consisting of malate
dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol dehydrogenase, .alpha.-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
.beta.-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase.
[0731] 66. The targetable construct of embodiment 64, wherein said
immunomodulator is selected from the group consisting of a
cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic
growth factor, a colony stimulating factor (CSF), an interferon
(IFN), erythropoietin, thrombopoietin and a combination
thereof.
[0732] 67. The targetable construct of embodiment 64, wherein said
immunomodulator is selected from the group consisting of IL-1,
IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF,
interferon-.gamma., -.alpha., -.beta. or -.gamma., TNF-.alpha., and
"S1 factor.
[0733] 68. The targetable construct of embodiment 64, wherein said
oligonucleotide is an anti-sense oligonucleotide.
[0734] 69. The targetable construct of embodiment 68, wherein said
anti-sense oligonucleotide is bcl-2 or p53.
[0735] 70. A solid support to which the targetable construct of
embodiment 57 is bound.
[0736] 71. A biosensor comprising the targetable construct of
embodiment 57.
[0737] 72. A targetable complex comprising four arms capable of
binding a target epitope, said tetravalent targetable complex
comprising:
[0738] (a) a targetable construct comprising (i) a molecular
scaffold and (ii) two pairs of a carrier epitope; and
[0739] (b) two molecules of a bi-specific antibody, each antibody
comprising (i) two arms, each arm comprising a binding site for
said carrier epitope, and (ii) two arms, each comprising a binding
site for said target epitope,
[0740] wherein one or more of the following applies:
[0741] (I) said targetable complexes have a Kd for said target
epitope from about 0.1 nM to about 100 nM,
[0742] (II) mixing said targetable construct and said bi-specific
antibody at relative concentrations ranging from about 10.sup.-3 to
about 10.sup.3 results in a mixture in which greater than about 75%
of the complexes therein have a defined stoichiometry of two
molecules of said bi-specific antibody, and one molecule of said
targetable construct, and
[0743] (III) a pair of carrier epitopes is bound by said
bi-specific antibody in a 1:1 ratio.
[0744] 73. The targetable complex of embodiment 72, wherein mixing
said targetable construct and said bi-specific antibody at relative
concentrations ranging from about 10.sup.-3 to about 10.sup.3
results in a mixture in which greater than about 85% of the
multimeric complexes have a defined stoichiometry of two molecules
of said bi-specific antibody, and one molecule of said targetable
construct.
[0745] 74. The targetable complex of embodiment 72, wherein mixing
said targetable construct and said bi-specific antibody at relative
concentrations ranging from about 10.sup.-3 to about 10.sup.3
results in a mixture in which greater than about 95% of the
multimeric complexes have a defined stoichiometry of two molecules
of said bi-specific antibody, and one molecule of said targetable
construct.
[0746] 75. The targetable complex of embodiment 72, wherein mixing
said targetable construct and said bi-specific antibody at relative
concentrations ranging from about 10.sup.-3 to about 10.sup.3
results in a mixture in which greater than about 99% of the
multimeric complexes have a defined stoichiometry of two molecules
of said bi-specific antibody, and one molecule of said targetable
construct.
[0747] 76. The targetable complex of embodiment 72, wherein said Kd
for said target epitope is from about 0.1 nM to 10 nM.
[0748] 77. The targetable complex of embodiment 76, wherein said Kd
for said target epitope is from about 0.5 nM to about 5 nM.
[0749] 78. The targetable complex of embodiment 77, wherein said Kd
for said target epitope is about 1 nM.
[0750] 79. The targetable complex of embodiment 72, wherein said
molecular scaffold is a peptide or peptide derivative.
[0751] 80. The targetable complex of embodiment 72, wherein said
targetable construct is IMP 246.
[0752] 81. The targetable complex of embodiment 72, wherein said
targetable construct comprises two constructs that are conjugated
to each other, wherein each construct comprises a molecular
scaffold and a pair of carrier epitopes.
[0753] 82. The targetable complex of embodiment 81, wherein each of
said constructs is independently selected from the group consisting
of IMP 156, IMP 192 and IMP 222.
[0754] 83. The targetable complex of embodiment 72, wherein said
carrier epitope is a hapten.
[0755] 84. The targetable complex of embodiment 72, wherein said
carrier epitope is a chelator, or a complex between a chelator and
a metal ion.
[0756] 85. The targetable complex of embodiment 84, wherein said
chelator is selected from the group consisting of DTPA, DOTA,
benzyl DTPA, NOTA, and TETA.
[0757] 86. The targetable complex of embodiment 72, wherein said
bi-specific antibody is selected from the group consisting of
[hMN].sub.2-[734scFv].sub.2 and
[hMN.sup.(1253A)].sub.2-[734scFv].sub.2.
[0758] 87. The targetable complex of embodiment 72, wherein at
least one arm comprising a binding site for said carrier epitope is
734scFv.
[0759] 88. The targetable complex of embodiment 72, wherein said
targetable construct further comprises a bioactive moiety.
[0760] 89. The targetable complex of embodiment 88, wherein said
bioactive moiety is selected from the group consisting of a drug, a
prodrug, an enzyme, a radionuclide, a hormone, an immunomodulator,
an oligonucleotide, an image enhancing agent and a toxin. a
hormone, an immunomodulator, an oligonucleotide; a radionuclide, an
image enhancing agent and a toxin.
[0761] 90. The targetable construct of embodiment 89, wherein said
enzyme is selected from the group consisting of malate
dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol dehydrogenase, .alpha.-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
.beta.-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase.
[0762] 91. The targetable construct of embodiment 89, wherein said
immunomodulator is selected from the group consisting of a
cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic
growth factor, a colony stimulating factor (CSF), an interferon
(IFN), erythropoietin, thrombopoietin and a combination
thereof.
[0763] 92. The targetable construct of embodiment 89, wherein said
immunomodulator is selected from the group consisting of IL-1,
IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF,
interferon-.gamma., -.alpha., -.beta. or -.gamma., TNF-.alpha., and
"S1 factor.
[0764] 93. The targetable construct of embodiment 89, wherein said
oligonucleotide is an anti-sense oligonucleotide.
[0765] 94. The targetable construct of embodiment 93, wherein said
anti-sense oligonucleotide is bcl-2 or p53.
[0766] 95. A solid support to which the targetable complex of
embodiment 72 is bound.
[0767] 96. A biosensor comprising the targetable complex of
embodiment 72.
[0768] 97. A targetable complex, said targetable complex
comprising:
[0769] (a) a targetable construct comprising (i) a molecular
scaffold and (ii) two pairs of carrier epitopes, wherein the first
of said two pairs of carrier epitopes is specifically bound by a
first bi-specific antibody, and the second of said two pairs of
carrier epitopes is specifically bound by a second bi-specific
antibody, wherein said targetable construct forms a targetable
complex when combined with
[0770] (b) a first bi-specific antibody, said first bi-specific
antibody comprising (i) two copies of a first arm comprising a
binding site for said carrier epitope, and (ii) two copies of a
second arm comprising a binding site for a first target epitope,
and
[0771] (c) a second bi-specific antibody, said second bi-specific
antibody comprising (i) two copies of a first arm comprising a
binding site for said carrier epitope, and (ii) two copies of a
second arm comprising a binding site for a second target
epitope;
[0772] wherein said first bi-specific antibody and said second
bi-specific antibody can be the same or different, said pairs of
carrier epitopes can be the same or different, and said target
epitopes can be the same or different, and wherein one or more of
the following applies
[0773] (I) said targetable complexes have a Kd for said target
epitope from about 0.1 nM to about 100 nM,
[0774] (II) mixing said targetable construct and said bi-specific
antibody at relative concentrations ranging from about 10.sup.-3 to
about 10.sup.3 results in a mixture in which greater than about 75%
of the complexes therein have a defined stoichiometry of two
molecules of said bi-specific antibody, and one molecule of said
targetable construct, and
[0775] (III) each of said pairs of carrier epitopes is bound by one
of said bi-specific antibodies in a 1:1 ratio.
[0776] 98. The targetable complex of embodiment 97, wherein mixing
said targetable construct and said bi-specific antibody at relative
concentrations ranging from about 10.sup.-3 to about 10.sup.3
results in a mixture in which greater than about 85% of the
multimeric complexes have a defined stoichiometry of two molecules
of said bi-specific antibody, and one molecule of said targetable
construct.
[0777] 99. The targetable complex of embodiment 97, wherein mixing
said targetable construct and said bi-specific antibody at relative
concentrations ranging from about 10.sup.-3 to about 10.sup.3
results in a mixture in which greater than about 95% of the
multimeric complexes have a defined stoichiometry of two molecules
of said bi-specific antibody, and one molecule of said targetable
construct.
[0778] 100. The targetable complex of embodiment 97, wherein mixing
said targetable construct and said bi-specific antibody at relative
concentrations ranging from about 10.sup.-3 to about 10.sup.3
results in a mixture in which greater than about 99% of the
multimeric complexes have a defined stoichiometry of two molecules
of said bi-specific antibody, and one molecule of said targetable
construct.
[0779] 101. The targetable complex of embodiment 97, wherein said
Kd for said target epitope is from about 0.1 nM to 10 nM.
[0780] 102. The targetable complex of embodiment 101, wherein said
Kd for said target epitope is from about 0.5 nM to about 5 nM.
[0781] 103. The targetable complex of embodiment 102, wherein said
Kd for said target epitope is about 1 nM.
[0782] 104. The targetable complex of embodiment 97, wherein said
molecular scaffold is a peptide or peptide derivative.
[0783] 105. The targetable complex of embodiment 97, wherein said
targetable construct is IMP 246.
[0784] 106. The targetable complex of embodiment 97, wherein said
targetable construct comprises two constructs that are conjugated
to each other, wherein each construct comprises a molecular
scaffold and a pair of carrier epitopes.
[0785] 107. The targetable complex of embodiment 107, wherein each
of said constructs is independently selected from the group
consisting of IMP 156, IMP 192 and IMP 222.
[0786] 108. The targetable complex of embodiment 97, wherein said
carrier epitope is a hapten.
[0787] 109. The targetable complex of embodiment 97, wherein said
carrier epitope is a chelator, or a complex between a chelator and
a metal ion.
[0788] 110. The targetable complex of embodiment 110, wherein said
chelator is selected from the group consisting of DTPA, DOTA,
benzyl DTPA, NOTA, and TETA.
[0789] 111. The targetable complex of embodiment 97, wherein said
bi-specific antibody is selected from the group consisting of
[hMN].sub.2-[734scFv].sub.2 and
[hMN.sup.(1253A)].sub.2-[734scFv].sub.2.
[0790] 112. The targetable complex of embodiment 97, wherein at
least one arm comprising a binding site for said carrier epitope is
734scFv.
[0791] 113. The targetable complex of embodiment 97, wherein said
targetable construct further comprises a bioactive moiety.
[0792] 114. The targetable complex of embodiment 113, wherein said
bioactive moiety is selected from the group consisting of a drug, a
prodrug, an enzyme, a radionuclide, a hormone, an immunomodulator,
an oligonucleotide, an image enhancing agent and a toxin.
[0793] 115. The targetable construct of embodiment 114, wherein
said enzyme is selected from the group consisting of malate
dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol dehydrogenase, .alpha.-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
.beta.-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase.
[0794] 116. The targetable construct of embodiment 114, wherein
said immunomodulator is selected from the group consisting of a
cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic
growth factor, a colony stimulating factor (CSF), an interferon
(IFN), erythropoietin, thrombopoietin and a combination
thereof.
[0795] 117. The targetable construct of embodiment 114, wherein
said immunomodulator consists essentially of IL-1, IL-2, IL-3,
IL-6, IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF,
interferon-.gamma., -.alpha., -.beta. or -.gamma., TNF-.alpha., and
"S1 factor.
[0796] 118. The targetable construct of embodiment 114, wherein
said oligonucleotide is an anti-sense oligonucleotide.
[0797] 119. The targetable construct of embodiment 118, wherein
said anti-sense oligonucleotide is bcl-2 or p53.
[0798] 120. A solid support to which the targetable complex of
embodiment 97 is bound.
[0799] 121. A biosensor comprising the targetable complex of
embodiment 97.
[0800] 122. A targetable complex, said targetable complex
comprising:
[0801] (A) a targetable construct comprising a molecular scaffold
and X pairs of carrier epitopes, wherein each of said pairs of
carrier epitopes is specifically bound by one of X bi-specific
antibodies, each bi-specific antibody comprising (i) two copies of
a first arm comprising a binding site for a carrier epitope, and
(ii) two copies of a second arm comprising a binding site for one
of Y target epitopes, and
[0802] (B) X bi-specific antibodies;
[0803] wherein:
[0804] (1) X is a whole integer >3,
[0805] (2) Y is a whole integer >1,
[0806] (3) said X bi-specific antibodies can be the same or a
mixture of different bi-specific antibodies,
[0807] (4) said X pairs of carrier epitopes can be the same or a
mixture of different carrier epitopes, and
[0808] (5) when Y>2, said Y target epitopes can be the same or a
mixture of different target epitopes;
[0809] wherein one or more of the following applies:
[0810] (a) said targetable complex has a Kd of from about 0.1 nM to
about 100 nM for at least one of said target epitopes,
[0811] (b) a pair of carrier epitopes is simultaneously bound by
said two copies of a first arm comprising a binding site for said
carrier epitope, wherein said two copies of a first arm comprising
a binding site for said carrier epitope are part of said
bi-specific antibody.
[0812] 123. The targetable construct of embodiment 122, wherein
said Kd for at least one of said target epitopes is from about 0.1
nM to 10 nM.
[0813] 124. The targetable construct of embodiment 123, wherein
said Kd for at least one of said target epitopes is from about 0.5
nM to about 5 nM.
[0814] 125. The targetable construct of embodiment 124, wherein
said Kd for for at least one of target epitope is about 1 nM.
[0815] 126. The targetable construct of embodiment 122, wherein
said molecular scaffold is a peptide or peptide derivative.
[0816] 127. The targetable construct of embodiment 122, wherein at
least one of said carrier epitopes is a hapten.
[0817] 128. The targetable construct of embodiment 122, wherein
said targetable construct further comprises a bioactive moiety.
[0818] 129. The targetable complex of embodiment 123, wherein said
bioactive moiety is selected from the group consisting of a drug, a
prodrug, an enzyme, a hormone, an immunomodulator, an
oligonucleotide, a radionuclide, an image enhancing agent and a
toxin.
[0819] 130. The targetable construct of embodiment 129, wherein
said enzyme is selected from the group consisting of malate
dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol dehydrogenase, .alpha.-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
.beta.-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase.
[0820] 131. The targetable construct of embodiment 129, wherein
said immunomodulator is selected from the group consisting of a
cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic
growth factor, a colony stimulating factor (CSF), an interferon
(IFN), erythropoietin, thrombopoietin and a combination
thereof.
[0821] 132. The targetable construct of embodiment 129, wherein
said immunomodulator selected from the group consisting of IL-1,
IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF,
interferon-.gamma., -.alpha., -.beta. or -.gamma., TNF-.alpha., and
"S1 factor.
[0822] 133. The targetable construct of embodiment 129, wherein
said oligonucleotide is an anti-sense oligonucleotide.
[0823] 134. The targetable construct of embodiment 133, wherein
said anti-sense oligonucleotide is bcl-2 or p53.
[0824] 135. A solid support to which the targetable construct of
embodiment 122 is bound.
[0825] 136. A biosensor comprising the targetable construct of
embodiment 122.
[0826] 137. The targetable construct or the targetable complex of
any of embodiments 1, 31, 57, 72, 97 or 122, wherein said
targetable construct or said targetable complex has a half-life in
a defined set of conditions of from about 24 hours to about 500
days.
[0827] 138. The targetable construct or the targetable complex of
embodiment 137, wherein said targetable construct or said
targetable complex has a half-life in a defined set of conditions
of from about 24 hours to about 10 days.
[0828] 139. The targetable construct or the targetable complex of
embodiment 137, wherein said targetable construct or said
targetable complex has a half-life in a defined set of conditions
of from about 24 hours to about 72 hours.
[0829] 140. The targetable construct or the targetable complex of
embodiment 137, wherein said defined set of conditions is a set of
physiological conditions.
[0830] 141. The targetable construct or the targetable complex of
embodiment 137, wherein said half-life in a defined set of
conditions is a serum half-life in vitro.
[0831] 142. The targetable construct or the targetable complex of
any of embodiments 1, 31, 57, 72, 97 or 122, wherein said target
epitope is associated with a hyperproliferative disease.
[0832] 143. The targetable construct or targetable complex of
embodiment 143, wherein said target epitope is a tumor associated
antigen associated with a type of cancer selected from the group
consisting of acute lymphoblastic leukemia, acute myelogenous
leukemia, biliary cancer, breast cancer, cervical cancer, chronic
lymphocytic leukemia, chronic myelogenous leukemia, colorectal
cancer, endometrial cancer, esophageal, gastric, head and neck
cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid,
non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, glioma,
melanoma, liver cancer, prostate cancer, and urinary bladder
cancer.
[0833] 144. The targetable construct or targetable complex of
embodiment 142, wherein said target epitope is a tumor associated
antigen selected from the group consisting of A3, antigen specific
for A33 antibody, BrE3, CD1, CD1a, CD3, CD5, CD15, CD19, CD20,
CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, NCA
95, NCA90, HCG and its subunits, CEA, CSAp, EGFR, EGP-1, EGP-2,
Ep-CAM, Ba 733, HER2/neu, KC4, KS-1, KS1-4, Le-Y, MAGE, MUC1, MUC2,
MUC3, MUC4, PAM-4, PSA, PSMA, RS5, S100, TAG-72, p53, tenascin,
IL-6, insulin growth factor-1 (IGF-1), Tn antigen,
Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, 17-1
A, an angiogenesis marker, a cytokine, an immunomodulator, an
oncogene marker, an oncogene product, and other tumor associated
antigens.
[0834] 145. A method of treating a hyperproliferative disease,
comprising administering the targetable construct or targetable
complex of embodiment 142 to a subject in need thereof.
[0835] 146. The method of embodiment 148, further comprising
administering at least one additional agent suitable for the
treatment of said hyperproliferative disease.
[0836] 147. The targetable construct or the targetable complex of
any of embodiments embodiments 1, 31, 57, 72, 97 and 122, wherein
said target epitope is associated with a diseased caused by a
pathogen.
[0837] 148. The targetable construct or targetable complex of
embodiment 147, wherein said pathogen is selected from the group
consisting of a bacterium, an intracellular pathogen, a fungus, a
parasite and a virus.
[0838] 149. The targetable construct or targetable complex of
embodiment 147, wherein said pathogen is a bacteria selected from
the group consisting of Streptococcus agalactiae, Legionella
pneumophilia, Streptococcus pyogenes, Escherichia coli, Salmonella
typhimurium, Neisseria gonorrhoeae, Neisseria meningitidis,
Pneumococcus sp., Hemophilis influenzae B, Yersina pestis,
Mycobacteria sp., Mycobacterium leprae, Mycobacterium tuberculosis,
Treponema pallidum, Pseudomonas aeruginosa, Francisella tularensis,
Brucella sp., Brucella abortus, Bacillus anthracis, Clostridium
botulinum, and Clostridium tetani.
[0839] 150. The targetable construct or targetable complex of
embodiment 147, wherein said pathogen is an intracellular pathogen
selected from the group consisting of Chlamydia sp., Rickettsia
sp., Leishmania sp., Kokzidioa sp., Borrelia burgdorfei, Plasmodia
sp., Plasmodium falciparum, Trypanosoma sp., Trypanosoma brucei and
Trypanosoma cruzi.
[0840] 151. The targetable construct or targetable complex of
embodiment 147, wherein said pathogen is a fungus selected from the
group consisting of Candida sp., Candida albicans, Aspergillus sp.,
Mucor sp., Rhizopus sp., Fusarium sp., Penicillium marneffei,
Microsporum sp., Trichophyton mentagrophytes, Histoplasma
capsulatum, Blastomyces dermatitidis and Coccidioides immitis.
[0841] 152. The targetable construct or targetable complex of
embodiment 147, wherein said pathogen is a virus selected from the
group consisting of hepatitis type A, hepatitis type B, hepatitis
type C, influenza, varicella, adenovirus, HSV-I, HSV-II,
rinderpest, rhinovirous, echovirus, rabies virus, Ebola virus,
rotavirus, respiratory syncytial virus, papilloma virus, papova
virus, CMV, echinovirus, arbovirus, huntavirus, coxsackie virus,
mumps virus, measles virus, rubella virus, polio virus, HIV-I,
HIV-II, Sendai virus, feline leukemia virus, Reovirus, poliovirus,
human serum parvo-like virus, SV40, RSV, MMTV, Varicella-Zoster
virus, Dengue virus, rubella virus, measles virus, adenovirus,
human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia
virus, VSV, Variola virus, Sindbis virus, lymphocytic
choriomeningitis virus, Rinderpest virus, wart virus and blue
tongue virus.
[0842] 153. A method of treating a pathogenic disease, comprising
administering the targetable construct or targetable complex of
embodiment 147 to a subject in need thereof
[0843] 154. A method for ablating non-malignant cells or tissue in
a patient, said method comprising treating the patient with the
targetable construct or targetable complex of any of embodiments 1,
26, 47, 57, 77 or 97, wherein said non-malignant cells or tissue
are selected from the group consisting of ectopic tissue, retained
tissue, normal organ tissue and bone marrow.
[0844] 155. A method of treating a disease in a subject, comprising
administering to said subject the targetable construct or
targetable complex of any of embodiments 1, 31, 57, 72, 97 or 122,
in an amount effective to modulate a biochemical process, wherein
said target epitope is comprised within, displayed by or released
from one or more cells, tissues, organs or systems of a subject
comprising said disease.
[0845] 156. The method of embodiment 155, wherein said bi-specific
antibody is a naked antibody.
[0846] 157. The method of embodiment 155, wherein modulating said
one or more biochemical processes causes, enhances, limits or
prevents cellular quiescence, necrosis, apoptosis or a complement
cascade, mutagenesis or carcinogenesis.
[0847] 158. The targetable construct or the targetable complex of
any of embodiments embodiments 1, 31, 57, 72, 97 or 122, wherein
said target epitope is associated with a cardiovascular
disorder.
[0848] 159. A method of treating a cardiovascular disorder,
comprising administering the targetable construct or targetable
complex of embodiment 158 to a subject in need thereof.
[0849] 160. The method of embodiment 159, further comprising
administering at least one additional agent suitable for the
treatment of said cardiovascular disorder.
[0850] 161. The targetable construct or the targetable complex of
any of embodiments embodiments 1, 31, 57, 72, 97 or 122, wherein
said target epitope is associated with an autoimmune disorder.
[0851] 162. A method of treating an autoimmune disorder, comprising
administering the targetable construct or targetable complex of
embodiment 161 to a subject in need thereof.
[0852] 163. The method of embodiment 162, wherein autoimmune
disorder is selected from the group consisting of acute idiopathic
thrombocytopenic purpura, chronic idiopathic thrombocytopenic
purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis,
systemic lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular syndromes, bullous pemphigoid, diabetes mellitus,
Henoch-Schonlein purpura, post-streptococcalnephritis, erythema
nodosum, Takayasu's arteritis, Addison's disease, rheumatoid
arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis,
erythema multiforme, IgA nephropathy, polyarteritis nodosa,
ankylosing spondylitis, Goodpasture's syndrome,
thromboangitisubiterans, Sjogren's syndrome, primary biliary
cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma,
chronic active hepatitis, polymyositis/dermatomyositis,
polychondritis, pamphigus vulgaris, Wegener's granulomatosis,
membranous nephropathy, amyotrophic lateral sclerosis, tabes
dorsalis, giant cell arteritis/polymyalgia, pemiciousanemia,
rapidly progressive glomerulonephritis and fibrosing
alveolitis.
[0853] 164. The method of embodiment 162, further comprising
administering at least one additional agent suitable for the
treatment of said hyperproliferative disease.
[0854] 165. A pharmaceutical composition comprising the targetable
construct or targetable complex of any of embodiments 1, 31, 57,
72, 97 or 122.
[0855] 166. A method of treating a disease in a subject, comprising
administering to said subject the pharmaceutical composition of
embodiment 165.
[0856] 167. A kit comprising the targetable construct or targetable
complex of any of embodiments 1, 31, 57, 72, 97 or 122.
[0857] 168. A method of detecting a substance in a sample,
comprising contacting said sample with the targetable construct or
targetable complex of any one of embodiments any of embodiments 1,
31, 57, 72, 97 or 122, wherein said substance is or comprises a
target epitope.
[0858] 169. A method of detecting a substance in a sample, wherein
said substance is or comprises a target epitope, comprising
contacting said sample with the biosensor of any of embodiments 1,
30, 56, 71, 96 or 121, and detecting a signal therefrom.
[0859] 170. The method of embodiment 169, wherein said sample is a
biological sample.
[0860] 171. The method of embodiment 170, wherein said biological
sample is is selected from the group consisting of a sample of
serum, blood, plasma, lymph, urine, feces, skin, intraocular fluid,
synovial fluid, phlegm, cartilage and bone, and a biopsy
sample.
[0861] 172. A method of detecting a substance in an environment,
wherein said substance is or comprises a target epitope, comprising
exposing the biosensor of any of 1, 30, 56, 71, 96 or 121 to said
environment, and detecting a signal therefrom.
[0862] 173. A solid support comprising the targetable construct or
targetable complex of any of embodiments 1, 31, 57, 72, 97 or
122.
[0863] 174. The solid support of embodiment 173, wherein said solid
support is selected from the group consisting of a dipstick, a
bead, an interior surface of a well in a multiwell plate, and a
membrane.
[0864] 175. A method of purifying a substance, wherein said
substance is or comprises a target epitope, said method comprising
contacting a sample comprising said substance to the solid support
of embodiment 173.
[0865] 176. A method of detecting a substance in a sample, wherein
said substance is or comprises a target epitope, said method
comprising contacting said sample with the solid support of
embodiment 173.
[0866] 177. A dipstick comprising the solid support of embodiment
173.
[0867] 178. A method of detecting a substance in a fluid sample,
wherein said substance is or comprises a target epitope, said
method comprising contacting said fluid sample with the dipstick of
embodiment 177.
[0868] 179. A multiwell plate comprising the targetable construct
or targetable complex of any of embodiments 1, 31, 57, 72, 97 or
122.
[0869] 180. A method of detecting a substance in a sample, wherein
said substance is or comprises a target epitope, said method
comprising contacting said sample with the multiwell plate of
embodiment 179.
[0870] 181. An immunoassay for a substance, wherein said substance
is or comprises a target epitope, said method comprising contacting
said substance with the multiwell plate of embodiment 179.
[0871] 182. A membrane comprising the targetable construct or
targetable complex of any of embodiments 1, 31, 57, 72, 97 or
122.
[0872] 183. A method of detecting a substance, wherein said
substance is or comprises a target epitope, said method comprising
contacting said substance with the membrane of embodiment 182.
[0873] 184. A bead comprising the targetable construct or
targetable complex of any of embodiments 1, 31, 57, 72, 97 or
122.
[0874] 185. A method of purifying a substance, wherein said
substance is or comprises a target epitope, said method comprising
contacting said substance with the bead of embodiment 184.
[0875] 186. A method of binding a substance to a solid support,
wherein said substance is or comprises a target epitope, said
method comprising contacting said substance with the solid support
of embodiment 173.
[0876] 187. A method of removing all or some of a substance from a
composition, wherein said substance is or comprises a target
epitope, said method comprising contacting said composition with
the solid support of embodiment 173.
[0877] 188. The method of embodiment 187, wherein said composition
is a fluid.
[0878] 189. The method of embodiment 187, wherein said composition
is a biological sample.
[0879] 190. The method of embodiment 189, wherein said sample is a
biological sample which is selected from the group consisting of
serum, blood, plasma, lymph, urine, feces, sweat, intraocular
fluid, semen, synovial fluid, mucus, exudent, bone marrow and a
biopsy sample.
[0880] 191. A method of removing all or some of a substance from a
tissue in a patient, wherein said substance is or comprises a
target epitope, said method comprising contacting said tissue with
the solid support of embodiment 173 and reintroducing said tissue
into said patient.
[0881] 192. The method of embodiment 191, wherein said tissue is
selected from the group consisting of serum, blood, plasma, lymph,
intraocular fluid, bone marrow.
[0882] 193. The method of embodiment 191, wherein said substance is
or is part of a toxin, a pathogen, a hyperproliferative cell and an
infected cell.
[0883] 194. A device for treating a fluid of a patient in order to
remove a substance therefrom and reintroducing said fluid into said
patient, wherein said substance is or comprises a target epitope,
said device comprising the solid support of embodiment 173.
[0884] 195. A method of treating a patient, said method comprising
removing a fluid from a patient, passing said fluid through the
device of embodiment 194, and reintroducing said fluid into said
patient.
[0885] 196. The method of embodiment 187, further comprising
separating said substance from said solid support.
[0886] 197. The method of embodiment 187, wherein said substance is
an undesirable substance.
[0887] 198. The method of embodiment 187, wherein said substance is
a compound of interest.
[0888] 199. The method of embodiment 187, wherein said method is
part of a manufacturing process.
[0889] 200. The method of embodiment 159, wherein said
cardiovascular disease is an atherosclerotic plaque, ischemia,
fibrin clot, vascular clot, and myocardial infarction.
[0890] 201. The method of embodiment 200, wherein said vascular
clot is an embolus or thrombosis.
[0891] 202. The method of embodiment 155, wherein said tissue is
hypoplastic, absent, anatomically displaced or ectopic.
[0892] 203. The method of embodiment 155, wherein said disease or
disorder is a neurodegenerative or metabolic disease.
[0893] 204. The method of embodiment 203, wherein said metabolic
disease is amyloidosis and said targetable construct binds
amyloid.
[0894] 205. The method of embodiment 203, wherein said
neurodegenerative disease is Alzheimer's disease.
[0895] 206. A method of diagnosing/detecting a disease in a
subject, comprising administering to said subject the targetable
construct or targetable complex of any of embodiments 1, 31, 57,
72, 97 or 122, in an amount effective to modulate a biochemical
process, wherein said target epitope is comprised within, displayed
by or released from one or more cells, tissues, organs or systems
of a subject comprising said disease.
[0896] 207. The method of embodiment 206, wherein said method is
suitable for detecting a cardiovascular lesion.
[0897] 208. The method of embodiment 207, wherein said method is
suitable for photodynamic diagnosis.
[0898] 209. The method of embodiment 208, wherein said targetable
construct comprises a photosensitizer selected from the group
consisting of dihematoporphyrin, benzoporphyrin monoacid ring A,
tin etiopurpurin, sulfonated aluminum phthalocyanine, and lutetium
texaphyrin.
* * * * *
References