U.S. patent application number 17/562727 was filed with the patent office on 2022-07-21 for methods for dna-depending targeting of a cell permeant antibody.
This patent application is currently assigned to Yale University. The applicant listed for this patent is Yale University. Invention is credited to Peter GLAZER, James E. HANSEN.
Application Number | 20220227885 17/562727 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220227885 |
Kind Code |
A1 |
HANSEN; James E. ; et
al. |
July 21, 2022 |
METHODS FOR DNA-DEPENDING TARGETING OF A CELL PERMEANT ANTIBODY
Abstract
The invention provides methods for selective targeting of live
cells, which have undergone or are undergoing radiation or
chemotherapy, at a site of interest with a cell-penetrating
polypeptide. In one embodiment of the invention, the method
comprises contacting the live cells with a cell-penetrating
polypeptide comprising cell-penetrating determinants so that the
cell-penetrating polypeptide binds extracellular DNA near or around
the live cells so as to form a complex or association therewith
such that the complex or associated polypeptide-DNA so bound bind
the live cells and penetrates the live cells thereby selectively
targeting live cells at a site of interest with a cell-penetrating
polypeptide.
Inventors: |
HANSEN; James E.; (Guilford,
CT) ; GLAZER; Peter; (Guilford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yale University |
New Haven |
CT |
US |
|
|
Assignee: |
Yale University
New Haven
CT
|
Appl. No.: |
17/562727 |
Filed: |
December 27, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16414636 |
May 16, 2019 |
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17562727 |
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15047575 |
Feb 18, 2016 |
10383945 |
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16414636 |
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62117694 |
Feb 18, 2015 |
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International
Class: |
C07K 16/44 20060101
C07K016/44; C07K 16/18 20060101 C07K016/18; A61K 47/68 20060101
A61K047/68 |
Goverment Interests
[0002] This invention was made with government support by the
United States Department of Veterans Affairs. The government has
certain rights in the invention.
Claims
1.-96. (canceled)
97. A method for inhibiting tumor cells associated with ischemia,
cellular/tissue necrosis or cellular/tissue apoptosis in vivo, by
selective targeting of the tumor cells with a cell-penetrating
polypeptide, comprising administering the cell-penetrating
polypeptide to a subject in need thereof, wherein the
cell-penetrating polypeptide is a 3E10 antibody or a fragment
thereof.
98. The method of claim 97, wherein the fragment is a single chain
Fv (scFv) fragment of mAb 3E10 antibody.
99. (canceled)
100. The method of claim 98, wherein the 3E10 scFv is formulated in
a pharmaceutically acceptable composition.
101. The method of claim 100, wherein the composition is
administered to a subject in need thereof by injection.
102. The method of claim 101, wherein the injection of the 3E10
scFv results in localization of the 3E10 scFv to tumor cell
nuclei.
103. The method of claim 102, wherein the 3E10 scFv is detectable
by immunohistochemistry (IHC) in tumor cell nuclei for up to 24
hours after injection.
104. The method of claim 101, wherein the 3E10 scFv does not
localize in major organs to the extent that the 3E10 scFv localizes
in tumor cell nuclei.
105. The method of claim 104, wherein the major organs comprise one
or more of heart, kidney, skeletal muscle, and liver.
106. The method of claim 97, wherein the tumor cells are associated
with a glioma.
Description
[0001] The subject application claims the priority of U.S. Ser. No.
62/117,694, filed Feb. 18, 2015, the disclosure of which, in its
entirety, is hereby incorporated by reference into this
application.
[0003] Throughout this application various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
BACKGROUND OF THE INVENTION
[0004] A select group of lupus anti-DNA autoantibodies penetrate
into living cells (1), and one unusual lupus autoantibody that
penetrates cell nuclei without causing any apparent harm to normal
cells or tissues, 3E10 (2), has been developed as a molecular
delivery vehicle. Specifically, a 3E10 single chain variable
fragment (scFv) with an enhancing mutation in CDR1 that increases
DNA binding and efficiency of nuclear penetration has been used to
carry cargo proteins including p53, Hsp70, and other antibody
fragments into cell nuclei in vitro and in vivo (3-6). 3E10 scFv
also has activity by itself and has been shown to inhibit DNA
repair, sensitize cancer cells to DNA-damaging therapy, and to be
toxic to BRCA2-deficient cancer cells (7). 3E10 scFv has potential
to be used in molecular therapy approaches to diseases ranging from
cancer to ischemic conditions such as stroke, and a greater
understanding of the details of the mechanism by which it
penetrates cell nuclei is important to further delineating the
scope of its therapeutic applications.
[0005] Mutations in 3E10 that interfere with its ability to bind
DNA also render the antibody incapable of nuclear penetration. In
addition, 3E10 scFv has previously been shown capable of
penetrating into cell nuclei in an ENT2-dependent manner, with
efficiency of nuclear uptake greatly impaired in ENT2-deficient
cells (8). Taken together, these findings suggest a link between
cellular uptake of DNA and nuclear penetration by 3E10 scFv.
Interestingly, when a 3E10 scFv-Hsp70 fusion protein (Fv-Hsp70) was
administered intravenously to rats three hours after ligation of
middle cerebral arteries to induce stroke, Fv-Hsp70 was found to
selectively localize to regions of ischemic brain (9).
[0006] The invention involves the discovery of the mechanism by
which some anti-DNA antibodies or fragments thereof penetrate the
cell for use in better treating disease, disorders and
conditions.
SUMMARY OF THE INVENTION
[0007] The invention provides methods for selective targeting of
live cells, which have undergone or are undergoing radiation or
chemotherapy, at a site of interest with a cell-penetrating
polypeptide. In one embodiment of the invention, the method
comprises contacting the live cells with a cell-penetrating
polypeptide comprising cell-penetrating determinants so that the
cell-penetrating polypeptide binds extracellular DNA near or around
the live cells so as to form a complex or association therewith
such that the complex or associated polypeptide-DNA so bound bind
the live cells and penetrates the live cells thereby selectively
targeting live cells at a site of interest with a cell-penetrating
polypeptide.
[0008] Additionally, the invention provides method for selective
targeting of live cells at or near the proximity of a cellular
injury with a cell-penetrating polypeptide which comprises
cell-penetrating determinants joined to, or combined with, a
therapeutic agent. In one embodiment, the method comprising
administering the cell-penetrating polypeptide at or near the
proximity of the injury so that it binds extracellular DNA from the
cellular injury so as to form a complex or association therewith
such that the complex or associated polypeptide-DNA-therapeutic
agent so bound bind the live cells and penetrates the live cells
thereby selectively targeting live cells at a site of interest with
the cell-penetrating polypeptide.
[0009] The invention further provides methods for selective
targeting of live cells at or near the proximity of a cellular
injury with a cell-penetrating polypeptide. In an embodiment of the
invention, the cell-penetrating determinants are joined to, or
combined with, a therapeutic agent. The method may comprise
contacting the live cells with a composition having (a) a
cell-penetrating polypeptide which comprises cell-penetrating
determinants and (b) extracellular DNA so that the cell-penetrating
polypeptide binds extracellular DNA near or around the live cells
so as to form a complex or association therewith such that the
complex or associated polypeptide-DNA so bound bind the live cells
and penetrates the live cells thereby selectively targeting live
cells at a site of interest with the cell-penetrating
polypeptide.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1. 3E10 scFv penetrates most efficiently into living
cells surrounding a dead cell. GM02605 fibroblasts were washed with
serum free media and then treated with 10 .mu.M 3E10 scFv for one
hour, followed by anti-Myc immunostaining to detect nuclear
penetration by 3E10 scFv. Nuclear penetration by 3E10 scFv was
restricted to cells in close proximity to a dead cell, suggesting
that a factor released by dead cells promotes nuclear uptake of the
fragment.
[0011] FIG. 2. Extracellular DNA facilitates penetration of 3E10
scFv into cell nuclei. GM02605 fibroblasts were washed with serum
free media and then treated with control buffer alone or 10 .mu.M
3E10 scFv in the presence of control buffer, cell lysate,
DNA-depleted cell lysate, or purified DNA for one hour, followed by
anti-Myc immunostaining to detect nuclear penetration by 3E10 scFv.
Nuclear penetration into .about.100% of the cells was only observed
in the presence of cell lysate or purified DNA.
[0012] FIG. 3. 3E10 scFv localizes to tumor cell nuclei in vivo.
Immunodeficient mice bearing subcutaneous U87 human glioma
xenografts were treated by intraperitoneal injection of control
buffer or 3E10 scFv. Mice were sacrificed 4 or 24 hours after
treatment, and tumors and select normal tissues were immunostained
for the presence of 3E10 scFv. (A) Four hours after treatment 3E10
scFv was detected in the nuclei of the U87 tumor cells but was not
detected in tissues of major organs including heart, kidney,
skeletal muscle, and liver. These results are consistent with
enhanced uptake of 3E10 scFv into sites of high cell turnover where
DNA is released from dying cells. (B) Twenty-four hours after
treatment 3E10 scFv was still detectable in the tumors,
demonstrating the stability of the uptake into tumor nuclei.
DETAILED DESCRIPTION OF THE INVENTION
[0013] It is to be understood that the present disclosure is not
limited to particular embodiments described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting, since the scope of the
present disclosure will be limited only by the appended claims.
[0014] The detailed description of the present disclosure is
divided into various sections only for the reader's convenience and
disclosure found in any section may be combined with that in
another section. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which the present
disclosure belongs.
Definitions
[0015] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a compound" includes a plurality of
compounds.
[0016] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the present disclosure belongs.
As used herein the following terms have the following meanings.
[0017] As used herein, the term "about" when used before a
numerical designation, e.g., temperature, time, amount,
concentration, and such other, including a range, indicates
approximations which may vary by (+) or (-) 10%, 5% or 1%.
[0018] As used herein, the term "administration" may be effected in
one dose, continuously or intermittently or by several subdoses
which in the aggregate provide for a single dose. Dosing can be
conducted throughout the course of treatment. Methods of
determining the most effective means and dosage of administration
are known to those of skill in the art and will vary with the
composition used for therapy, the purpose of the therapy, the
target cell being treated and the subject being treated. Single or
multiple administrations can be carried out with the dose level and
pattern being selected by the treating physician. Suitable dosage
formulations and methods of administering the agents are known in
the art. Route of administration can also be determined and method
of determining the most effective route of administration are known
to those of skill in the art and will vary with the composition
used for treatment, the purpose of the treatment, the health
condition or disease stage of the subject being treated and target
cell or tissue. Non-limiting examples of route of administration
include intratumoral delivery, peritumoral delivery,
intraperitoneal delivery, intrathecal delivery, intramuscular
injection, subcutaneous injection, intravenous delivery, nasal
spray and other mucosal delivery (e.g. transmucosal delivery),
intra-arterial delivery, intraventricular delivery, intrasternal
delivery, intracranial delivery, intradermal injection,
electroincorporation (e.g., with electroporation), ultrasound, jet
injector, oral and topical patches.
[0019] A "therapeutic agent," as used herein, may be a molecule, or
compound that is useful in treatment of a disease or condition. A
"therapeutically effective amount," "therapeutically effective
concentration" or "therapeutically effective dose" is the amount of
a compound that produces a desired therapeutic effect in a subject,
such as preventing or treating a target condition, alleviating
symptoms associated with the condition, producing a desired
physiological effect, or allowing imaging or diagnosis of a
condition that leads to treatment of the disease or condition. The
precise therapeutically effective amount is the amount of the
composition that will yield the most effective results in terms of
efficacy of treatment in a given subject. This amount will vary
depending upon a variety of factors, including, but not limited to,
the characteristics of the therapeutic compound (including
activity, pharmacokinetics, pharmacodynamics, and bioavailability),
the physiological condition of the subject (including age, sex,
disease type and stage, general physical condition, responsiveness
to a given dosage, and type of medication), the nature of the
pharmaceutically acceptable carrier or carriers in the formulation,
and the route of administration. One skilled in the clinical and
pharmacological arts will be able to determine a therapeutically
effective amount through routine experimentation, namely by
monitoring a subject's response to administration of a compound and
adjusting the dosage accordingly. For additional guidance, see
Remington: The Science and Practice of Pharmacy 21.sup.(st)
Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott
Williams & Wilkins, Philadelphia, Pa., 2005.
[0020] As used herein, "in combination" or "in combination with,"
when used herein in the context of multiple agents, therapeutics,
or treatments, means in the course of treating the same disease or
condition in a subject administering two or more agents, drugs,
treatment regimens, treatment modalities or a combination thereof.
This includes simultaneous administration (or "coadministration"),
administration of a first agent prior to or after administration of
a second agent, as well as in a temporally spaced order of up to
several days apart. Such combination treatment may also include
more than a single administration of any one or more of the agents,
drugs, treatment regimens or treatment modalities. Further, the
administration of the two or more agents, drugs, treatment
regimens, treatment modalities or a combination thereof may be by
the same or different routes of administration.
[0021] "Treating" or "treatment" of a condition, disease or
disorder may refer to preventing the condition, disease or
disorder, slowing the onset or rate of development of the
condition, disease or disorder, reducing the risk of developing the
condition, disease or disorder, preventing or delaying the
development of symptoms associated with the condition, disease or
disorder, reducing or ending symptoms associated with the
condition, disease or disorder, generating a complete or partial
regression of the condition, disease or disorder, or some
combination thereof. Examples of diseases or disorders include
colorectal cancer, osteosarcoma, non-small cell lung cancer, breast
cancer, ovarian cancer, glial cancer, solid tumors, metastatic
tumor, acute lymphoblastic leukemia, acute myelogenous leukemia,
adrenocortical carcinoma, Kaposi sarcoma, lymphoma, anal cancer,
astrocytomas, basal cell carcinoma, bile duct cancer, bladder
cancer, bone cancer, brain tumor, breast cancer, bronchial tumor,
cervical cancer, chronic lymphocytic leukemia, chronic myelogenous
leukemia, chronic myeloproliferative disorders, colon cancer,
colorectal cancers, ductal carcinoma in situ, endometrial cancer,
esophageal cancer, eye cancer, intraocular, retinoblastoma,
metastatic melanoma, gallbladder cancer, gastric cancer,
gastrointestinal carcinoid tumor, gastrointestinal stromal tumors,
glioblastoma, glioma, hairy cell leukemia, head and neck cancer,
hepatocellular carcinoma, hepatoma, Hodgkin lymphoma,
hypopharyngeal cancer, Langerhans cell histiocytosis, laryngeal
cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma
in situ, lung cancer, non-small cell lung cancer, small cell lung
cancer, lymphoma, AIDS-related lymphoma, Burkitt lymphoma,
non-Hodgkin lymphoma, cutaneous T-cell lymphoma, melanoma, squamous
neck cancer, mouth cancer, multiple myeloma, myelodysplastic
syndromes, myelodysplastic/myeloproliferative neoplasms, nasal
cavity and paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, oral cavity cancer, oropharyngeal cancer,
osteosarcoma, ovarian cancer, pancreatic carcinoma, papillary
carcinomas, parathyroid cancer, penile cancer, pharyngeal cancer,
pheochromocytoma, pineal parenchymal tumors, pineoblastoma,
pituitary tumor, pleuropulmonary blastoma, primary central nervous
system lymphoma, prostate cancer, rectal cancer, renal cell cancer,
salivary gland cancer, sarcoma, Ewing sarcoma, soft tissue sarcoma,
squamous cell carcinoma, Sezary syndrome, skin cancer, Merkel cell
carcinoma, testicular cancer, throat cancer, thymoma, thymic
carcinoma, thyroid cancer, urethral cancer, endometrial cancer,
uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
Waldenstrom macroglobulinemia, and Wilms tumor.
[0022] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer",
"cancerous", "cell proliferative disorder", "proliferative
disorder" and "tumor" are not mutually exclusive as referred to
herein. Tumor or cancer status may also be assessed by sampling for
the number, concentration or density of tumor or cancer cells,
alone or with respect to a reference. In accordance with the
practice of the invention, inhibiting a tumor may be measured in
any way as is known and accepted in the art, including complete
regression of the tumor(s) (complete response); reduction in size
or volume of the tumor(s) or even a slowing in a previously
observed growth of a tumor(s), e.g., at least about a 10-30%
decrease in the sum of the longest diameter (LD) of a tumor, taking
as reference the baseline sum LD (partial response); mixed response
(regression or stabilization of some tumors but not others); or no
apparent growth or progression of tumor(s) or neither sufficient
shrinkage to qualify for partial response nor sufficient increase
to qualify for progressive disease, taking as reference the
smallest sum LD since the treatment started (stable disease).
[0023] Examples of cytotoxic agents include, but are not limited to
ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium
bromide, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicine, dihydroxy anthracenedione, actinomycin D,
diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A
chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,
restrictocin, phenomycin, enomycin, curcin, crotin, calicheamicin,
Saponaria officinalis inhibitor, maytansinoids, and glucocorticoid
and other chemotherapeutic agents, as well as radioisotopes such as
.sup.212Bi, .sup.131I, .sup.131In, .sup.90Y, and .sup.186Re.
Suitable detectable markers include, but are not limited to, a
radioisotope, a fluorescent compound, a bioluminescent compound,
chemiluminescent compound, a metal chelator or an enzyme.
[0024] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment or man-made environment in which the antibody is
synthesized and/or assembled. An "isolated" antibody is deficient
in and, preferably, eliminated of contaminant components of its
natural environment or man-made environment in which the antibody
is synthesized and/or assembled. Contaminant components of its
natural environment or man-made environment are materials which
would interfere with diagnostic or therapeutic uses for the
antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. "Isolated" antibody does
not require absolute purity. In some embodiments, the antibody will
be purified (1) to greater than 95% by weight of antibody as
determined by the Lowry method, and most preferably more than 99%
by weight, or (2) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using Coomassie blue or, preferably, silver
stain. Other purification methods are well known and contemplated
herein.
[0025] The term "vector," is intended to refer to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. Examples of vectors include, but are not limited
to, plasmids, (e.g., a circular double stranded DNA loop into which
additional DNA segments may be ligated or introduced), phage
vectors, and viral vectors (e.g., wherein additional DNA segments
may be ligated or introduced into the viral genome). Certain
vectors are capable of directing the expression of genes to which
they are operatively linked. Such vectors are referred to herein as
"recombinant expression vectors" (or simply, "recombinant
vectors").
[0026] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides, and/or their analogs, or any
substrate that can be incorporated into a polymer by DNA or RNA
polymerase, or by a synthetic reaction.
[0027] The terms "antibody" and "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal
antibodies (for example, full length or intact monoclonal
antibodies), polyclonal antibodies, multivalent antibodies,
multispecific antibodies (e.g., bispecific antibodies so long as
they exhibit the desired biological activity) and may also include
certain antibody fragments (as described in greater detail herein).
An antibody can be human, humanized and/or affinity matured.
[0028] The term "variable region" or "variable domain" refers to a
region or domain, which is characterized by the presence of certain
portions of the antibody differing extensively in sequence among
antibodies and is used in the binding of each particular antibody
to a particular antigen. The "variable region" or "variable domain"
confers binding specificity to the antibody. Sequence variability
is not evenly distributed throughout the variable domain or
variable region of an antibody. Rather, sequence variability is
concentrated in three segments called complementarity-determining
regions (CDRs) or hypervariable regions both in the light-chain and
the heavy-chain variable domains. The more highly conserved
portions of variable domains are called the framework (FR). The
variable domains of native heavy and light chains each comprise
four FR regions, largely adopting a n-sheet configuration,
connected by three CDRs, which form loops connecting, and in some
cases forming part of, the n-sheet structure. The CDRs in each
chain are held together in close proximity by the FR regions and,
with the CDRs from the other chain, contribute to the formation of
the antigen-binding site of antibodies (see Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, National
Institute of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody-dependent cellular toxicity.
[0029] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. In a two-chain Fv
species, this region consists of a dimer of one heavy- and one
light-chain variable domain in tight, non-covalent association. In
a single-chain Fv species, one heavy- and one light-chain variable
domain may be covalently linked by a flexible peptide linker such
that the light and heavy chains can associate in a "dimeric"
structure analogous to that in a two-chain Fv species.
[0030] The Fab fragment contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0031] "Antibody fragments" comprise only a portion of an intact
antibody, wherein the portion preferably retains at least one,
preferably most or all, of the functions normally associated with
that portion when present in an intact antibody. Examples of
antibody fragments include Fab, Fab', F(ab').sub.2, and Fv
fragments; diabodies; linear antibodies; single-chain antibody
molecules; single chain Fv fragment (scFv) and multispecific
antibodies formed from antibody fragments. In one embodiment, an
antibody fragment comprises an antigen binding site of the intact
antibody and thus retains the ability to bind antigen. In another
embodiment, an antibody fragment, for example, comprises the Fc
region, retains at least one of the biological functions normally
associated with the Fc region when present in an intact antibody,
such as FcRn binding, antibody half-life modulation, ADCC function
and complement binding. In one embodiment, an antibody fragment is
a monovalent antibody that has an in vivo half-life substantially
similar to an intact antibody. For example, such an antibody
fragment may comprise on antigen binding arm linked to an Fc
sequence capable of conferring in vivo stability to the
fragment.
[0032] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues.
[0033] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. Humanized antibodies may be human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or nonhuman primate having the desired
specificity, affinity, and capacity. The humanized antibody may
optionally also comprise at least a portion of an immunoglobulin
constant region (Fc), e.g. that of a human immunoglobulin.
[0034] "Chimeric" antibodies have a portion of the heavy and/or
light chain identical with or homologous to corresponding sequences
in antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855
(1984)).
[0035] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For example, one embodiment of 3E10 mAb is a 3E10 scFv having the
primary structure shown in FIG. 4-1 through 4-4 of U.S. Patent
Application Publication No.: US 2013/0266570 A1, published 10 Oct.
2013.
[0036] An "antigen" is a target to which an antibody can
selectively bind. The target antigen may be polypeptide,
carbohydrate, nucleic acid, lipid, hapten or other naturally
occurring or synthetic compound.
[0037] In accordance with the practice of the invention,
"extracellular DNA" is DNA free of a cell or cell-free DNA.
Extracellular DNA may be introduced or administered by methods
known in the art such as, for example, microinjection of DNA into
extracellular space or outside of a cell or cells, cell or cells in
a tissue, or cell or cells in an organ, so long as the DNA is not
introduced or administered into a cell. The delivered DNA in the
extracellular space or outside of a cell may be in any physical
state, including in a solution, as a solid, as a colloidal form, as
a semi-crystalline state, as a nanoparticle or combination thereof.
The delivered DNA may be isolated DNA from nature, either total,
fractionated, intact or sheared, or synthesized by the hand of man
such as through synthetic chemistry or in vitro enzymatic methods
as known in the art. Alternatively, the extracellular DNA may be
produced in situ by dying cells that release its nucleic acid
content. In one embodiment, extracellular DNA is produced by
radiation, chemotherapeutic agent, toxin, or by any condition that
promotes cell death. In one embodiment, the invention contemplates
the use of radiation or a chemotherapeutic agent at a site of
interest or a site of injury to produce extracellular DNA or
additional extracellular DNA. In another embodiment, extracellular
DNA may be produced in situ through freezing, heat, laser, hypoxic
condition, a poison, laceration, force and trauma.
[0038] A "bispecific antibody" of the invention includes antibodies
with not only binding specificities for two targets but also
include antibodies with additional determinants, which may be
derived from immunoglobulin sequences or non-immunoglobulin
sequences, with specificities for other target(s). For example, a
bispecific antibody includes heteroconjugates with binding
specificities for at least two different targets. For example a
heteroconjugates includes a hybrid antibody created from linking
two different antibodies or antibody fragments or a hybrid of an
antibody or antibody fragment linked to a lectin or lectin fragment
or another determinant with an intracellular binding specificity or
a cell-penetrating ability, so long as the heteroconjugates have
binding specificities for at least two targets. A bispecific
antibody may further include heteroconjugates in which a bispecific
antibody is coupled to a therapeutic agent (e.g., chemotherapeutic
agent or toxin or cytoprotective agent) or an imaging agent (e.g.,
radioisotope). A bispecific antibody may be produced by recombinant
DNA methods in which coding sequences of immunoglobulin genes are
manipulated to produce the bispecific antibody. The coding
sequences of the immunoglobulin genes may be used in its entirety,
mutated at specific sequences or codons, or used partially by
truncating the coding sequences to produce the bispecific antibody
or components that results in production of a bispecific antibody.
In some embodiments, a bispecific antibody includes an intact
antibody or a Fv fragment, Fab, Fab' or F(ab').sub.2 fragment or a
diabody, linear antibody, single-chain antibody molecule or scFv
antibody fragment coupled chemically or recombinantly, disulfide
bridges or by other means to a second determinant which
specifically recognizes at least a different target than the target
recognized by the intact antibody or the Fv, Fab, Fab' or
F(ab').sub.2 fragment or the diabody, linear antibody, single-chain
antibody molecule or scFv antibody fragment. The second determinant
includes a second intact antibody or a Fv fragment, Fab, Fab' or
F(ab').sub.2 fragment or a diabody, linear antibody, single-chain
antibody molecule or scFv antibody fragment different from the
binding specificity of the first antibody or the first Fv, Fab,
Fab' or F(ab').sub.2 fragment or a the first diabody, linear
antibody, single-chain antibody molecule or scFv antibody
fragment.
[0039] In accordance with the practice of the invention, the second
determinant may recognize a target that is located inside the cell,
e.g., in the cytoplasm or in the cell nucleus. In another
embodiment, the second determinant recognizes a target that is
normally an intracellular protein and not normally on the surface
of a cell or not normally secreted by the cell. In a further
embodiment, the second determinant recognizes an E3
ubiquitin-protein ligase, a tumor suppressor-interacting protein, a
binding partner of a tumor suppressor protein, an oncoprotein, or a
DNA repair protein, wherein the second determinant fails to
recognize any protein that normally resides on the cell surface. In
yet a further embodiment, the second determinant recognizes a
transcription factor, a transcriptional repressor, a
transcriptional co-factor, a nuclear receptor, a steroid receptor,
a methylase, an acetylase, a deacetylase, RNA polymerase, a kinase,
a phosphatase, an intracellular signaling molecule (not a cell
surface signaling molecule), a cell cycle regulatory protein, a
protease, a DNA repair protein, a recombinase, a chromosomal
protein, an apoptotic protein, a SUMO ligase, a ubiquitin ligase, a
metabolic protein, an organelle protein, a nuclear protein, a
nucleolar protein, a mitochondrial protein, a ligand, a ribosomal
protein, an enzyme, a cytoskeletal protein, a chromosomal protein,
a structural protein, a intracellular soluble protein, an
intracellular shuttling protein or a regulatory protein so long as
the second determinant fails to recognize any protein that normally
resides on the cell surface.
[0040] A bispecific antibody includes chimeric antibodies,
recombinant antibodies, humanized antibodies or human antibodies or
their derivatives. A bispecific antibody includes antibodies of the
invention in which one or more of the complementarity determining
region (CDR) of the invention is used to screen for additional
antibodies or agents that can compete with the binding of the 3E10
antibody. Peptide, phage display, cDNA, or chemical libraries may
be used for such a screen.
[0041] Examples of 3E10 bispecific antibodies, e.g., 3E10 scFv-3G5
scFv bispecific antibody and 3E10 scFv-PAb421 scFv bispecific
antibody, are disclosed in U.S. Ser. No. 13/844,318, filed Mar. 15,
2013, which is incorporated by reference herein. Additional
examples of anti-DNA monoclonal antibody 3E10 (also referred to
herein as a 3E10 antibody or mAb 3E10) include an antibody produced
by ATCC PTA 2439 or a functional fragment or variant thereof or an
antibody having the specificity of mAb 3E10 (Chan G, et al., Int.
J. Cancer 2016 138(1):182-6; Weisbart R H, et al., Sci. Rep. 2015
5:12022; Noble P W, et al., Cancer Res. 2015 75(11):2285-91; Hansen
J E, et al., Sci. Transl. Med. 2012 24; 4(157):157ra142; Weisbart R
H, et al., Mol. Cancer Ther. 2012 11(10):2169-73; Heinze E, et al.,
Oncol. Lett. 2011 2(4):665-668; Zhan X, et al., Stroke. 2010
41(3):538-43; Hansen J E, et al., J. Biol. Chem. 2007
282(29):20790-3; Hansen J E, et al., Cancer Res. 2007
67(4):1769-74; Hansen J E, et al., Brain Res. 2006 1088(1):187-96;
Hansen J E, et al., Scientific World Journal. 2005 5:782-8;
Weisbart R H, et al., J. Drug Target. 2005 13(2):81-7; Weisbart R
H, et al., Int. J. Oncol. 2004 25(6):1867-73; Weisbart R H, et al.,
Int. J. Oncol. 2004 25(4):1113-8; Weisbart R H, et al., Cancer
Lett. 2003 195(2):211-9; Weisbart R H, et al., Mol. Immunol. 2003
39(13):783-9; Weisbart R H, et al., J. Immunol. 2000
164(11):6020-6; Spertini F, et al., J Rheumatol. 1999
26(12):2602-8; Weisbart R H, et al., J. Autoimmun. 1998
11(5):539-46; Zack D J, et al., J. Immunol. 1996 157(5):2082-8;
Zack D J, et al., Mol. Immunol. 1995 32 (17-18):1345-53; Zack D J,
et al., J. Immunol. 1995 154(4):1987-94; Zack D J, et al., Immunol.
Cell Biol. 1994 72(6):513-20; and Weisbart R H, et al., J. Immunol.
1990 144(7):2653-8). The full 3E10 antibody has been previously
described (Weisbart R H, et al., J. Immunol. 1990 144(7):
2653-2658; ATCC Accession No. PTA 2439 hybridoma; SEQ ID NOS.: 2
and 4 of PCT International Publication No.: WO 2010/148010 A1,
published 23 Dec. 2010) as well as its nucleic acid sequence and
protein sequence (Figures 3 and 4 of Zack D J, et al., J. Immunol.
1995 154(4): 1987-1994; FIGS. 3 and 4 of US Patent Application
Publication No.: US 2008/0292618 A1; FIGS. 1 and 2 of PCT
International Publication No.: WO 2010/138769 A1, published 2 Dec.
2010; GenBank Accession Numbers: L16982 for mAb 3E10 VH chain and
L34051 for mAb 3E10 V.kappa. light chain). Location of the
complement-determining regions (e.g., CDR1, CDR2 and CDR3) along
with the framework regions (i.e., FR1, FR2, FR3, and FR4) of the
3E10 variable heavy chain and light chain domains are provided in
Figures 3 and 4 of Zack D J, et al., J. Immunol. 1995 154(4):
1987-1994; FIGS. 3 and 4 of US Patent Application Publication No.:
US 2008/0292618 A1; FIGS. 1 and 2 of PCT International Publication
No. WO 2010/138769 A1, published 2 Dec. 2010. Particularly useful
variant is substitution of aspartic acid (D) at amino acid position
31 of the heavy chain variable region (VH) of 3E10 antibody with
asparagine (N), the D31N variant, which increases binding to ssDNA
and dsDNA (Zack D J, et al., J. Immunol. 1995 154(4): 1987-1994)
and enhances cell and nuclear penetration (Zack D J, et al., J.
Immunol. 1996 157(5): 2082-2088; Weisbart W H, et al., J.
Autoimmunity 11(5): 539-546). A preferred embodiment of 3E10
antibody or its fragment is 3E10 antibody or its fragment or
derivative with aspartic acid to asparagine change at amino acid 31
of the VH chain, the D31N variant.
[0042] A "subject" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to, farm
animals (such as cows), pets (such as cats, dogs and horses),
primates, mice and rats.
[0043] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result.
[0044] A "therapeutically effective amount" of a substance/molecule
of the invention, agonist or antagonist may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the substance/molecule, agonist or
antagonist to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the substance/molecule, agonist or
antagonist are outweighed by the therapeutically beneficial
effects. A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.
[0045] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes, chemotherapeutic agents e.g. methotrexate,
adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide),
doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or
other intercalating agents, enzymes and fragments thereof such as
nucleolytic enzymes, antibiotics, and toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant
or animal origin, including fragments and/or variants thereof, and
the various antitumor or anticancer agents disclosed below. Other
cytotoxic agents are described below. A tumoricidal agent causes
destruction of tumor cells.
[0046] According to the present invention, where administration
includes a pharmaceutical formulation, preferably the formulation
is a unit dosage containing a daily dose or unit, daily sub-dose or
an appropriate fraction thereof, of the active ingredient (also
referred to herein as a therapeutic agent). In one embodiment, the
active ingredient comprises a cell-penetrating polypeptide of the
invention. In one embodiment, the active ingredient comprises a
cell-penetrating polypeptide-conjugate, such as a cell-penetrating
polypeptide chemically crosslinked to a chemical, peptide or
protein with a desired biological activity, a cell-penetrating
polypeptide modified with a radioisotope or modified with a
chelator bound to a radioisotope, or a cell-penetrating polypeptide
linked to a peptide or protein (and produced) through recombinant
DNA methods. In another embodiment, the active ingredient comprises
a fusion protein of a cell-penetrating polypeptide and a second
protein or peptide with a desired biological activity. In one
embodiment, the desired biological activity may be an activity that
induces cell death or is cell protective. In one embodiment, the
active ingredient is DNA and/or its degradation product(s). In one
embodiment, the active ingredient is extracellular DNA and/or its
degradation product(s).
[0047] The compositions of the invention can be administered by any
parenteral route, in the form of a pharmaceutical formulation
comprising the active ingredient, optionally in the form of a
nontoxic organic, or inorganic, acid, or base, addition salt, in a
pharmaceutically acceptable dosage form. Depending upon the
disorder and patient to be treated, as well as the route of
administration, the compositions may be administered at varying
doses.
[0048] In human therapy, compositions of the invention may be
administered alone but may generally be administered in admixture
with a suitable pharmaceutical excipient diluent or carrier
selected with regard to the intended route of administration and
standard pharmaceutical practice.
[0049] In embodiments of the present invention in which
polypeptides or polynucleotides of the invention are administered
parenterally, such administration can be, for example,
intravenously, intra-arterially, intraperitoneally, intrathecally,
intraventricularly, intracisternally, intracranially,
intramuscularly or subcutaneously, or they may be administered by
infusion techniques. They are best used in the form of a sterile
aqueous solution which may contain other substances, for example,
enough salts or glucose to make the solution isotonic with blood.
The aqueous solutions should be suitably buffered (preferably to a
pH of from 3 to 9), if necessary. The preparation of suitable
parenteral formulations under sterile conditions is readily
accomplished by standard pharmaceutical techniques well-known to
those skilled in the art.
[0050] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
METHODS OF THE INVENTION
[0051] The invention is directed to the discovery that certain
anti-DNA antibodies, including but not limited to, 3E10 monoclonal
antibody or other anti-DNA antibodies that by their dependence on a
salvage pathway for cell and nuclear penetration, require
extracellular DNA or its degradation product in order to penetrate
cells in a salvage pathway-dependent process, such as a nucleoside
salvage pathway or ENT-2 nucleoside transporter pathway. This
requirement for extracellular DNA or its degradation product for
cell and nuclear penetration provides an opportunity for "targeted"
therapies with these anti-DNA antibodies by introducing or
producing extracellular DNA or its degradation product near or
around live cells at a site of interest being targeted by these
cell-penetrating anti-DNA antibodies. "Targeted" therapies, using
such anti-DNA antibodies or their conjugates, reduce any potential
systemic toxicity as well as increase effectiveness of the
antibodies and their conjugates.
[0052] In one embodiment, the invention provides a method for
selective targeting of live cells at a site of interest with a
cell-penetrating polypeptide which comprises: (a) introducing or
producing extracellular DNA and/or its degradation product(s) near
or around the live cells at the site of interest; (b) introducing
the cell-penetrating polypeptide comprising cell-penetrating
determinants near or around the live cells, before, after or
concurrently with the DNA of step (a); (c) contacting extracellular
DNA or its degradation product near or around the live cells with a
cell-penetrating polypeptide comprising cell-penetrating
determinants so that the cell-penetrating polypeptide binds
extracellular DNA or its degradation product near or around the
live cells so as to form a complex; (d) contacting one of the live
cells with the complex in (c) so as to bind and penetrate the live
cell; and (e) permitting additional complexes to form as in (c) and
contacting additional cells with said complexes so as to bind and
penetrate additional live cells at the site of interest; thereby
selectively targeting live cells at the site of interest with a
cell-penetrating polypeptide.
[0053] In an embodiment of the invention, the extracellular DNA
and/or its degradation product(s) is introduced or produced near or
around the live cells at about less than 20 mm from the cells at
the site of interest; less than 10 mm from cells at the site of
interest; a range of between 0.5 mm to 5 mm from the cells at the
site of interest; a range of between 0.5 mm to 20 mm from the cells
at the site of interest; range of between 0.5 mm to 0.1 mm from the
cells at the site of interest; range of less than 0.1 mm from the
cells at the site of interest; range of between 100 .mu.m to 10
.mu.m from the cells at the site of interest; or directly into the
site of interest (e.g., directly into the tumor mass or cancer). In
another embodiment, the extracellular DNA and/or its degradation
product(s) is introduced near or around the live cells in close
proximity to a target cell, tissue or injury.
[0054] The invention provides methods for selective targeting of
live cells, which have undergone or are undergoing radiation or
chemotherapy, at a site of interest with a cell-penetrating
polypeptide. In one embodiment of the invention, the method
comprises contacting the live cells with a cell-penetrating
polypeptide comprising cell-penetrating determinants so that the
cell-penetrating polypeptide binds extracellular DNA near or around
the live cells so as to form a complex or association therewith
such that the complex or associated polypeptide-DNA so bound binds
the live cells and penetrates the live cells thereby selectively
targeting live cells at a site of interest with a cell-penetrating
polypeptide. Examples of cell-penetrating polypeptides include
cell-penetrating antibodies such as 3E10, 5C5, 5C6 and 4H2
(Weisbart R H, et al., J. Immunol. 1990 144(7): 2653-2658; Zack D
J, et al., J. Immunol. 1995 154(4): 1987-1994; Weidle U H, et al.,
Cancer Genomics Proteomics 2013 10: 239-250; Weisbart R H, et al.,
Sci. Rep. 2015 5: 12022; Noble P W, et al., Sci. Rep. 2014 4:5958;
Colburn K K, et al., J. Rheumatol. 2003 30(5):993-7). Examples of
cell-penetrating determinants include, but are not limited to,
cell-penetrating determinants from antibodies such as 3E10, 5C5,
5C6 and 4H2.
[0055] In one embodiment, the site of interest is an injury site.
Examples of injury sites include but are not limited to an
intracranial injury, brain injury, heart injury (e.g., myocardial
infarction), skin injury, liver injury, gastrointestinal injury,
lung injury, eye injury, kidney injury, pancreas injury, peritoneal
injury, bone injury, nasopharyngeal injury, uterine injury,
cervical injury, breast injury, organ injury, tissue injury, burn
or radiation injury. An injury may include a cellular injury
involving tissue or organ injury. Examples of cellular injury
include any of chemical injury, excess reactive oxygen species,
burn, hypothermia, ischemia, hypoxia, blunt force trauma, stress,
heat shock, cold shock, hypothermia, mechanical stress, hypoxia,
ischemia, cellular swelling, DNA damage, DNA fragmentation,
membrane damage, organelle damage, damage due to heat, damage due
to cold, damage due to radiation, damage due to chemical exposure,
damage due to dehydration, mitochondrial damage, activation of
apoptotic pathway, damage due to an infection, damage due to
acidification, damage due to protein misfolding, damage due to
intracellular protein aggregation, damage due to laser, damage due
to aspiration, damage due to vacuum, damage due to cellular stress,
injury due to damage to cell membrane, damage due to changes in
osmotic pressure, or any cellular malfunction that results in cell
death or results in altered cell proliferation that is deleterious,
such as a cancer or other diseases or disorders referred to
herein.
[0056] Additionally, the invention provides method for selective
targeting of live cells at or near the proximity of a cellular
injury with a cell-penetrating polypeptide which comprises
cell-penetrating determinants which polypeptide is optionally
joined to, or combined with, a therapeutic agent. In one
embodiment, the method comprising administering the
cell-penetrating polypeptide at or near the proximity of the injury
so that it binds extracellular DNA from the cellular injury so as
to form a complex or association therewith such that the complex or
associated polypeptide-DNA-therapeutic agent so bound bind the live
cells and penetrates the live cells thereby selectively targeting
live cells at a site of interest with the cell-penetrating
polypeptide.
[0057] In one embodiment, introducing the cell-penetrating
polypeptide comprising cell-penetrating determinants before, after
or concurrently with the DNA of step (a) is at a site other than
near or around the live cells at the site of interest, such as the
introduction of the cell-penetrating polypeptide via an intravenous
injection to permit systemic circulation of the introduced
cell-penetrating polypeptide.
[0058] The invention also provides methods for inhibiting cellular
injury in a subject. In one embodiment the method comprises
administering directly to the live cells at or near a site of
cellular injury of the subject a cell-penetrating polypeptide
comprising cell-penetrating determinants joined to a therapeutic
agent. The method further comprises the step of contacting
extracellular DNA or its degradation product with the
cell-penetrating polypeptide so that the cell-penetrating
polypeptide binds extracellular DNA or its degradation product near
or around the live cells so as to form a complex. The live cell and
the complex come in contact so that the complex can penetrate the
live cell. Additional complexes are permitted to form and contact
additional cells at the site of injury so that that the complexes
penetrate additional live cells at the site of cellular injury;
thereby, inhibiting cellular injury in the subject.
[0059] The invention also provides methods for inhibiting a cell or
inducing cell death in a subject comprising administering directly
to the live cells at or near a site of injury of the subject a
cell-penetrating polypeptide comprising cell-penetrating
determinants which polypeptide is optionally joined to a
therapeutic agent. The method further comprises the step of
contacting extracellular DNA or its degradation product near or
around the live cells with the cell-penetrating polypeptide so that
the cell-penetrating polypeptide binds extracellular DNA or its
degradation product near or around the live cells thereby forming a
complex which can bind and penetrate the live cell which induces
cell death or inhibition. Additional complexes are permitted to
form and contact additional cells at the site of injury so that
that the complexes penetrate additional live cells live cells at
the site of injury thereby inducing cell death or inhibition at or
near a site of injury with the cell-penetrating polypeptide.
[0060] In one embodiment, "introducing or producing extracellular
DNA and/or its degradation product(s) near or around the live cells
at the site of interest" comprises administering DNA and/or its
degradation product(s). In one embodiment, DNA is administered into
the extracellular space or outside of a cell (e.g., not inside a
cell) by any method known in the art, including injection,
microinjection, microprojectile and implantation. In another
embodiment, "introducing, producing or permitting presence of
extracellular DNA and/or its degradation product(s) near or around
the live cells at the site of interest" comprises a man-made
intervention to produce localized cellular damage, such as through
radiation (e.g., low dose radiation), chemotherapeutic agent,
cytotoxic drug, toxin, hypoxia, blunt force trauma, hypothermia,
burn or an infectious agent, so as to cause cell death and release
of chromosomal DNA.
[0061] In one embodiment, the cell-penetrating polypeptide is
administered or introduced at a site of interest. In another
embodiment, the cell-penetrating polypeptide is administered or
introduced (e.g., directly administered or introduced) near or
around a site of interest. In an embodiment of the invention, a
site of interest is a site of injury, e.g., cellular injury. In
another embodiment, the cell penetrating polypeptide is
administered or introduced at a site of cellular injury. In another
embodiment, the cell-penetrating polypeptide is administered or
introduced at a site away from the injury. Merely by way of
example, the cell-penetrating polypeptide may be administered or
introduced in a subject by subcutaneous injection, intramuscular
injection or intravenous injection. In another example, the
cell-penetrating polypeptide is administered or introduced to a
subject wherein the cell-penetrating polypeptide circulates
systemically.
[0062] In one embodiment, the extracellular DNA is administered or
introduced at a site of interest. It may be coadministered with the
cell-penetrating polypeptide. Alternatively, the extracellular DNA
may be separately administered with the cell-penetrating
polypeptide, e.g., administered before or after the administration
of the cell-penetrating polypeptide. In one embodiment of the
invention, the extracellular DNA is administered or introduced at a
site of injury. In another embodiment, the extracellular DNA is
administered or introduced at a site of cellular injury. In one
embodiment, the extracellular DNA is administered or introduced
(e.g., directly administered or introduced) near or around the live
cells at a site of interest, e.g., a site of injury. In another
embodiment, extracellular DNA is administered or introduced near or
around the live cells at a site of cellular injury. In one
embodiment, the extracellular DNA may be produced in situ through
the use of cell damaging agents. In one embodiment, the
extracellular DNA is produced by the act of man at a site of
interest in a subject.
[0063] In another embodiment, the invention provides a method for
inhibiting cellular injury in a subject comprising: (a)
administering directly to the live cells at or near a site of
cellular injury of the subject a cell-penetrating polypeptide
comprising cell-penetrating determinants joined to a therapeutic
agent; (b) contacting extracellular DNA or its degradation product
with the cell-penetrating polypeptide so that the cell-penetrating
polypeptide binds extracellular DNA or its degradation product near
or around the live cells so as to form a complex; (c) contacting
one of the live cells with the complex in (b) so as to bind and
penetrate the live cell; and (d) permitting additional complexes to
form as in (b) and contacting additional cells with said complexes
so as to bind and penetrate additional live cells at the site of
cellular injury; thereby, inhibiting cellular injury in the
subject
[0064] In a further embodiment, the invention provides a method for
selective targeting of live cells at or near a site of cellular
injury with a cell-penetrating polypeptide which comprises
cell-penetrating determinants joined to a therapeutic agent, the
method comprising: (a) contacting the live cells with a composition
comprising (i) a cell-penetrating polypeptide which comprises
cell-penetrating determinants joined to a therapeutic agent and
(ii) extracellular DNA or its degradation product so that the
cell-penetrating polypeptide binds extracellular DNA or its
degradation product near or around the live cells so as to form a
complex such that the complex so formed binds one of the live cells
and penetrates the live cell; and (b) permitting additional
complexes to form as in (a) and contacting additional cells with
said complexes so as to bind and penetrate additional live cells at
the site of cellular injury, thereby selectively targeting live
cells at a site of cellular injury with a cell-penetrating
polypeptide.
[0065] In yet a further embodiment, the invention provides a method
for inducing cell death in a subject comprising: (a) administering
directly to the live cells at or near a site of injury of the
subject a cell-penetrating polypeptide comprising cell-penetrating
determinants joined to a therapeutic agent; (b) contacting
extracellular DNA or its degradation product near or around the
live cells with the cell-penetrating polypeptide so that the
cell-penetrating polypeptide binds extracellular DNA or its
degradation product near or around the live cells so as to form a
complex; (c) contacting one of the live cells with the complex in
(b) so as to bind and penetrate the live cell which induces cell
death; and (d) permitting additional complexes to form as in (b)
and contacting additional cells with said complexes so as to bind
and penetrate additional live cells inducing additional cell death
at the site of injury; thereby inducing cell death at or near a
site of injury with the cell-penetrating polypeptide.
[0066] In yet another embodiment, the methods of the invention
further provides the step of administering DNA and/or its
degradation product (s) to the injury site or to a site of interest
in an extracellular space to facilitate further selective
targeting. In one embodiment, the introduced or administered DNA is
a double-stranded DNA. In another embodiment, the introduced or
administered DNA is a single-stranded DNA. In one embodiment, the
introduced or administered DNA is isolated or purified DNA isolated
from a cell (e.g., DNA from a cell lysate or purified from a cell),
a virus or a bacteriophage. In an embodiment of the invention, the
introduced or administered DNA may have modified bases (e.g.,
5-methyl-cytosine). In an embodiment of the invention, the
introduced or administered DNA is synthesized DNA such as a
chemically synthesized oligonucleotide with or without modified
bases. In an embodiment of the invention, the DNA so introduced or
administered is not purified away from non-DNA nucleic acid (e.g.,
RNA), nucleotide, nucleoside or purine or pyrimidine base. In an
embodiment of the invention, the DNA so introduced or administered
is purified away from non-DNA nucleic acid, nucleotide, nucleoside
or purine or pyrimidine base.
[0067] In an embodiment of the invention, the administered DNA is a
polymer of thymidine monophosphate (dTMP) or poly (dT). In a
further embodiment, the administered DNA comprises a thymidine or
dT or thymine-deoxyribose. For example, in one embodiment a 3E10
antibody or fragment thereof has a higher binding affinity to
single stranded DNA comprising a dT in the DNA sequence. As a
further example, the single stranded DNA so introduced or
administered comprises a poly (dT) sequence.
[0068] The DNA may be single-stranded DNA having a length of about
40,000 bases to about 2 bases or dinucleotide. In a further
embodiment, the DNA may have a length of about more than 1,000
bases. In another embodiment, the DNA may have a length of about
more than 2000 bases. In yet another embodiment, the DNA may have a
length of about less than 2000 bases. In another embodiment, the
DNA may have a length of about 50 bases to 500 bases. In yet
another embodiment, the DNA may have a length of about less than
100 bases.
[0069] The DNA may be double stranded DNA having a length of about
less than 20 kilobase pairs (kb pairs) to about 5 bp. In one
embodiment, the DNA may have a length of about less than 2000 base
pair (bp). In one embodiment, the DNA may have a length of about
more than 2000 base pair (bp). In another embodiment, the DNA may
have a length of about 50 bp to 500 bp. In yet another embodiment,
the DNA may have a length of about less than 100 bp. In one
embodiment, the DNA is purified calf thymus double stranded DNA
sheared to an average length of 2000 bp.
[0070] In one embodiment, the DNA is non-infectious DNA (e.g.,
complete viral genome) or enzymatic DNA (e.g., DNAzyme).
Preferably, the DNA is non-infectious DNA.
[0071] In one embodiment, the DNA is partially degraded DNA in the
DNA-antibody complex or DNA-cell-penetrating polypeptide complex
before cell penetration by the antibody or cell-penetrating
polypeptide.
[0072] In one embodiment, the DNA and/or its degradation product(s)
may be single-stranded, double-stranded, triple-stranded, or
four-stranded or a combination thereof.
[0073] In an embodiment of the invention, the dose of the DNA
and/or its degradation product(s) so introduced or administered may
be between about 100 .mu.g to 1 .mu.g, between about 1 .mu.g to 100
ng DNA, between about 100 ng to 10 ng DNA, between about 10 ng to 1
ng DNA, between about 1 ng to 100 pg DNA, between about 100 .mu.g
to 10 pg DNA, or between 10 pg to 1 pg DNA. In one embodiment, the
dose is less than 1 ng DNA. In yet a further embodiment, the dose
may be may be less than about 100 pg DNA. Factors to be considered
in choice of actual dose include condition and tissue/organ being
treated, number of cells at the site of interest to be treated,
size of the tumor, size of the injury or ischemia, diffusibility of
DNA and/or its degradation product(s) from the site of interest,
tumor, injury site or ischemic site, and volume of the
extracellular space at and around the site of interest. Doses will
need to be adjusted based on the desired outcome. Multiple doses
may need to be introduced or administered.
[0074] In one embodiment, the dose of the DNA and/or its
degradation product(s) is DNA. In another embodiment, the dose of
the DNA and/or its degradation product(s) is its degradation
product(s). In one embodiment, the DNA degradation product(s) may
be directly obtained from degradation DNA. In another embodiment,
the DNA degradation product(s) may be equivalent to the degradation
product(s) directly produced from the DNA. In another embodiment,
the DNA degradation product(s) nucleotide, nucleoside, pyrimidine
base and/or purine base. In another embodiment, the DNA degradation
product(s) is produced in situ from the introduced or administered
DNA. In yet another embodiment, the dose of the DNA and/or its
degradation product(s) is or comprises both DNA and its degradation
product(s).
[0075] The DNA and/or its degradation product(s) introduced or
administered may be in a liquid formulation or a solid formulation.
In one embodiment of a liquid formulation, the liquid formulation
comprises DNA and/or its degradation product(s) and an aqueous
carrier, preferably isotonic, at a DNA and/or its degradation
product(s) concentration of less than 20 mg/ml, a DNA and/or its
degradation product(s) concentration between 10 mg/ml to 1 mg/ml, a
DNA concentration between 1 mg/ml to 100 .mu.g/ml, a DNA and/or its
degradation product(s) concentration between 100 .mu.g/ml to 10
.mu.g/ml, a DNA and/or its degradation product(s) concentration
between 10 .mu.g/ml to 1 .mu.g/ml, a DNA and/or its degradation
product(s) concentration between 1 .mu.g/ml to 100 ng/ml, a DNA
and/or its degradation product(s) concentration between 100 ng/ml
to 10 ng/ml, or a DNA and/or its degradation product(s)
concentration between 10 ng/ml to 1 ng/ml. In one embodiment, the
DNA and/or its degradation product(s) concentration is less than 1
.mu.g/ml. In one embodiment, the DNA and/or its degradation
product(s) concentration is greater than 1 .mu.g/ml. In a solid
formation, the DNA and/or its degradation product(s) may be in the
form of free acid or preferably in a salt. In one embodiment, the
solid formulation is an immediate-release formulation comprising
DNA and/or its degradation product(s) and a suitable carrier. In
one embodiment, the solid formulation is a sustained-release
formulation comprising DNA and/or its degradation product(s) and a
suitable carrier. In one embodiment, the solid formulation is a
combination of an immediate-release formulation and
sustained-release formulation.
[0076] In one embodiment, the liquid formulation comprises a
cell-penetrating polypeptide and DNA and/or its degradation
product(s). In one embodiment, the solid formulation comprises a
cell-penetrating polypeptide and DNA and/or its degradation
product(s). In a further embodiment, the cell-penetrating
polypeptide and DNA is in a cell-penetrating polypeptide-DNA
complex or association. In another embodiment, the cell-penetrating
polypeptide and DNA degradation product(s) is in a cell-penetrating
polypeptide-DNA degradation product complex or association.
[0077] For example, in one embodiment, a site of interest is an
injury site that may be created through the use of a cell-damaging
agent. Examples of cell-damaging agents include, but are not
limited to, a radioisotope, cytotoxic agent or radiation. Suitable
examples of radioisotope include .sup.46Sc, .sup.47Sc, .sup.48Sc,
.sup.67Cu, .sup.67Ga, .sup.72Ga, .sup.73Ga, .sup.90Y, .sup.67Cu,
.sup.109Pd, .sup.111Ag, .sup.111In, .sup.125I, .sup.131I,
.sup.149Pm, .sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.186Re,
.sup.188Re, .sup.211At, .sup.211Bi, .sup.212Bi, .sup.212Pb,
.sup.213Bi, .sup.214Bi and .sup.225Ac. Suitable cytotoxic agents
include ricin, ricin A-chain, doxorubicin, daunorubicin,
paclitaxel, taxol, ethidium bromide, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicine, dihydroxy
anthracenedione, actinomycin D, diphtheria toxin, Pseudomonas
exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain,
alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin,
enomycin, curcin, crotin, calicheamicin, Saponaria officinalis
inhibitor, maytansinoids, and glucocorticoid. Examples of radiation
include microwave, infrared, ultraviolet, X-ray, gamma ray, alpha
particle radiation, beta ray and ionizing radiation.
[0078] In one embodiment, the injury site is created through the
use of a cell-damaging agent wherein the cell-damaging agent is a
DNA damaging agent. In one embodiment, the DNA damaging agent is
doxorubicin. In one embodiment, the injury site is created through
the use of a cell-damaging agent wherein the cell damaging agent is
a DNA damaging agent other than doxorubicin. In one embodiment, the
injury site is created through the use of a cell-damaging agent
other than a DNA-damaging agent. In one embodiment, the injury site
is created through the use of a cell-damaging agent other than a
DNA-damaging agent is taxol.
[0079] In one embodiment, the injury site is created through the
use of radiation. Examples of radiation include microwave,
infrared, ultraviolet, X-ray, gamma ray, alpha particle radiation,
beta ray and ionizing radiation. In one embodiment, the injury site
is created through means other than through the use of radiation.
Examples of radiation include microwave, infrared, ultraviolet,
X-ray, gamma ray, alpha particle radiation, beta ray and ionizing
radiation.
[0080] In one embodiment, the site of interest comprises
extracellular DNA. In another embodiment, the site of interest is
devoid or substantially devoid of extracellular DNA. In one
embodiment, the site of interest is a site within an in vitro cell
or organ culture. In one embodiment, the site of interest is an in
vivo site. In one embodiment, the site of interest is a site within
a subject or a mammal.
[0081] The invention further provides methods for selective
targeting of live cells at or near the proximity of a cellular
injury with a cell-penetrating polypeptide. In an embodiment of the
invention, the cell-penetrating determinants are joined to, or
combined with (e.g., as an admix), a therapeutic agent. The method
may comprise contacting the live cells with a composition having
(a) a cell-penetrating polypeptide which comprises cell-penetrating
determinants and (b) extracellular DNA so that the cell-penetrating
polypeptide binds extracellular DNA near or around the live cells
so as to form a complex or association therewith such that the
complex or associated polypeptide-DNA so bound bind the live cells
and penetrate the live cells thereby selectively targeting live
cells at a site of interest with the cell-penetrating
polypeptide.
[0082] The invention also provides method for inhibiting a tumor
associated with ischemia, cellular/tissue necrosis or
cellular/tissue apoptosis by selective targeting of live cells at a
site of interest by any of the methods of the invention.
[0083] Examples of tumors include colorectal cancer, osteosarcoma,
non-small cell lung cancer, breast cancer, ovarian cancer, glial
cancer, solid tumors, metastatic tumor, acute lymphoblastic
leukemia, acute myelogenous leukemia, adrenocortical carcinoma,
Kaposi sarcoma, lymphoma, anal cancer, astrocytomas, basal cell
carcinoma, bile duct cancer, bladder cancer, bone cancer, brain
tumor, breast cancer, bronchial tumor, cervical cancer, chronic
lymphocytic leukemia, chronic myelogenous leukemia, chronic
myeloproliferative disorders, colon cancer, colorectal cancers,
ductal carcinoma in situ, endometrial cancer, esophageal cancer,
eye cancer, intraocular, retinoblastoma, metastatic melanoma,
gallbladder cancer, gastric cancer, gastrointestinal carcinoid
tumor, gastrointestinal stromal tumors, glioblastoma, glioma, hairy
cell leukemia, head and neck cancer, hepatocellular carcinoma,
hepatoma, Hodgkin lymphoma, hypopharyngeal cancer, Langerhans cell
histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver
cancer, lobular carcinoma in situ, lung cancer, non-small cell lung
cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma,
Burkitt lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma,
melanoma, squamous neck cancer, mouth cancer, multiple myeloma,
myelodysplastic syndromes, myelodysplastic/myeloproliferative
neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal
cancer, neuroblastoma, oral cavity cancer, oropharyngeal cancer,
osteosarcoma, ovarian cancer, pancreatic carcinoma, papillary
carcinomas, parathyroid cancer, penile cancer, pharyngeal cancer,
pheochromocytoma, pineal parenchymal tumors, pineoblastoma,
pituitary tumor, pleuropulmonary blastoma, primary central nervous
system lymphoma, prostate cancer, rectal cancer, renal cell cancer,
salivary gland cancer, sarcoma, Ewing sarcoma, soft tissue sarcoma,
squamous cell carcinoma, Sezary syndrome, skin cancer, Merkel cell
carcinoma, testicular cancer, throat cancer, thymoma, thymic
carcinoma, thyroid cancer, urethral cancer, endometrial cancer,
uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
Waldenstrom macroglobulinemia, and Wilms tumor. In one embodiment,
the tumor is a glioma. In one embodiment, the tumor is a tumor
other than a glioma.
[0084] In one embodiment, the tumor is associated with an
amplification or over activity of an oncogene. In one embodiment,
the tumor is associated with a loss or under activity of a tumor
suppressor gene. In one embodiment, the tumor suppressor gene is
BRCA2. In one embodiment, the tumor is associated with a loss or
under activity of a tumor suppressor gene, other than BRCA2. In one
embodiment, the tumor is associated with amplification or over
activity of an oncogene and loss or under activity of a tumor
suppressor gene. In one embodiment, the tumor is associated with a
loss or mutation of a gene for a DNA repair enzyme. In one
embodiment, the gene for a DNA repair enzyme is RAD51 or its
homolog. In one embodiment, the tumor is free of a loss or mutation
of a gene for a DNA repair enzyme. In one embodiment, the tumor is
free of a mutation in the RAD51 gene or its homolog.
[0085] The invention provides methods for increasing uptake and
enhancing efficacy of a cell-penetrating polypeptide comprising an
anti-DNA antibody or a derivative or variant thereof in targeting
tumor or cancer cells comprising (a) inducing additional
extracellular DNA release at or near the tumor or cancer cells
through the use of a cell-damaging agent or introducing additional
extracellular DNA or artificial DNA at or near the tumor or cancer
cells, (b) administering the cell-penetrating polypeptide, (c)
allowing the cell-penetrating polypeptide to form additional
complexes with the additional extracellular DNA or artificial DNA,
and (d) permitting the additional complexes in (c) to contact the
tumor or cancer cells, thereby increasing uptake and enhancing
efficacy of a cell-penetrating polypeptide comprising an anti-DNA
antibody or a derivative or variant thereof in targeting tumor or
cancer cells.
[0086] Also, the invention provides methods for enhancing the
effects of chemotherapy or radiation therapy by selectively
targeting live cells by any of the methods of the invention.
[0087] In accordance with the practice of the invention, any of the
methods of the invention may be an adjunct therapy to a
chemotherapy or a radiation therapy. The chemotherapy or the
radiation therapy may be administered concurrently or before
selectively targeting the live cells by any of the methods of the
invention.
[0088] The invention further provides method for protecting cells
(cytoprotection) from a disease or disorder associated with a
hydrogen peroxide toxicity or reactive oxygen species (ROS)
toxicity by selective targeting of live cells at the injury site by
any of the methods of the invention. The disease or disorder may be
a brain injury, heart injury, skin injury, or radiation injury and
may be an acute injury. Examples of brain injury include but are
not limited to brain trauma, spinal cord injury, peripheral nerve
injury, or stroke. A heart injury may include but not limited to a
myocardial infarction. Examples of skin injury include but are not
limited to wound, burn, or decubitus ulcer. A radiation injury may
include but not limited to burn or poison. In another embodiment,
the disease or disorder may be acute renal failure, acute organ
failure, liver injury, bowel infarction, peripheral vascular
disease, pulmonary failure, or a cancer.
[0089] The cell-penetrating polypeptide may be a therapeutic agent.
In one embodiment, the therapeutic agent may be the anti-DNA
antibody of the invention, e.g., 3E10 mAb or its fragment or its
variant, free of any other pharmaceutically active agent. In
another embodiment, the cell-penetrating polypeptide may comprise a
therapeutic agent linked or coupled to an anti-DNA antibody of the
invention. The therapeutic agent linked or coupled to an anti-DNA
antibody of the invention may be a cytotoxic agent, a
cytoprotective agent, or another antibody or its fragment with an
intracellular-binding determinant. The intracellular-binding
determinant may be to an oncoprotein, tumor suppressor gene, a
transcription factor, a cell signaling molecule, a nuclear
receptor, a steroid receptor, a cell signaling molecule, a protein
kinase, a phosphatase, an acetylase, a ligase, a methylase, a
protease, an enzyme, a shuttling protein, a nuclear protein, a
nucleolar protein, transcription components, a soluble protein, a
cytoskeletal protein and a membrane protein.
[0090] Additionally, the cell-penetrating polypeptide may be an
anti-DNA antibody (e.g., polyclonal, monoclonal, chimeric,
bispecific and humanized antibodies). In one embodiment, the
anti-DNA antibody binds DNA (including single stranded or double
stranded DNA). Examples of suitable anti-DNA antibodies include,
but are not limited to, 3E10 antibody, H7 antibody, H9 antibody,
H72 antibody, H205 antibody, H317 antibody F14-6 antibody, SN22
antibody, SN50 antibody, SN111 antibody, SN112 antibody, SN575
antibody, SN604 antibody, SN608 antibody, F4.1 antibody, J20.8
antibody, F14.6 antibody, and 9D7 antibody or a derivative or
variant thereof (Vlahakos D, Foster M H, Ucci A A, Barrett K J,
Datta S K, and Madaio M P (1992) "Murine monoclonal anti-DNA
antibodies penetrate cells, bind to nuclei, and induce glomerular
proliferation and proteinuria in vivo." J. Am. Soc. Nephrol.
2(8):1345-54; Ruiz-Arguelles A, Perez-Romano B, Llorente L,
Alarcon-Segovia D, and Castellanos J M (1998) "Penetration of
anti-DNA antibodies into immature live cells." J. Autoimmun.
11(5):547-56; Avrameas A, Ternynck T, Nato F, Buttin G, and
Avrameas S (1998) "Polyreactive anti-DNA monoclonal antibodies and
a derived peptide as vectors for the intracytoplasmic and
intranuclear translocation of macromolecules" Proc. Natl. Acad.
Sci. U.S.A. 95(10):5601-5606; Song Y C, Sun G H, Lee T P, Huang J
C, Yu C L, Chen C H, Tang S J, and Sun K H (2008) "Arginines in the
CDR of anti-dsDNA autoantibodies facilitate cell internalization
via electrostatic interactions." Eur. J. Immunol. 38(143178-90).
Sequence of CDR for F4.1 antibody, J20.8 antibody and F14.6
antibody may be found in Avrameas A, Ternynck T, Nato F, Buttin G,
and Avrameas S (1998) "Polyreactive anti-DNA monoclonal antibodies
and a derived peptide as vectors for the intracytoplasmic and
intranuclear translocation of macromolecules" Proc. Natl. Acad.
Sci. U.S. A. 95(10):5601-5606. Sequence of CDR for 9D7 antibody may
be found in Song Y C, Sun G H, Lee T P, Huang J C, Yu C L, Chen C
H, Tang S J, and Sun K H (2008) "Arginines in the CDR of anti-dsDNA
autoantibodies facilitate cell internalization via electrostatic
interactions." Eur. J. Immunol. 38(11):3178-90. In one embodiment,
the anti-DNA antibody is an isolated monoclonal antibody or a
derivative or variant thereof. In an embodiment, the isolated
monoclonal antibody or a derivative or variant thereof is taken up
by live cells in the presence of extracellular nucleic acid, DNA or
artificial DNA. In an embodiment, the isolated monoclonal antibody
or a derivative or variant thereof allows for targeted uptake of
cells with an increased concentration of extracellular nucleic
acid, DNA or artificial DNA. In an embodiment, the cells targeted
for uptake of the isolated monoclonal antibody or a derivative or
variant are diseased or tumor cells. In an embodiment, the isolated
monoclonal antibody or a derivative or variant is targeted to a
live cell or any cell within a group of live cells at a site within
an animal or human by targeted delivery of a cytotoxic agent or
radiation to induce limited cell death and subsequent release of
DNA around or near the live cell or around, near or in a group of
live cells. In one embodiment, the isolated monoclonal antibody or
a derivative or variant is targeted to a live cell or any cell
within a group of live cells at a site within an animal or human by
targeted delivery of a nucleic acid, DNA or artificial DNA. In one
embodiment, such targeted delivery of a nucleic acid, DNA or
artificial DNA may be achieved by an implant or transplant of an
object, substance, cells or cell-based material containing nucleic
acid, DNA or artificial DNA. The implant or transplant may release
nucleic acid, DNA or artificial DNA in a single burst or over an
extended period. In a preferred embodiment, the anti-DNA antibody
is an isolated monoclonal antibody 3E10 as produced by a hybridoma
having ATCC accession number PTA 2439 or a derivative or variant
thereof. In one embodiment, the variant has a change in the amino
acid sequence of a 3E10 scFv, wherein the change in the amino acid
sequence does not abolish or prevent cell penetration.
[0091] In one embodiment, the cell-penetrating polypeptide is a
3E10 bispecific antibody having an Fv fragment with the
cell-penetrating determinant which is a 3E10 Fv and a second Fv
fragment with an intracellular target-binding determinant which is
a 3G5 Fv.
[0092] In another embodiment, the bispecific antibody comprises an
Fv fragment with a cell-penetrating determinant of anti-DNA
monoclonal antibody 3E10 and a second Fv fragment with an
intracellular target-binding determinant for MDM2.
[0093] In one embodiment, the cell-penetrating polypeptide is a
3E10 bispecific antibody having an Fv fragment with the
cell-penetrating determinant which is a 3E10 Fv and a second Fv
fragment with an intracellular target-binding determinant which is
a PAb421 Fv.
[0094] In another embodiment, the bispecific antibody comprises an
Fv fragment with a cell-penetrating determinant of anti-DNA
monoclonal antibody 3E10 and a second Fv fragment with an
intracellular target-binding determinant for p53.
[0095] Examples of 3E10 bispecific antibodies, e.g. 3E10 scFv and
3G5 scFv as well 3E10 scFv and PAb421 scFv, are disclosed in U.S.
Ser. No. 13/844,318, filed Mar. 15, 2013, which is incorporated by
reference herein.
[0096] The 3E10 bispecific antibodies of the invention may further
comprise one or more amino acid sequence comprising Ala-Gly-Ile-His
(AGIH) at the amino terminus of one or both of the Fv regions.
Cell-penetrating polypeptides may further comprise one or more
amino acid sequence comprising Ala-Gly-Ile-His (AGIH) at the amino
terminus.
[0097] In another embodiment, the cell-penetrating polypeptide
comprises a 3E10 Fv attached to a heat shock protein (Hsp).
Examples of heat shock proteins include but are not limited to,
human Hsp-70 (Hunt and Morimoto PNAS Vol, 82, pp. 64-55-6459,
Figures 2 and 3); HspA (e.g., HspA1A, H5pA1B, HspA1L, HspA2, HspA5,
HspA6, HspA7, HspA8, HspA9, HspA12A, HspA12B, HspA13, HspA14); HspH
(e.g., HspH1, HspH2, HspH3, and HspH4); Hsp40 (e.g., DnaJA (e.g.
DNAJA1, DNAJA2, DNAJA3, and DNAJA4), DnaJB (e.g., DNAJB1, DNAJB2,
DNAJB3, DNAJB4, DNAJB5, DNAJB6, DNAJB7, DNAJB8, DNAJB9, DNAJB11,
DNAJB12, DNAJB13, and DNAJB14), DnaJC (e.g., DNAJC1, DNAJC2,
DNAJC3, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC7, DNAJC8, DNAJC9,
DNAJC10, DNAJC11, DNAJC12, DNAJC13, DNAJC14, DNAJC15, DNAJC16,
DNAJC17, DNAJC18, DNAJC19, DNAJC20, DNAJC21, DNAJC22, DNAJC23,
DNAJC24, DNAJC25, DNAJC26, DNAJC27, DNAJC28, and DNAJC30) and HSPB
(HSPB1, HSPB2, HSPB3, HSPB4, HSPB5, HSPB6, HSPB7, HSPB8, HSPB9,
HSPB10 and HSPB11) (Kampinga et al., Cell Stress and Chaperones
(2009) 14:105-111).
[0098] In a further embodiment, the cell-penetrating polypeptide is
a fusion protein comprising a 3E10 Fv joined to a Hsp-70 or portion
thereof, and optionally, the 3E10 Fv comprising an amino acid
sequence AGIH at its amino terminus. Examples of 3E10 fusion
proteins are disclosed in U.S. Ser. No. 13/815,829, filed Mar. 15,
2013, which is incorporated by reference herein.
[0099] In yet another embodiment, the cell-penetrating polypeptide
is a fusion protein comprising a 3E10 Fv joined to Hsp-27 or
portion thereof, and optionally, the 3E10 Fv comprising an amino
acid sequence AGIH at its amino terminus.
[0100] The invention also provides a further embodiment, wherein
the cell-penetrating polypeptide is a fusion protein comprising a
3E10 Fv attached or joined to a Hsp-90 or portion thereof, and
optionally, the 3E10 Fv comprising an amino acid sequence AGIH at
its amino terminus.
[0101] Additionally, the invention also provides an embodiment,
wherein the cell-penetrating polypeptide is a fusion protein
comprising a 3E10 Fv attached or joined to GRP78 or portion
thereof, and optionally, the 3E10 Fv comprising an amino acid
sequence AGIH at its amino terminus.
[0102] The invention also provides a further embodiment, wherein
the cell-penetrating polypeptide is a fusion protein comprising a
3E10 Fv attached or joined to an E3 ubiquitin-protein ligase, a
tumor suppressor-interacting protein, a binding partner of a tumor
suppressor protein, an oncoprotein, or a DNA repair protein or
portion thereof, and optionally, the 3E10 Fv comprising an amino
acid sequence AGIH at its amino terminus. Fusion proteins may be
produced by recombinant DNA methods in which coding sequences
isolated from at least two different sources are assembled in a
single nucleic acid molecule so as to allow the production of a
single polypeptide for the fusion protein, also called chimeric
protein.
[0103] The invention also provides a further embodiment, wherein
the cell-penetrating polypeptide is a fusion protein comprising a
3E10 Fv attached or joined to a transcription factor, a
transcriptional repressor, a transcriptional co-factor, a nuclear
receptor, a steroid receptor, a methylase, an acetylase, a
deacetylase, RNA polymerase, a kinase, a phosphatase, an
intracellular signaling molecule (not a cell surface signaling
molecule), a cell cycle regulatory protein, a protease, a DNA
repair protein, a recombinase, a chromosomal protein, an apoptotic
protein, a SUMO ligase, a ubiquitin ligase, a metabolic protein, an
organelle protein, a nuclear protein, a nucleolar protein, a
mitochondrial protein, a ligand, a ribosomal protein, an enzyme, a
cytoskeletal protein, a chromosomal protein, a structural protein,
a intracellular soluble protein, an intracellular shuttling protein
or a regulatory protein or portion thereof, so long as the second
determinant fails to recognize any protein that normally resides on
the cell surface. In a further embodiment, the fusion protein
comprises an amino acid sequence AGIH at its amino terminus.
[0104] In one embodiment, the cell-penetrating polypeptide is a
fusion protein comprising a 3E10 Fv attached or joined to a nuclear
transcription factor, or portion thereof, that is both a tumor
suppressor factor and regulatory of T-regulatory cell. In one
embodiment, the nuclear transcription factor that is both a tumor
suppressor factor and regulatory of T-regulatory cell is Foxp3. In
one embodiment, the cell-penetrating polypeptide is a fusion
protein comprising a 3E10 Fv attached or joined to Foxp3 or portion
thereof. In one embodiment, the cell-penetrating polypeptide is a
fusion protein comprising a 3E10 Fv attached or joined to Foxp3 or
portion thereof as described in Heinze E, et al., Oncol Lett. 2011
2(4): 665-668.
[0105] In one embodiment, the cell-penetrating polypeptide is a
fusion protein comprising a 3E10 Fv attached or joined to a tumor
suppressor or portion thereof. In one embodiment, the tumor
suppressor factor is a p53 tumor suppressor protein. In one
embodiment, the cell-penetrating polypeptide is a fusion protein
comprising a 3E10 Fv attached or joined to p53 tumor suppressor or
portion thereof. In one embodiment, the cell-penetrating
polypeptide is a fusion protein comprising a 3E10 Fv attached or
joined to p53 tumor suppressor or portion thereof as described in
Weisbart R H, et al., Cancer Lett. 2003 195(2):211-9.
[0106] In yet a further embodiment, the cell-penetrating
polypeptide is a 3E10 Fv attached to an Hsp-70 or portion thereof,
the 3E10 Fv comprising an amino acid sequence AGIH at its amino
terminus.
[0107] In an additional embodiment, the cell-penetrating
polypeptide is a 3E10 Fv attached to Hsp-27 or portion thereof, the
3E10 Fv comprising an amino acid sequence AGIH at its amino
terminus.
[0108] The invention also provides an embodiment, wherein the
cell-penetrating polypeptide is a 3E10 Fv attached to a Hsp-90 or
portion thereof, the 3E10 Fv comprising an amino acid sequence AGIH
at its amino terminus.
[0109] In an additional embodiment, the cell-penetrating
polypeptide is a 3E10 Fv attached to glucose regulated protein 78
kDa (GRP78) or portion thereof, the 3E10 Fv comprising an amino
acid sequence AGIH at its amino terminus.
[0110] For example, chimeric antibodies of the invention may be
immunoglobulin molecules that comprise a human and non-human
portion. The antigen combining region (variable region) of a
chimeric antibody can be derived from a non-human source (e.g.
murine) and the constant region of the chimeric antibody which
confers biological effector function to the immunoglobulin can be
derived from a human source. The chimeric antibody should have the
antigen binding specificity of the non-human antibody molecule and
the effector function conferred by the human antibody molecule.
[0111] In general, the procedures used to produce chimeric
antibodies can involve the following steps: [0112] a) identifying
and cloning the correct gene segment encoding the antigen binding
portion of the antibody molecule; this gene segment (known as the
VDJ, variable, diversity and joining regions for heavy chains or
VJ, variable, joining regions for light chains or simply as the V
or variable region) may be in either the cDNA or genomic form;
[0113] b) cloning the gene segments encoding the constant region or
desired part thereof; [0114] c) ligating the variable region with
the constant region so that the complete chimeric antibody is
encoded in a form that can be transcribed and translated; [0115] d)
ligating this construct into a vector containing a selectable
marker and gene control regions such as promoters, enhancers and
poly(A) addition signals; [0116] e) amplifying this construct in
bacteria; [0117] f) introducing this DNA into eukaryotic cells
(transfection) most often mammalian lymphocytes; [0118] g)
selecting for cells expressing the selectable marker; [0119] h)
screening for cells expressing the desired chimeric antibody; and
[0120] i) testing the antibody for appropriate binding specificity
and effector functions.
[0121] A chimeric antibody may include a "humanized" antibody in
which one or more of the complementary-determining region (CDR)
from the variable region of a non-human antibody (such as a mouse
monoclonal antibody) may be used to replace the corresponding CDR
in a human antibody, such that the resulting chimeric antibody has
the framework region of a human antibody and one or more CDR of a
non-human antibody. The chimeric or humanized antibody may be
produced by recombinant DNA methods and may be a whole antibody, an
antibody fragment, a bi-specific antibody, a single chain Fv
antibody or combinations thereof.
[0122] Antibodies of several distinct antigen binding specificities
have been manipulated by these protocols to produce chimeric
proteins [e.g. anti-TNP: Boulianne et al., Nature 312:643 (1984);
and anti-tumor antigens: Sahagan et al., J. Immunol. 137:1066
(1986)]. Likewise, several different effector functions have been
achieved by linking new sequences to those encoding the antigen
binding region. Some of these include enzymes [Neuberger et al.,
Nature 312:604 (1984)], immunoglobulin constant regions from
another species and constant regions of another immunoglobulin
chain [Sharon et al., Nature 309:364 (1984); Tan et al., J.
Immunol. 135:3565-3567 (1985)]. Additionally, procedures for
modifying antibody molecules and for producing chimeric antibody
molecules using homologous recombination to target gene
modification have been described (Fell et al., Proc. Natl. Acad.
Sci. USA 86:8507-8511 (1989)).
[0123] In accordance with the practice of the invention, the DNA
(e.g., extracellular DNA) may be a single-stranded,
double-stranded, triple-stranded, or four-stranded or a combination
thereof. Further, the extracellular DNA may comprise a
phosphodiester bond, a phosphorothioate bond or a methylphosphonate
bond or a combination thereof. For example, the DNA (e.g.,
extracellular DNA) may comprise a 5'-to-3' linkage, an inverted
5'-to-5' linkage or an inverted 3'-to-3' linkage or a combination
thereof. The DNA may be isolated from nature or synthesized in a
laboratory.
[0124] In one embodiment, the degradation product of DNA may be
bound by the anti-DNA antibody of the invention. DNA degradation
products include lower molecular weight DNAs, which may be
single-stranded, double-stranded, triple-stranded, or four-stranded
or a combination thereof. Further, DNA degradation products include
nucleotides, nucleosides and nucleobases. In one embodiment of the
invention, the DNA degradation product includes a thymine base,
thymidine or a thymidine monophosphate (dTMP). In one embodiment,
the thymidine monophosphate may be a 3' dTMP, 5' dTMP or cyclic 3',
5' dTMP.
[0125] In one embodiment of the invention, the DNA degradation
product comprises a guanine base, deoxyguanosine or a
deoxyguanosine monophosphate (dGMP) or a combination thereof. In
one embodiment, the dGMP may be a 3' dGMP, 5' dGMP or cyclic 3', 5'
dGMP.
[0126] Further, in one embodiment of the invention, the DNA (e.g.,
extracellular DNA) comprises an artificial DNA. For example, the
artificial DNA may comprise a DNA mimetic. In one embodiment, the
DNA mimetic comprises a pseudopeptide backbone. Merely as examples,
the pseudopeptide backbone may comprise any of an ethylglycine, a
propylglycine, an ethyl-.beta.-alanine, a propionyl linker, a retro
inverso linker, a (S,S)-cyclohexyl linker, a (R,R)-cyclohexyl
linker, an L-ornithine, a 2-me-ethyl-glycine, an ethyl-lysine, a
L-proline, a n-proline, a glycine backbone/ethyl linker, a
L-4-trans-amino proline, a L-4-cis-amino proline, a D-4-trans-amino
proline, a .beta.-alanine/proline, a glycylglycine/ethyl linker, a
glycine/ethyl linker, a proline-glycine, a .beta.-amino-alanine,
E-OPA, Z-OPA, APNA, a serinol-ethyl-methyl linker, a
serinol-ethyl-ethyl linker, an .alpha.-methyl-serinol-ethyl-ethyl
linker, an aminopentan, a hydroxyethyl phosphono glycine, an
aminoethyl phosphono glycine, a lysine, an aminoethyl prolyl or a
serinyl methylene or combination thereof.
[0127] Further in accordance with the practice of the invention,
the cell-penetrating polypeptide may be whole antibodies or
derivatives thereof (e.g., fragments thereof (e.g., Fv, Fab',
F(ab').sub.2) or recombinant proteins including recombinant
variable regions of immunoglobulin molecules (e.g., scFv,
bispecific antibody with scFv fragments)) containing the antigen
binding domain and/or one or more complement determining regions of
these antibodies that penetrate or are internalized into the cell
upon or after binding. These cell-penetrating polypeptides can be
from any source, e.g., rat, dog, cat, pig, horse, mouse or human.
It is intended that the term "penetrate" or "internalize" means
that the cell-penetrating polypeptide is taken into the cell.
Further, some of the antibodies induce inhibition of cancer cell
growth. The cell-penetrating polypeptide may be conjugated to a
therapeutic agent.
[0128] In accordance with the practice of the invention, an
antibody fragment includes at least a portion of the variable
region of the immunoglobulin molecule that binds to its target,
i.e., the antigen binding region. Some of the constant region of
the immunoglobulin may also be included.
[0129] In an embodiment of the invention, cell penetration is
dependent on a salvage pathway. For example, the nucleoside salvage
pathway may be a pathway mediated by equilibrative nucleoside
transporters (ENTs) or SLC29 family of integral membrane proteins.
Examples of an equilibrative nucleoside transporter (ENT) or a
member of the SLC29 family of integral membrane proteins is a
transporter for purine and pyrimidine nucleosides and nucleobases
or a metabolite thereof. The transporter for purine and pyrimidine
nucleosides and nucleobases or a metabolite thereof may be an
equilibrative nucleoside transporter ENT2.
[0130] In an embodiment of the invention, cell penetration is
dependent on binding to a nucleic acid. In an embodiment of the
invention, cell penetration is dependent on binding to DNA. In an
embodiment of the invention, cell penetration is dependent on
binding to an artificial DNA.
[0131] In an embodiment of the invention, cell penetration is
mediated by an anti-DNA antibody, antibody fragment or derivative
or variant which binds DNA and a cell surface molecule. In the case
of the cell surface molecule, binding by the anti-DNA antibody,
antibody fragment or derivative or variant to a cell surface
molecule may either be direct or indirect binding. An example of a
cell surface molecule is a cell surface polypeptide, carbohydrate,
lipid, phospholipid, polycation or polyanion. An example of a
polypeptide cell surface molecule is an equilibrative nucleoside
transporter ENT2.
[0132] In an embodiment of the invention, cell penetration by the
complex formed between the cell-penetrating polypeptide and DNA or
its degradation product comprises cellular entry by the
cell-penetrating polypeptide. The fate of the bound DNA or its
degradation product is not known. The term "bind" in the phrase "to
bind and penetrate the live cell" (or "to bind and penetrate
additional live cells") by a complex comprising a cell-penetrating
polypeptide and DNA or its degradation product refers to
association of the complex with the live cell at the cell surface.
The term "penetrate" in the above phrase refers to penetration of
the cell by the cell-penetrating polypeptide; whereas, the fate of
the DNA or its degradation product in the complex related to
cell-penetration is not known and remains to be determined.
[0133] In one embodiment of the invention, following cell
penetration, the anti-DNA antibody, antibody fragment or derivative
or variant of the invention further penetrates or accumulates in
the nucleus, e.g., 3E10 antibody.
[0134] Contemplated in the invention is the use of subcellular
localization sequences linked to the cell-penetrating polypeptide
of the invention so as to direct the cell-penetrating polypeptide
to a desired cellular compartment following cell entry. Subcellular
localization sequences are short polypeptides known in the art and
may be linked to the cell-penetrating polypeptide by recombinant
methods. Subcellular compartments which may be targeted with
subcellular localization sequences include nucleus, nucleolus,
cytoplasm, mitochondria, endoplasmic reticulum, Golgi, and
peroxisome.
[0135] As used herein recombinant variable regions of
immunoglobulin molecules refers to variable regions of Ig molecules
which are produced by molecular biological means. Sequences
encoding variable domain of the heavy and light chains may be
isolated from T-cells, B-cells, leukemic cells, lymphoma cells, or
immunoglobulin gene expressing cells, cloned into expression vector
systems, and introduced into a host cell to produce "recombinant
variable regions of immunoglobulin molecules." Alternatively, the
sequences may be recombinantly produced or obtained from genomic
DNA. Recombinant antibodies produced in this manner consists of an
antibody or antibody fragment with the antigen binding specificity
dependent on the variable region, comprising framework sequences
and CDRs. Such recombinant antibodies may be formed from a
polypeptide chain containing a variable region from a light chain
and a polypeptide chain containing a variable region from a heavy
chain or alternatively both the light chain and heavy chain
variable regions could be found within a polypeptide in which a
linker is used to link by recombinant DNA methods the coding
sequences for the two variable chain regions, such as in the case
of single chain Fv fragment (scFv).
[0136] When recombinant variable regions of immunoglobulin
molecules are formed from two separate polypeptides, one for the
light chain variable region and other for the heavy chain variable
region, the recombinant Ig molecules may be an intact antibody as
is normally produced by an organism from which the coding sequences
were isolated or it could be a fragment. Antibody fragments could
be produced either by recombinant DNA methods allowing tailored
antibodies not dependent on specific protease cleavage sites or by
proteolytic cleavage of the recombinant antibodies such as by IdeS,
pepsin, or papain to produce Fab, F(ab') or F(ab').sub.2 fragments.
The "recombinant variable regions of immunoglobulin molecules" may
include the entire constant region or a portion of the constant
region. In addition, the constant region of one antibody may be
replaced by recombinant DNA method with the constant region of a
different antibody if desired.
[0137] Single-chain antibodies or Fv consist of an antibody light
chain variable domain or region ("V.sub.L") and heavy chain
variable region ("V.sub.H") connected by a short peptide linker.
The peptide linker allows the structure to assume a conformation
which is capable of binding to antigen].
[0138] As used herein, a "conservative amino acid substitution" is
the replacement of one amino acid with another of a similar type
such that the binding specificity of the antibody is preserved.
Amino acids of a similar type can be classified into several groups
in which one amino acid within a group may be able to substitute
for another member of the group: [0139] (1) non-polar aliphatic
amino acids, such as alanine, glycine, isoleucine, leucine and
valine with alanine and glycine more related to each other and
isoleucine, leucine and valine more related to each other based on
size; [0140] (2) neutral polar amino acids, such as serine,
cysteine, threonine, glutamine and asparagine, and to a lesser
extent methionine; [0141] (3) cyclic amino acid, such as proline;
[0142] (4) aromatic amino acids, such as phenylalanine, tyrosine,
and tryptophan; [0143] (5) basic amino acids, such as histidine,
lysine and arginine; [0144] (6) acidic amino acids, such as
aspartic acid, glutamic acid, asparagine and glutamine; [0145] (7)
aspartic acid and asparagine; [0146] (8) glutamic acid and
glutamine; and [0147] (9) alanine, glycine, serine and cysteine
[0148] Discussions of conservative amino acid substitution may be
found in the patent literature.
[0149] Moreover, the present invention includes nucleic acids with
silent mutation or silent mutations. A silent mutation is a
mutation in the DNA which does not result in a change to the amino
acid sequence of a protein or results in a change to the amino acid
sequence of a protein but not its functionality. Degeneracy of the
genetic code allows multiple codons to code for the same amino
acid, allowing silent mutations to occur without changing the
protein sequence. Such silent mutations are well-known and may be
recited readily from publically available and accepted codon
tables. In the case of silent mutations in which the amino acid
sequence is changed but not the function of the protein, such
silent mutations are generally mutations in which one amino acid of
a certain chemical/physical characteristics is substituted with
another of a similar type. Such mutations may involve conservative
amino acid substitutions and may be detected through evolutionary
changes but is best determine empirically.
[0150] The invention additionally provides a method for detecting
an area or zone of cellular turnover. The method comprising (a)
contacting cells at a potential area or zone of cellular turnover
with a cell-penetrating polypeptide comprising 3E10 scFv or
cell-penetrating determinants of a lupus autoantibody or a fragment
or variant thereof so that the cell-penetrating polypeptide binds
extracellular DNA present at an area or zone of cellular turnover;
(b) permitting cellular uptake of the cell-penetrating polypeptide
of (a) at the area or zone of cellular turnover; and (c) detecting
the presence of the cell-penetrating polypeptide inside the cell
body, thereby detecting an area or zone of cellular turnover. In a
further embodiment, the method further comprises identifying a
center of cellular turnover wherein the center marked by presence
of a lysed cell produces a gradient of extracellular DNA such that
(a) presence of greatest amount of extracellular DNA near the
center results in greatest uptake of the cell-penetrating
polypeptide by live cells near the center of cellular turnover and
(b) presence of lesser amount of extracellular DNA away from the
center results in lower uptake of the cell-penetrating polypeptide
by live cells away from the center of cellular turnover, thereby
identifying the center of cellular turnover.
[0151] In accordance with the practice of the invention, the cells
or tissue may be from a mammal or derived from a mammal. Examples
of mammals include but are not limited to mouse, rat, hamster, cat,
dog, rabbit, bovine, pig, sheep, goat, horse, monkey or human.
[0152] Cancer immunotherapy using anti-DNA antibodies, alone or
combined with extracellular DNA, may follow the teachings generated
from various approaches which have been successfully employed with
respect to other types of cancer. For example, one way to apply
antitumor monoclonal antibodies clinically is to administer them in
unmodified form, using monoclonal antibodies of the invention which
display antitumor activity and/or internalizing ability and/or in
animal models. The anti-tumor activity of a particular anti-DNA
mAb, or combination of anti-DNA mAbs, is preferably evaluated in
vivo using a suitable animal model. Xenogenic cancer models,
wherein human cancer explants or passaged xenograft tissues are
introduced into immune compromised animals, such as nude or SCID
mice, are particularly appropriate and are known. Examples of
xenograft models of human prostate cancer (capable of
recapitulating the development of primary tumors, micrometastasis,
and the formation of osteoblastic metastases characteristic of late
stage disease) are well known. The examples herein provide detailed
experimental protocols for evaluating the anti-tumor potential of
anti-DNA mAb preparations in vivo. Other in vivo assays are
contemplated, such as those which measure regression of established
tumors, interference with the development of metastasis, and the
like.
[0153] The method of the invention contemplates the administration
of single anti-DNA mAbs as well as combinations, or "cocktails", of
different individual mAbs such as those recognizing different
epitopes. Such mAb cocktails may have certain advantages inasmuch
as they contain mAbs which bind to different epitopes and/or
exploit different effector mechanisms or combine directly cytotoxic
mAbs with mAbs that rely on immune effector functionality. Such
mAbs in combination may exhibit synergistic therapeutic effects. In
addition, the administration of anti-DNA mAbs may be combined with
other therapeutic agents, including but not limited to various
chemotherapeutic agents, androgen-blockers, and immune modulators.
The anti-DNA mAbs may be administered in their "naked" or
unconjugated form, or may have therapeutic agents conjugated to
them.
[0154] The anti-DNA monoclonal antibodies used in the practice of
the method of the invention may be formulated into pharmaceutical
compositions comprising a carrier suitable for the desired delivery
method. Suitable carriers include any material which when combined
with the anti-DNA mAbs retains the anti-tumor function of the
antibody or cytoprotective function of an antibody conjugate, if
cytoprotective effect is desired, and is non-reactive with the
subject's immune systems. Examples include, but are not limited to,
any of a number of standard pharmaceutical carriers such as sterile
phosphate buffered saline solutions, bacteriostatic water, and the
like (see, generally, Remington's Pharmaceutical Sciences 16.sup.th
Edition, A. Osal., Ed., 1980).
[0155] The anti-DNA antibody formulations may be administered via
any route capable of delivering the antibodies to the tumor site.
Potentially effective routes of administration include, but are not
limited to, intravenous, intraperitoneal, intramuscular,
intratumoral, intradermal, and the like. The preferred route of
administration is by intravenous injection. A preferred formulation
for intravenous injection comprises the anti-DNA mAbs in a solution
of preserved bacteriostatic water, sterile unpreserved water,
and/or diluted in polyvinylchloride or polyethylene bags containing
0.9% sterile Sodium Chloride for Injection, USP. The anti-DNA mAb
preparation may be lyophilized and stored as a sterile powder,
preferably under vacuum, and then reconstituted in bacteriostatic
water containing, for example, benzyl alcohol preservative, or in
sterile water prior to injection.
[0156] Treatment may involve the repeated administration of the
anti-DNA antibody preparation via an acceptable route of
administration such as intravenous injection (IV), at an effective
dose. Dosages will depend upon various factors generally
appreciated by those of skill in the art, including without
limitation the type of cancer and the severity, grade, or stage of
the cancer, the binding affinity and half-life of the mAb or mAbs
used, the extent of circulating shed DNA, the desired steady-state
antibody concentration level, frequency of treatment, and the
influence of chemotherapeutic agents used in combination with the
treatment method of the invention. Typical daily doses may range
from about 0.1 to 100 mg/kg. Doses in the range of 10-500 mg mAb
per week may be effective and well tolerated, although even higher
weekly doses may be appropriate and/or well tolerated. The
principal determining factor in defining the appropriate dose is
the amount of a particular antibody necessary to be therapeutically
effective in a particular context. Repeated administrations may be
required in order to achieve tumor inhibition or regression or,
alternatively, cytoprotection if cytoprotection is desired. Initial
loading doses may be higher. The initial loading dose may be
administered as an infusion. Periodic maintenance doses may be
administered similarly, provided the initial dose is well
tolerated.
[0157] In another embodiment, the invention provides methods for
selectively inhibiting a live cell by reacting any one or a
combination of the immunoconjugates of the invention with the cell
in an amount sufficient to inhibit the cell. Such amounts include
an amount to kill the cell or an amount sufficient to inhibit cell
growth or proliferation. As discussed supra the dose and dosage
regimen will depend on the nature of the disease or disorder to be
treated, its population, the site to which the antibodies are to be
directed, the characteristics of the particular immunotoxin, and
the patient. For example, the amount of immunoconjugate can be in
the range of 0.1 to 200 mg/kg of patient weight.
[0158] In another embodiment, the invention provides a method for
increasing uptake and enhancing efficacy of a cell-penetrating
polypeptide comprising an anti-DNA antibody or a fragment or a
variant thereof in targeting tumor or cancer cells comprising (a)
inducing additional extracellular DNA release at or near the tumor
or cancer cells through the use of a cell-damaging agent or
introducing additional extracellular DNA or artificial DNA at or
near the tumor or cancer cells; (b) administering the
cell-penetrating polypeptide; (c) allowing the cell-penetrating
polypeptide to form additional complexes with the additional
extracellular DNA or artificial DNA; and (d) permitting the
additional complexes in (c) to contact the tumor or cancer cells,
thereby increasing uptake and enhancing efficacy of a
cell-penetrating polypeptide comprising an anti-DNA antibody or a
fragment or a variant thereof in targeting tumor or cancer
cells.
[0159] In another embodiment, the invention provides a method for
diagnosing or identifying a site of cell or tissue injury, an
ischemic site with necrotic or apoptotic cells, a tumor site with
necrotic or apoptotic cells, a cancer site with necrotic or
apoptotic cells, or a site of cellular turnover comprising: (a)
administering a cell-penetrating polypeptide comprising
cell-penetrating determinants so as to interrogate one or more
sites with the cell-penetrating polypeptide; (b) detecting the
presence of the cell-penetrating polypeptide within the nucleus of
a live cell; and (c) determining if a cluster of live cells with
the cell-penetrating polypeptide is present at the interrogated
sites, wherein presence of a cluster of live cells with the
cell-penetrating polypeptide is indicative of a site of cell or
tissue injury, an ischemic site with necrotic or apoptotic cells, a
tumor site with necrotic or apoptotic cells, a cancer site with
necrotic or apoptotic cells, or a site of cellular turnover,
thereby, diagnosing or identifying a site of cell or tissue injury,
an ischemic site with necrotic or apoptotic cells, a tumor site
with necrotic or apoptotic cells, a cancer site with necrotic or
apoptotic cells, or a site of cellular turnover.
[0160] In another embodiment, the invention provides a method for
increasing or enhancing cytoprotection at an ischemic site
comprising: (a) administering DNA to the site in an extracellular
space so as to permit increased targeting of a cell-penetrating
polypeptide comprising 3E10 scFv or cell-penetrating determinants
of a lupus autoantibody or a fragment or variant thereof, and a
cytoprotective agent; (b) administering the cell-penetrating
polypeptide of (a); (c) contacting the extracellular DNA of (a) or
its degradation product at an ischemic site with a cell-penetrating
polypeptide of (a) so that the cell-penetrating polypeptide binds
extracellular DNA or its degradation product at an ischemic site so
as to form a complex; (d) contacting a live cell at risk for dying
at an ischemic site with the complex in (c) so as to bind and
penetrate the live cell; and (e) permitting additional complexes to
form as in (c) and contacting additional live cells with said
complexes so as to bind and penetrate additional live cells at risk
for dying at an ischemic site; thereby, delivering additional
cytoprotective agent to a cell at risk of dying and delivering
cytoprotective agent to more cells at risk of dying at an ischemic
site, thus, increasing or enhancing cytoprotection at an ischemic
site.
[0161] In one embodiment, the cytoprotective agent is a heat shock
protein, stress protein or chaperone protein. The heat shock
protein, stress protein or chaperone protein may be any of Hsp-70,
HspA1A, HspA1B, HspA1L, HspA2, HspA5, HspA6, HspA7, HspA8, HspA9,
HspA12A, HspA12B, HspA13, HspA14, HspH1, HspH2, HspH3, and HspH4,
Hsp40, DNAJA1, DNAJA2, DNAJA3, DNAJA4, DNAJB1, DNAJB2, DNAJB3,
DNAJB4, DNAJB5, DNAJB6, DNAJB7, DNAJB8, DNAJB9, DNAJB11, DNAJB12,
DNAJB13, DNAJB14, DNAJC1, DNAJC2, DNAJC3, DNAJC4, DNAJC5B, DNAJC5G,
DNAJC6, DNAJC7, DNAJC8, DNAJC9, DNAJC10, DNAJC11, DNAJC12, DNAJC13,
DNAJC14, DNAJC15, DNAJC16, DNAJC17, DNAJC18, DNAJC19, DNAJC20,
DNAJC21, DNAJC22, DNAJC23, DNAJC24, DNAJC25, DNAJC26, DNAJC27,
DNAJC28, DNAJC30, HSPB1, HSPB2, HSPB3, HSPB4, HSPB5, HSPB6, HSPB7,
HSPB8, HSPB9, HSPB10, HSPB11, hsp90, hsp84, hsp27, hsp20, GRP78,
alpha B crystallin, hsp60, hsp100, GRP94, GRP170, AIPL1, FKBP1A,
FKBP1B, FKBP2, FKBP3, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L,
FKBP10, FKBP11, FKBP12, FKBP14, FKBP15, FKBP38, FKBP52 and
LOC541473.
[0162] In one embodiment, an ischemic site is associated with a
condition selected from the group consisting of cardiac ischemia,
myocardial infarction, ischemic colitis, mesenteric ischemia, brain
ischemia, acute ischemic stroke, transient ischemic attack,
vascular dementia, stroke, acute limb ischemia, cyanosis, gangrene,
an embolism, a thrombosis, an atherosclerosis artery, a trauma,
venous outflow obstruction, acute arterial ischemia, an aneurysm,
mitral valve disease, chronic atrial fibrillation,
cardiomyopathies, an occlusion, pulmonary embolus, acute arterial
occlusion, peripheral arterial disease, a thromboembolism, a
compression, a shearing, a laceration, arterial dissection,
iatrogenic arterial injury, thoracic outlet syndrome,
atherosclerosis, hypoglycemia, tachycardia, hypotension, septic
shock, heart failure, superior mesenteric artery syndrome, sickle
cell disease, induced g-force, frostbite, improper cold compression
therapy, tourniquet application, increased glutamate receptor
stimulation, arteriovenous malformation, peripheral artery
occlusive disease, rupture of significant blood vessel, anemia,
cardiac arrhythmia, cardiorespiratory arrest, subarachnoid
hemorrhage, intracerebral hemorrhage, cerebral infarction, focal
brain ischemia, global brain ischemia, pulmonary infarction, lung
infarction, splenic infarction, limb infarction, deep vein
thrombosis, phlebitis, skeletal muscle infarction, diabetes
mellitus, avascular necrosis, testicular torsion, testicular
infarction, central retinal artery infarction, sepsis,
antiphospholipid syndrome, giant-cell arteritis, hemia, volvulus,
hepatic ischemia, dehydration and infection.
Compositions
[0163] The invention provides a pharmaceutical composition
comprising an anti-DNA antibody, fragment or derivative or variant
thereof and, optionally, a suitable carrier. The invention also
provides a composition or pharmaceutical composition comprising a
cell-penetrating polypeptide which comprises cell-penetrating
determinants and extracellular DNA or artificial DNA. The invention
also provides a composition or pharmaceutical composition
comprising a cell-penetrating polypeptide which comprises
cell-penetrating determinants and extracellular DNA or artificial
DNA, and optionally, a suitable carrier. The antibody or fragment
or derivative or variant thereof may be conjugated or linked to a
therapeutic drug or a cytotoxic agent. The antibody or fragment or
derivative or variant thereof may be conjugated or linked to a
hapten, an epitope tag or an imaging agent. The antibody or
fragment or derivative thereof may be conjugated or linked to a
cytoprotective agent. The antibody or fragment or derivative
thereof may be conjugated or linked to a chemical compound, a
peptide or a protein.
[0164] Suitable carriers for pharmaceutical compositions include
any material which when combined with the nucleic acid or other
molecule of the invention retains the molecule's activity and is
non-reactive with the subject's immune systems. Examples include,
but are not limited to, any of the standard pharmaceutical carriers
such as a phosphate buffered saline solution, water, emulsions such
as oil/water emulsion, and various types of wetting agents. Other
carriers may also include sterile solutions, tablets including
coated tablets and capsules. Typically such carriers contain
excipients such as starch, milk, sugar, certain types of clay,
gelatin, stearic acid or salts thereof, magnesium or calcium
stearate, talc, vegetable fats or oils, gums, glycols, or other
known excipients. Such carriers may also include flavor and color
additives or other ingredients. Compositions comprising such
carriers are formulated by well-known conventional methods. Such
compositions may also be formulated within various lipid
compositions, such as, for example, liposomes as well as in various
polymeric compositions, such as polymer microspheres.
Anti-DNA Antibodies
[0165] The invention provides anti-DNA antibodies for use in the
methods and compositions of the invention. In one embodiment, the
anti-DNA antibodies which may be used in the invention includes any
of H7 Antibody, H9 Antibody, H72 Antibody, H205 Antibody, H317
Antibody F14-6 Antibody, SN22 Antibody, SN50 Antibody, SN111
Antibody, SN112 Antibody, SN575 Antibody, SN604 Antibody, Sn608
Antibody, F4.1 Antibody, J20.8 Antibody, F14.6 Antibody, and 9D7
antibody or a derivative or variant thereof (Vlahakos D, Foster Mh,
Ucci Aa, Barrett Kj, Datta Sk, And Madaio Mp (1992) "Murine
Monoclonal Anti-Dna Antibodies Penetrate Cells, Bind To Nuclei, And
Induce Glomerular Proliferation And Proteinuria In Vivo." J. Am.
Soc. Nephrol. 2(8):1345-54; Ruiz-Arguelles A, Perez-Romano B,
Llorente L, Alarcon-Segovia D, And Castellanos Jm (1998)
"Penetration Of Anti-DNA Antibodies Into Immature Live Cells." J.
Autoimmun. 11(5):547-56; Avrameas A, Ternynck T, Nato F, Buttin G,
And Avrameas S (1998) "Polyreactive Anti-Dna Monoclonal Antibodies
And A Derived Peptide As Vectors For The Intracytoplasmic And
Intranuclear Translocation Of Macromolecules" Proc. Natl. Acad.
Sci. U.S.A. 95(10):5601-5606; Song Yc, Sun Gh, Lee Tp, Huang Jc, Yu
Cl, Chen Ch, Tang Sj, And Sun Kh (2008) "Arginines In The CDR Of
Anti-dsDNA Autoantibodies Facilitate Cell Internalization Via
Electrostatic Interactions." Eur. J. Immunol. 38(11):3178-90).
Sequence Of CDR For F4.1 Antibody, J20.8 Antibody And F14.6
Antibody May Be Found In Avrameas A, Ternynck T, Nato F, Buttin G,
And Avrameas S (1998) "Polyreactive Anti-DNA Monoclonal Antibodies
And A Derived Peptide As Vectors For The Intracytoplasmic And
Intranuclear Translocation Of Macromolecules" Proc. Natl. Acad.
Sci. U.S.A. 95(10):5601-5606. Sequence Of CDR For 9d7 Antibody May
Be Found In Song Yc, Sun Gh, Lee Tp, Huang Jc, Yu Cl, Chen Ch, Tang
Sj, And Sun Kh (2008) "Arginines In The CDR Of Anti-DsDNA
Autoantibodies Facilitate Cell Internalization Via Electrostatic
Interactions." Eur. J. Immunol. 38(11):3178-90. In One Embodiment,
The Anti-DNA Antibodies Which May Also Be Used In The Invention
Includes 5c5 Monoclonal Antibody, 5c6 Monoclonal Clonal Antibody
And 4h2 Monoclonal Antibody (Weisbart Rh, Et Al., J. Immunol. 1990
144(7): 2653-2658; Zack Dj, Et Al., J. Immunol. 1995 154(4):
1987-1994; Weidle Uh, Et Al., Cancer Genomics Proteomics 2013 10:
239-250; Weisbart Rh, Et Al., Sci. Rep. 2015 5: 12022; Noble Pw, Et
Al., Sci. Rep. 2014 4:5958; Colburn Kk, Et Al., J. Rheumatol. 2003
30(5):993-7).
3E10 Antibodies
[0166] The invention further provides an example of an anti-DNA
antibody which is a 3E10 antibody (e.g., polyclonal, monoclonal,
chimeric, and humanized antibodies) for use in the methods and
compositions of the invention. Anti-3E10 antibodies that are
particularly contemplated include monoclonal antibodies as well as
fragments thereof (e.g., recombinant proteins, such as scFv)
containing the antigen binding domain and/or one or more complement
determining regions of these antibodies. 3E10 Mab is a murine
monoclonal antibody and its nucleic acid sequence and amino acid
sequence provided in: Figures 3 and 4 Of Zack Dj, Et Al., J.
Immunol. 1995 154(4): 1987-1994; FIGS. 3 And 4 Of Us Patent
Application Publication No.: US 2008/0292618 A1; FIGS. 1 and 2 of
PCT International Publication No.: WO 2010/138769 A1, published 2
Dec. 2010; GenBank Accession Numbers: L16982 for mAb 3E10 Vh chain
and L34051 for mAb 3E10 v.kappa. light chain. location of the
complement-determining regions (e.g., CDR1, CDR2 and CDR3) Along
With The Framework Regions (i.e., FR1, FR2, FR3, and FR4) of the
3E10 variable heavy chain and light chain domains are provided in
figures 3 and 4 of Zack Dj, Et Al., J. Immunol. 1995 154(4):
1987-1994; FIGS. 3 and 4 of US Patent Application Publication No.:
US 2008/0292618 A1; FIGS. 1 and 2 of PCT International Publication
No. WO 2010/138769 A1, published 2 Dec. 2010. In a preferred
embodiment, the anti-3E10 antibody or its fragment is a variant,
such as the D31N 3E10 variant in which amino acid residue 31 of
3E10 variable heavy chain is mutated from an aspartic acid (D) to
an asparagine (N). This D31N variant of 3E10 antibody has increased
binding to ssDNA And dsDNA (Zack Dj, Et Al., J. Immunol. 1995
154(4): 1987-1994) and enhanced cell and nuclear penetration (Zack
Dj, Et Al., J. Immunol. 1996 157(5): 2082-2088; Weisbart Wh, et
al., J. Autoimmunity 1998 11(5): 539-546). In an embodiment of the
invention, the 3E10 antibody or its fragment or variant is a
derivative. In another embodiment, a derivative may be in the form
of an immunoconjugate. Such immunoconjugates are discussed
supra.
Humanized Antibodies
[0167] The present invention encompasses humanized antibodies.
Various methods for humanizing non-human antibodies are known in
the art.
[0168] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to one
method, humanized antibodies are prepared by a process of analysis
of the parental sequences and various conceptual humanized products
using three-dimensional models of the parental and humanized
sequences. Three-dimensional immunoglobulin models are commonly
available and are familiar to those skilled in the art.
Human Antibodies
[0169] Human antibodies of the invention can be constructed by
combining Fv clone variable domain sequences selected from
human-derived phage display libraries with known human constant
domain sequences. Alternatively, human monoclonal antibodies of the
invention can be made by the hybridoma method. Human myeloma and
mouse-human heteromyeloma cell lines for the production of human
monoclonal antibodies are well known in the art. Gene shuffling can
also be used to derive human antibodies from non-human, where the
human antibody has similar affinities and specificities to the
starting non-human antibody using a method called epitope
imprinting.
Bispecific Antibodies
[0170] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. Bispecific antibodies may be obtained from
intact antibodies or antibody fragments. Methods for making
bispecific antibodies are known in the art and described herein.
Antibodies with more than two valencies are contemplated. For
example, trispecific antibodies can be prepared.
Antibody Variants
[0171] In some embodiments, amino acid sequence modifications of
the antibodies described herein are contemplated. For example, it
may be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the antibody are prepared by introducing appropriate nucleotide
changes into the antibody nucleic acid, or by peptide synthesis.
Such modifications include, for example, deletions from, and/or
insertions into and/or substitutions of, residues within the amino
acid sequences of the antibody. Any combination of deletion,
insertion, and substitution is made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid alterations may be introduced in
the subject antibody amino acid sequence at the time that sequence
is made. Where the antibody comprises an Fc region, the
carbohydrate attached thereto may be altered. Another type of
variant is an amino acid substitution variant. These variants have
at least one amino acid residue in the antibody molecule replaced
by a different residue. The sites of greatest interest for
substitutional mutagenesis include the hypervariable regions, but
FR alterations are also contemplated. For example, in one preferred
embodiment of the 3E10 antibody, amino acid residue 31 of 3E10
variable heavy chain is mutated from an aspartic acid (D) to an
asparagine (N) to produce the D31N variant of 3E10 antibody. This
D31N variant of 3E10 antibody has increased binding to ssDNA and
dsDNA (Zack D J, et al., J. Immunol. 1995 154(4): 1987-1994) and
enhanced cell and nuclear penetration (Zack D J, et al., J.
Immunol. 1996 157(5): 2082-2088; Weisbart W H, et al., J.
Autoimmunity 11(5): 539-546).
[0172] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
Antibody Derivatives
[0173] The antibodies of the present invention can be further
modified to contain additional proteinaceous or nonproteinaceous
moieties that are known in the art. Proteinaceous or
nonproteinaceous moieties include, but are not limited to, chemical
functional groups such as hydrophilic linkers (e.g. polyethylene
glycol), crosslinking agents, metal chelators, epitope tags,
peptides such as AGIH tetrapeptide sequence, cytotoxic agents,
enzymes, cytoprotective agents, a second antibody (or fragment or
variant thereof), imaging agents or detectable markers. Suitable
examples of the imaging agent or detectable marker include a
radioisotope, a fluorophore, a fluorescent quencher, an enzyme, a
luminescent compound, a chemiluminescent compound, a bioluminescent
compound, a photon emitter, a heavy metal, a ferromagnetic agent, a
contrast agent, a metal chelator, and an epitope.
[0174] For example, derivatives of anti-DNA antibodies of the
invention (such as 3E10 antibody) may be a fusion protein
comprising the cell-penetrating determinants of the anti-DNA
antibody (such as 3E10 antibody) and a second biologically active
desired functional protein or peptide.
Nucleic Acid Molecules
[0175] In an embodiment, the invention provides a nucleic acid
molecule encoding the anti-DNA antibodies in the compositions of
the invention. The nucleic acid molecule may encode the anti-DNA
antibodies in the compositions of the invention.
[0176] The nucleic acids of the invention may comprise nucleotide
sequences and polypeptides encoding amino acid sequences which are
at least about 70% identical, preferably at least about 80%
identical, more preferably at least about 90% identical and most
preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%,
99%, 100%) to the reference nucleotide and amino acid sequences of
the present invention (i.e., see example herein) when the
comparison is performed by a BLAST algorithm wherein the parameters
of the algorithm are selected to give the largest match between the
respective sequences over the entire length of the respective
reference sequences. Polypeptides comprising amino acid sequences
which are at least about 70% similar, preferably at least about 80%
similar, more preferably at least about 90% similar and most
preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%,
99%, 100%) to the reference amino acid sequences of the present
invention when the comparison is performed with a BLAST algorithm
wherein the parameters of the algorithm are selected to give the
largest match between the respective sequences over the entire
length of the respective reference sequences, are also included in
the present invention.
[0177] The nucleic acid molecule may be a DNA molecule (e.g., cDNA)
encoding the bispecific composition of the invention. For example,
the invention provides for a DNA construct comprising a vector that
expresses the bispecific composition of the invention.
[0178] Additionally, the invention provides a vector which
comprises the nucleic acid molecule of the invention. The host
vector system comprises the vector of the invention in a suitable
host cell. Examples of suitable host cells include but are not
limited to bacterial cell and eukaryotic cells.
Vectors, Host Cells and Recombinant Methods
[0179] For recombinant production of an antibody of the invention,
the nucleic acid encoding it is isolated and inserted into a
replicable vector for further cloning (amplification of the DNA) or
for expression. DNA encoding the antibody is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). Many
vectors are available. The choice of vector depends in part on the
host cell to be used. Generally, preferred host cells are of either
prokaryotic or eukaryotic (generally mammalian) origin. It will be
appreciated that constant regions of any isotype can be used for
this purpose, including IgG, IgM, IgA, IgD, and IgE constant
regions, and that such constant regions can be obtained from any
human or animal species.
Immunoconjugates
[0180] The invention also provides immunoconjugates
(interchangeably termed "antibody-drug conjugates" or "ADC"),
comprising any of the anti-DNA antibodies described herein
conjugated to a cytotoxic agent such as a chemotherapeutic agent, a
drug, a growth inhibitory agent, a toxin (e.g., an enzymatically
active toxin of bacterial, fungal, plant, or animal origin, or
fragments thereof), or a radioactive isotope (i.e., a
radioconjugate). Immunoconjugates may also comprise any of the
anti-DNA antibodies described herein conjugated to a cytoprotective
agent such as a heat shock protein, stress protein or chaperone
protein.
[0181] Chemotherapeutic agents useful in the generation of
immunoconjugates are described herein. Conjugates of an antibody
and one or more small molecule toxins, such as a calicheamicin,
maytansinoids, dolastatins, aurostatins, a trichothecene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein. Other antitumor agents that
can be conjugated to the antibodies of the invention include BCNU,
streptozoicin, vincristine and 5-fluorouracil. Enzymatically active
toxins and fragments thereof which can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
Momordica charantia inhibitor, curcin, crotin, Saponaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes.
[0182] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate.
[0183] In an embodiment, heat shock protein, stress protein or
chaperone protein include any of Hsp-70, HspA1A, HspA1B, HspA1L,
HspA2, HspA5, HspA6, HspA7, HspA8, HspA9, HspA12A, HspA12B, HspA13,
HspA14, HspH1, HspH2, HspH3, and HspH4, Hsp40, DNAJA1, DNAJA2,
DNAJA3, DNAJA4, DNAJB1, DNAJB2, DNAJB3, DNAJB4, DNAJB5, DNAJB6,
DNAJB7, DNAJB8, DNAJB9, DNAJB11, DNAJB12, DNAJB13, DNAJB14, DNAJC1,
DNAJC2, DNAJC3, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC7, DNAJC8,
DNAJC9, DNAJC10, DNAJC11, DNAJC12, DNAJC13, DNAJC14, DNAJC15,
DNAJC16, DNAJC17, DNAJC18, DNAJC19, DNAJC20, DNAJC21, DNAJC22,
DNAJC23, DNAJC24, DNAJC25, DNAJC26, DNAJC27, DNAJC28, DNAJC30,
HSPB1, HSPB2, HSPB3, HSPB4, HSPB5, HSPB6, HSPB7, HSPB8, HSPB9,
HSPB10, HSPB11, hsp90, hsp84, hsp27, hsp20, GRP78, alpha B
crystallin, hsp60, hsp100, GRP94, GRP170, AIPL1, FKBP1A, FKBP1B,
FKBP2, FKBP3, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10,
FKBP11, FKBP12, FKBP14, FKBP15, FKBP38, FKBP52 and LOC541473.
[0184] Immunoconjugate of any anti-DNA antibody described herein
and a cytoprotective agent may be produced by any coupling method
known in the art, including chemical crosslinking and recombinant
methods.
[0185] The antibody or fragment thereof of the invention may be
labeled with a detectable marker or conjugated to a second
molecule, such as a therapeutic agent (e.g., a cytotoxic agent or
cytoprotective agent) thereby resulting in an immunoconjugate. For
example, the therapeutic agent includes, but is not limited to, an
anti-tumor drug, a toxin, a radioactive agent, a cytokine, a second
antibody, an enzyme, a substrate, a heat shock protein, a stress
protein or a chaperone protein. Further, the invention provides an
embodiment wherein the antibody of the invention is linked to an
enzyme that converts a prodrug into a cytotoxic drug. The invention
provides an embodiment wherein the antibody of the invention is
linked to an enzyme that participates in a denatured protein
response or protein refold. The invention provides an embodiment
wherein the antibody of the invention is linked to substrate that
participates in a detoxification process or an enzyme that
detoxifies, such as glutathione S-transferase, cytochrome P450
oxidase, UDP-glucuronosyltransferase or alcohol dehydrogenase.
[0186] Examples of cytotoxic agents include, but are not limited to
ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium
bromide, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicine, dihydroxy anthracenedione, actinomycin D,
diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A
chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,
restrictocin, phenomycin, enomycin, curcin, crotin, calicheamicin,
Saponaria officinalis inhibitor, maytansinoids, and glucocorticoid
and other chemotherapeutic agents, as well as radioisotopes such as
.sup.212Bi, .sup.131I, .sup.131In, .sup.90Y, and .sup.186Re.
Suitable detectable markers include, but are not limited to, a
radioisotope, a fluorescent compound, a bioluminescent compound,
chemiluminescent compound, a metal chelator or an enzyme.
Antibodies may also be conjugated to an anti-cancer pro-drug
activating enzyme capable of converting the pro-drug to its active
form.
[0187] Additionally, the recombinant protein of the invention
comprising the antigen-binding region of any of the monoclonal
antibodies of the invention can be used to treat cancer. In such a
situation, the antigen-binding region of the recombinant protein is
joined to at least a functionally active portion of a second
protein having therapeutic activity. The second protein can
include, but is not limited to, an enzyme, lymphokine, oncostatin,
toxin or a second antibody directed to a different antigen.
Suitable toxins include those described above.
[0188] Techniques for conjugating (e.g., by chemical means) or
joining (by recombinant means) therapeutic agents to antibodies are
well known (see, e.g., Arnon et al., "Monoclonal Antibodies For
Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal
Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56
(Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug
Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al.
(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody
Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in
Monoclonal Antibodies '84: Biological And Clinical Applications,
Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982)).
Pharmaceutical Formulations
[0189] Therapeutic formulations comprising an antibody of the
invention are prepared for storage by mixing the antibody having
the desired degree of purity with optional physiologically
acceptable carriers, excipients or stabilizers (Remington: The
Science and Practice of Pharmacy 20th edition (2000)), in the form
of aqueous solutions, lyophilized or other dried formulations.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, histidine and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0190] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose intended.
In one embodiment, the formulation contains an anti-DNA antibody or
fragments, derivatives or variants thereof, as described herein and
DNA (extracellular DNA) as the only therapeutic agents in the
formulation.
[0191] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington: The Science and Practice of Pharmacy 20th edition
(2000).
[0192] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes. When appropriate, chemical or
radiation sterilization method may be used.
[0193] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the
immunoglobulin of the invention, which matrices are in the form of
shaped articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma.-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated immunoglobulins remain
in the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0194] In one embodiment, the sustained-release formulation
comprises a cell-penetrating polypeptide and a carrier. In another
embodiment, the sustained-release formulation comprises a
cell-penetrating polypeptide and a preservative. In another
embodiment, the sustained-release formulation comprises DNA and/or
its degradation product(s) and a carrier. In another embodiment,
the sustained-release formulation comprises DNA and/or its
degradation product(s) and a preservative. In one embodiment, the
sustained-release formulation comprises a cell-penetrating
polypeptide and DNA and/or its degradation products. In one
embodiment, the sustained-release formulation comprises a
cell-penetrating polypeptide and DNA and/or its degradation
products and a carrier. In one embodiment, the sustained-release
formulation comprises a cell-penetrating polypeptide and DNA and/or
its degradation products. In one embodiment, the sustained-release
formulation comprises a cell-penetrating polypeptide and DNA and/or
its degradation products and a preservative.
[0195] The formulation may be an immediate-release formulation. In
one embodiment, the immediate-release formulation comprises a
cell-penetrating polypeptide and a carrier. In another embodiment,
the immediate-release formulation comprises a cell-penetrating
polypeptide and a preservative. In another embodiment, the
immediate-release formulation comprises DNA and/or its degradation
product(s) and a carrier. In another embodiment, the
immediate-release formulation comprises DNA and/or its degradation
product(s) and a preservative. In one embodiment, the
immediate-release formulation comprises a cell-penetrating
polypeptide and DNA and/or its degradation products. In one
embodiment, the immediate-release formulation comprises a
cell-penetrating polypeptide and DNA and/or its degradation
products and a carrier. In one embodiment, the immediate-release
formulation comprises a cell-penetrating polypeptide and DNA and/or
its degradation products. In one embodiment, the immediate-release
formulation comprises a cell-penetrating polypeptide and DNA and/or
its degradation products and a preservative.
[0196] In a further embodiment, the formation is a combination of
an immediate-release formulation and a sustained-release
formulation.
[0197] Use of immunologically reactive fragments, such as the Fab,
Fab', or F(ab).sub.2 fragments or scFv is often preferable,
especially in a therapeutic context, as these fragments are
generally less immunogenic than the whole immunoglobulin. Further,
bi-specific antibodies specific for two or more epitopes may be
generated using methods generally known in the art. Further,
antibody effector functions may be modified so as to enhance the
therapeutic effect of 3E10 antibodies on cancers. Homodimeric
antibodies may also be generated by cross-linking techniques known
in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565). The
invention also provides pharmaceutical compositions having the
monoclonal antibodies or anti-idiotypic monoclonal antibodies of
the invention, such as anti-idiotypic mAb 1C7 directed against
anti-DNA antibody including 3E10 mAb (Weisbart R H et al., J.
Immunol. 1990 144(7): 2653-2658). Such anti-idiotypic antibodies
may be useful in neutralizing activity of an anti-DNA antibody or
3E10 antibody or a fragment, variant or derivative thereof.
[0198] The following examples are intended to illustrate the
present invention, not to limit the scope of the invention in any
way.
Example 1
Methods and Materials
[0199] Production and purification of 3E10 scFv. 3E10 scFv used in
these studies was previously modified by a D31N mutation in CDR1 of
the variable region of the heavy chain that results in a 50-fold
increase in DNA-binding affinity and efficiency of nuclear
penetration. 3E10 scFv was produced in P. pastoris and purified as
described previously (10).
[0200] Cell lines and tissue culture. The GM02605 human fibroblast
cell line (Coriell Biorepository, Camden, N.J.) was selected for
these studies because it grows to confluence in 96-well tissue
culture plates with remarkably high viability (>99% viability
maintained over several days of growth as determined by propidium
iodide exclusion assay). Cells were grown in MEM with 10% FCS and
washed with MEM without serum before incubation with 10 .mu.M 3E10
scFv for one hour. Nuclear penetration by 3E10 scFv was then
examined by anti-Myc immunostaining as previously described
(10).
[0201] Cell lysate. COS-7 cell lysate was prepared by subjecting
cells to multiple freeze-thaw cycles in liquid nitrogen. Cell
debris was removed by centrifugation. DNA-depleted COS-7 cell
lysate was prepared by passing the lysate through a Centricon
cellulose filter with a molecular weight cut off of 10,000 Da.
[0202] DNA. Purified calf thymus DNA sheared to an average length
of 2000 bp was purchased from Invitrogen (Ultrapure, Invitrogen,
Carlsbad, Calif.).
[0203] Human glioma xenografts. U87 human glioma subcutaneous
xenografts were generated in nude mice. When tumors reached size of
100 mm.sup.3 mice were treated with intraperitoneal injection (IP)
of control PBS buffer or 0.8 mg 3E10 scFv in PBS. Mice were
sacrificed 4 or 24 hours after treatment, and tumors and selected
normal tissues were fixed in formalin and embedded in paraffin.
Tissues were then surveyed for nuclear penetration by 3E10 scFv by
immunohistochemistry (IHC) as previously described (11). Briefly,
sections were incubated with 9E10 anti-Myc (Invitrogen) primary
antibody directed at the C-terminal Myc tag in 3E10 scFv before
probing with secondary antibody and visualizing with
3,3'-diaminobenzidine and counterstaining with hematoxylin.
Results
[0204] A factor released by dead cells appears to enhance nuclear
penetration by 3E10 scFv. We sought to test the efficiency of
nuclear penetration by 3E10 scFv into cells in the absence of
extracellular DNA, and for these studies selected the GM02605 human
fibroblast cell line because it maintains a high degree of
viability (>99%) with minimal cell death even while maintained
in culture for several days. With low rates of turnover the
confounding effects of DNA released by dead cells are minimized.
The GM02605 cells were washed with serum free media and then
treated with 10 .mu.M 3E10 scFv for one hour, after which cells
were fixed and immunostained for presence of the fragment.
Remarkably, 3E10 scFv did not penetrate into most cells. Instead,
3E10 scFv was detected only in the nuclei of cells centered around
what appeared to be a dead cell, with a gradient effect observed
with diminishing amounts of intranuclear antibody detected with
increasing distance from the central dead cell. A representative
image demonstrating this effect is shown in FIG. 1. This
observation was consistent with a factor released locally by dying
cells enhancing nuclear penetration of 3E10 scFv into surrounding
cells.
[0205] Addition of cell lysate promotes homogenous nuclear uptake
of 3E10 scFv. To test the hypothesis that a factor released by dead
cells enhances nuclear uptake of 3E10 scFv we next compared the
efficiency of nuclear penetration of the fragment into the GM02605
fibroblasts in the presence or absence of a cell lysate. As shown
in FIG. 2, in the absence of the cell lysate minimal nuclear uptake
of 3E10 scFv was observed. However, the addition of cell lysate
facilitated nuclear penetration by 3E10 scFv into .about.100% of
the cells. These results further support the hypothesis that a
factor released by dead cells contained in cell lysate promotes
nuclear uptake of 3E10 scFv.
[0206] DNA-depleted cell lysate does not enhance nuclear uptake of
3E10 scFv. Based on our previous observations that 3E10 scFv binds
DNA and is unable to penetrate cells that are deficient in the ENT2
nucleoside transporter, we hypothesized that DNA is the critical
factor in cell lysate that promotes nuclear uptake of 3E10 scFv. To
test this, the GM06205 fibroblasts were treated with 10 .mu.M 3E10
scFv in the presence of cell lysate that had been filtered to
remove DNA content. In contrast to the complete cell lysate, the
DNA-depleted lysate did not enhance nuclear uptake of 3E10 scFv
(FIG. 2), which strongly supports the hypothesis that DNA is the
relevant factor contributing to nuclear penetration by the
fragment.
[0207] Addition of purified DNA promotes homogeneous nuclear uptake
of 3E10 scFv. To confirm that extracellular DNA enhances nuclear
penetration by 3E10 scFv, we next treated the GM02605 fibroblasts
with 3E10 scFv in the presence of purified DNA. As shown in FIG. 2,
addition of purified DNA to the media significantly enhanced the
efficiency of penetration by 3E10 scFv into cell nuclei. Taken
together, these data indicate that nuclear penetration by 3E10 scFv
is enhanced by the presence of extracellular DNA.
[0208] 3E10 scFv targets tumor cells in vivo. Based on the
observation that 3E10 scFv penetrates cell nuclei most efficiently
in the presence of extracellular DNA, we hypothesized that when
administered in vivo the fragment would accumulate most efficiently
into tissues in which there would be expected to be a higher
concentration of extracellular DNA due to high cellular turnover,
such as is associated with tumors. To test this, subcutaneous U87
human glioma xenografts were generated in immunodeficient mice, and
once tumors grew to size of 100 mm.sup.3 mice were treated with
intraperitoneal injection of control buffer or 3E10 scFv. Mice were
then sacrificed 4 or 24 hours after treatment, and tumors and
select normal tissues were immunostained for the presence of 3E10
scFv. As shown in FIG. 3A, four hours after treatment 3E10 scFv was
detected in the nuclei of the U87 xenograft cells, but was not
detected in tissues of major organs including heart, kidney,
skeletal muscle, and liver. 3E10 scFv was also detected in the
tumors 24 hours after treatment, demonstrating the stability of the
uptake into tumor nuclei (FIG. 3B). These results are consistent
with enhanced uptake of 3E10 scFv into sites of high cell turnover
where DNA is released from dying cells.
Discussion
[0209] 3E10 scFv has been explored as a therapeutic intracellular
transport system and has successfully mediated delivery of p53,
Hsp70, and the anti-MDM2 antibody 3G5 to target tissues in vivo. In
addition, 3E10 by itself has been shown to sensitize tumors in vivo
to DNA-damaging agents including ionizing radiation and
doxorubicin. Importantly, 3E10 and 3E10 scFv have never been found
to be significantly toxic to any normal tissues in any of these
previous in vivo studies. The present study was carried out to
further evaluate preliminary evidence that 3E10 scFv preferentially
targets ischemic tissue or areas of high cell turnover rates such
as malignant tissue. For example, we previously found that the
Fv-Hsp70 fusion protein protected rats from reperfusion injury of
ischemic brain even when administered 3 hours after ligation of the
middle cerebral artery, and in this study 3E10 scFv was shown to
localize in ischemic but not normoxic brain (9). These findings
were consistent with a previous study that showed targeting of an
anti-histone antibody to an area of cell death in vivo (12). We
also observed that the 3E10-3G5 bispecific antibody yielded a
profound suppression of MDM2-addicted tumors in vivo but showed a
remarkable absence of systemic toxicity (13), suggesting a
preferential localization of the agent to tumor cells.
[0210] In the present study we have now shown that penetration of
3E10 scFv into live cells in vitro requires the presence of
extracellular DNA. Of note, our findings are consistent with a
previous report that demonstrated the requirement of extracellular
DNA for penetration of an another anti-DNA antibody into living
T-cells, however only 10% of cells internalized antibody (14).
Moreover, when 3E10 scFv was administered systemically it was
observed to preferentially localize into U87 cancer cells implanted
subcutaneously, consistent with the fact that rapidly growing
cancers are ischemic and have a high level of cell turnover and
therefore the local environment should be enriched for
extracellular DNA. The selective targeting of 3E10 scFv to such
tissues may therefore explain in part the remarkable lack of
off-target toxicity of 3E10 alone and of 3E10 scFv-p53 and 3E10-3G5
administered systemically in our previous studies.
[0211] We previously showed that 3E10 scFv penetrates living cells
through the ENT2 nucleoside salvage pathway, and our results here
suggest that 3E10 scFv bound to DNA may be processed by membrane
nucleases and phosphatases into fragments that are accessible to
this pathway. Further studies are required to characterize these
membrane-related events, but the primary significance of our study
is the demonstration that 3E10 scFv has targeting specificity in
vivo to areas of tissue ischemia and high cell turnover. This
finding further establishes the potential to use 3E10 scFv in a
variety of clinical applications that include protecting organs
from reperfusion injury and cytotoxic applications for cancer
therapy. In addition, recognition of the requirement for
extracellular DNA for nuclear penetration by 3E10 scFv suggests
that combinations of 3E10 scFv with targeted approaches that
selectively increase cell turnover in tumors, such as locally
applied radiotherapy, might facilitate the subsequent penetration
of 3E10 scFv into all cells comprising a tumor mass due to enhanced
release of DNA by dying cells.
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