U.S. patent application number 15/187579 was filed with the patent office on 2017-05-18 for methods and compositions for bi-specific targeting of cd19/cd22.
The applicant listed for this patent is Jeff Lion, Daniel A. Vallera. Invention is credited to Jeff Lion, Daniel A. Vallera.
Application Number | 20170137513 15/187579 |
Document ID | / |
Family ID | 42740172 |
Filed Date | 2017-05-18 |
United States Patent
Application |
20170137513 |
Kind Code |
A1 |
Vallera; Daniel A. ; et
al. |
May 18, 2017 |
METHODS AND COMPOSITIONS FOR BI-SPECIFIC TARGETING OF CD19/CD22
Abstract
Methods and composition involving genetically engineered
targeting conjugates with reversed orientation of V.sub.L and
V.sub.H chains are provided. For example, in certain aspects
targeting conjugates comprising V.sub.L and V.sub.H chains of
anti-CD22 and anti-CD19 are described. In a further aspect, the
invention provides methods and targeting conjugates comprising
therapeutic agents or diagnostic agents for delivery to B
cells.
Inventors: |
Vallera; Daniel A.;
(Richfield, MN) ; Lion; Jeff; (Fresno,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vallera; Daniel A.
Lion; Jeff |
Richfield
Fresno |
MN
CA |
US
US |
|
|
Family ID: |
42740172 |
Appl. No.: |
15/187579 |
Filed: |
June 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13256812 |
Jan 27, 2012 |
9371386 |
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PCT/US2010/027012 |
Mar 11, 2010 |
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15187579 |
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61160530 |
Mar 16, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/565 20130101;
C07K 2317/76 20130101; A61K 38/19 20130101; C07K 2317/56 20130101;
A61K 47/6849 20170801; C07K 2317/622 20130101; C07K 2317/92
20130101; C07K 14/21 20130101; C12Y 204/02036 20130101; A61K
47/6827 20170801; A61P 35/02 20180101; A61K 38/00 20130101; C07K
2319/55 20130101; C07K 2317/31 20130101; A61K 2039/505 20130101;
A61K 38/164 20130101; C07K 16/2803 20130101; C07K 2317/73 20130101;
A61K 47/6851 20170801; C12N 9/1077 20130101; A61P 35/00
20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 14/21 20060101 C07K014/21; C12N 9/10 20060101
C12N009/10 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
numbers R01-CA36725 and R01-CA082154 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A conjugate comprising a therapeutic agent conjugated to a
targeting moiety comprising at least a first antigen-binding
fragment that binds a first antigen and a second antigen-binding
fragment that binds a second antigen, wherein the first
antigen-binding fragment comprises a first V.sub.L domain which is
linked at its carboxy terminus to a first V.sub.H domain
(V.sub.L-V.sub.H orientation), and/or the second antigen-binding
fragment comprises a second V.sub.L domain which is linked at its
carboxy terminus to a second V.sub.H domain (V.sub.L-V.sub.H
orientation).
2. The conjugate of claim 1, wherein said conjugate is further
defined as a fusion protein.
3. The conjugate of claim 2, wherein said fusion protein is
DT2219ARL having an amino acid sequence of SEQ ID NO:01.
4. The conjugate of claim 1, wherein said agent and targeting
moiety is chemically conjugated.
5. The conjugate of claim 1, wherein the first and/or second
antigen-binding fragment is an Fv fragment.
6. The conjugate of claim 1, wherein the first and/or second
antigen-binding fragment is an scFv fragment.
7. The conjugate of claim 1, wherein the first V.sub.L domain is
linked to the first V.sub.H domain via a first peptide linker.
8. The conjugate of claim 1, wherein the second V.sub.L domain is
linked to the second V.sub.H domain via a second peptide
linker.
9. The conjugate of claim 7, wherein the first peptide linker
comprises at least three charged resides selected from the group
consisting of lysine, arginine, glutamic acid, aspartic acid, and
histidine.
10. The conjugate of claim 8, wherein the second peptide linker
comprises at least three charged resides selected from the group
consisting of lysine, arginine, glutamic acid, aspartic acid, and
histidine.
11. The conjugate of claim 9 or 10, wherein the first or second
peptide linker is a ARL linker (GSTSGSGKPGSGEGSTKG; SEQ ID
NO:14).
12. The conjugate of claim 1, wherein the first antigen-binding
fragment is linked to the second antigen-binding fragment via a
third peptide linker.
13. The conjugate of claim 12, wherein the third peptide linker is
G4S.
14. The conjugate of claim 1, wherein the therapeutic agent
comprises a therapeutic peptide, wherein the therapeutic peptide is
linked at its carboxy terminus to the first or second
antigen-binding fragment.
15. The conjugate of claim 1, wherein the therapeutic agent
comprises a therapeutic peptide, wherein the therapeutic peptide is
linked at its amino terminus to the first or second antigen-binding
fragment.
16. The conjugate of claim 1, wherein the first antigen and second
antigen are different.
17. The conjugate of claim 1, wherein the first or second antigen
is selected from the group consisting of CD19, CD22, CD45, CD10,
CD5, CD79a, polymorphic HLA-DR.
18. The conjugate of claim 17, wherein the first antigen is CD19,
and the second antigen is CD22.
19. The conjugate of claim 1, wherein the first antigen-binding
fragment is linked at its carboxy terminus to the therapeutic agent
or the second antigen-binding fragment.
20. The conjugate of claim 1, wherein the second antigen-binding
fragment is linked at its carboxy terminus to the therapeutic agent
or the first antigen-binding fragment.
21. The conjugate of claim 1, wherein said agent is a cytotoxic
agent, a cytokine, an anti-angiogenic agent, a chemotherapeutic
agent, a pro-apoptosis agent, an enzyme, a hormone, a growth
factor, a peptide, a protein, an antibiotic, an antibody, a Fab
fragment of an antibody, an antigen, a survival factor, an
anti-apoptotic agent, a hormone antagonist, a virus, a
bacteriophage, a bacterium, a liposome, a cell, a nucleic acid or
an expression vector.
22. The conjugate of claim 1, wherein said agent is a cytotoxic
agent.
23. The conjugate of claim 22, wherein said cytotoxic agent
comprises a peptide, a polypeptide, or a small molecule.
24. The conjugate of claim 23, wherein said cytotoxic agent is
selected from the group consisting of gelonin, ricin, abrin,
diphtheria toxin, Pseudomonas exotoxin, Clostridium perfringens
enterotoxin, dodecandrin, tricosanthin, tricokirin, bryodin,
mirabilis antiviral protein, barley ribosome-inactivating protein
(BRIP), pokeweed antiviral protein (PAPs), saporin, luffin,
momordin, colicin, anthrax toxin, tetanus toxin, botulinum
neurotoxin, and fragments thereof.
25. The conjugate of claim 24, wherein said cytotoxic agent
comprises diphtheria toxin.
26. The conjugate of claim 25, wherein said cytotoxic agent
comprises the translocation enhancer region of diphtheria
toxin.
27. The conjugate of claim 25, wherein said cytotoxic agent
comprises the amino terminal 390 amino acids of diphtheria
toxin.
28. The conjugate of claim 24, wherein said cytotoxic agent
comprises Pseudomonas exotoxin KDEL (SEQ ID NO:05).
29. The conjugate of claim 24, wherein said cytotoxic agent
comprises Pseudomonas exotoxin KDEL 7 mutant.
30. The conjugate of claim 21, wherein said agent is an
anti-angiogenic agent selected from the group consisting of
thrombospondin, angiostatin, endostatin or pigment
epithelium-derived factor, angiotensin, laminin peptides,
fibronectin peptides, plasminogen activator inhibitors, tissue
metalloproteinase inhibitors, interferons, interleukin 12, platelet
factor 4, IP-10, Gro-.beta., 2-methoxyoestradiol,
proliferin-related protein, carboxiamidotriazole, CM101,
Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),
interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,
Linomide, thalidomide, pentoxifylline, genistein, TNP-470,
paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470,
platelet factor 4 or minocycline.
31. The conjugate of claim 21, wherein said agent is a cytokine
selected from the group consisting of interleukin 1 (IL-1), IL-2,
IL-5, IL-10, IL-11, IL-12, IL-18, interferon-.gamma. (IF-.gamma.),
IF-.alpha., IF-.beta., tumor necrosis factor-.alpha. (TNF-.alpha.),
or GM-CSF (granulocyte macrophage colony stimulating factor).
32. The conjugate of claim 1, further defined as being comprised in
a pharmaceutically acceptable carrier.
33. A pharmaceutical composition, comprising the conjugate of claim
1.
34. A method of treating a human patient having a B-cell
malignancy, comprising the step of administering to said patient
with the conjugate of claim 1, wherein the conjugate targets B
cells.
35. The method of claim 34, wherein said B-cell malignancy is
selected from the group consisting of B-cell subtype of
non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, acute
lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL),
chronic myelogenous leukemia (CML), hairy cell leukemia, and
prolymphocytic leukemia.
36. The method of claim 34, wherein said conjugate exerts an
anti-tumor activity.
37. The method of claim 36, wherein said anti-tumor activity is
selected from the group consisting of increasing tumor-free
survival, killing a tumor cell or tissue, inducing apoptosis of a
tumor cell or tissue, inhibiting tumor growth, inhibiting
metastatic spread, reducing tumor burden and inducing tumor
regression.
38. The method of claim 34, further comprising the step of treating
said subject with chemotherapy, radiotherapy, surgery, hormone
therapy or gene therapy.
39-40. (canceled)
41. The conjugate of claim 10, wherein the second peptide linker is
a ARL linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO:14).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 13/256,812 filed Jan. 27, 2012, now issued as
U.S. Pat. No. 9,371,386; which is a 35 USC .sctn.371 National Stage
application of International Application No. PCT/US2010/027012
filed Mar. 11, 2010, now expired; which claims the benefit under 35
USC .sctn.119(e) to U.S. Application Ser. No. 61/160,530 filed Mar.
16, 2009, now expired. The disclosure of each of the prior
applications is considered part of and is incorporated by reference
in the disclosure of this application.
BACKGROUND OF THE INVENTION
[0003] Field of the Invention
[0004] The present invention relates generally to the field of
immunology and tumor biology. More particularly, it concerns
compositions and methods involving bispecific antibodies for B-cell
malignancy therapeutics and/or diagnostics.
[0005] Background Information
[0006] The immune system of vertebrates consists of a number of
organs and cell types which have evolved to accurately recognize
foreign antigens, specifically bind to, and eliminate/destroy such
foreign antigens. Lymphocytes, among other cell types, are critical
to the immune system. Lymphocytes are divided into two major
sub-populations, T cells and B cells. Although inter-dependent, T
cells are largely responsible for cell-mediated immunity and B
cells are largely responsible for antibody production (humoral
immunity).
[0007] In humans, each B cell can produce an enormous number of
antibody molecules. Such antibody production typically ceases (or
substantially decreases) when a foreign antigen has been
neutralized. Occasionally, however, proliferation of a particular B
cell will continue unabated and may result in a cancer known as a
B-cell lymphoma. B-cell lymphomas, such as the B-cell subtype of
non-Hodgkin's lymphoma, are significant contributors to cancer
mortality. The response of B-cell malignancies to various forms of
treatment is mixed. For example, in cases in which adequate
clinical staging of non-Hodgkin's lymphoma is possible, field
radiation therapy can provide satisfactory treatment. Still, about
one-half of the patients die from the disease (Devesa et al.,
1987).
[0008] Acute leukemia is the most common childhood malignancy,
representing 30% of all cancer in American children under the age
of 15-19 years and 12% of cancer cases in those aged 15 to 19 years
old. In the United States, approximately 2500 new cases are
diagnosed annually; 80% of these are B lineage acute lymphoblastic
leukemia (B-ALU). Chemotherapy resistant blasts are a frequent
cause of treatment failure in all leukemia patients (List, 1996)
and alternative therapies are urgently needed.
[0009] The majority of chronic lymphocytic leukemias are of the
B-cell lineage (Freedman, 1990). This type of B-cell malignancy is
the most common leukemia in the Western world (Goodman et al.,
1996). The natural history of chronic lymphocytic leukemia falls
into several phases. In the early phase, chronic lymphocytic
leukemia is an indolent disease, characterized by the accumulation
of small mature functionally-incompetent malignant B cells having a
lengthened life span. Eventually, the doubling time of the
malignant B cells decreases and patients become increasingly
symptomatic. While treatment can provide symptomatic relief, the
overall survival of the patients is only minimally affected. The
late stages of chronic lymphocytic leukemia are characterized by
significant anemia and/or thrombocytopenia. At this point, the
median survival is less than two years (Foon et al., 1990). Due to
the very low rate of cellular proliferation, chronic lymphocytic
leukemia is resistant to cytotoxic drug treatment.
[0010] Traditional methods of treating B-cell malignancies,
including chemotherapy and radiotherapy, have limited utility due
to toxic side effects. Therefore, there remains a need to develop
novel treatments for B-cell malignancies with improved
efficacy.
SUMMARY OF THE INVENTION
[0011] The present invention is based in part on the finding that
the superior in vivo activity of a conjugate in treating B-cell
malignancy results from genetic alterations of antibody or
fragments thereof comprised in the conjugate, such as reverse
orienting V.sub.H-V.sub.L domains and adding aggregation
reducing/stabilizing linkers.
[0012] Thus, in accordance with certain aspects of the present
invention, there is provided a conjugate comprising a therapeutic
agent conjugated to a targeting moiety comprising at least a first
antigen-binding fragment that binds a first antigen and a second
antigen-binding fragment that binds a second antigen, wherein the
first antigen-binding fragment comprises a first V.sub.L domain
which is linked at its carboxy terminus to a first V.sub.H domain
(V.sub.L-V.sub.H orientation), and/or the second antigen-binding
fragment comprises a second V.sub.L domain which is linked at its
carboxy terminus to a second V.sub.H domain (V.sub.L-V.sub.H
orientation). Preferably, the conjugate is further defined as a
fusion protein, for example, DT2219ARL having an amino acid
sequence of SEQ ID NO:01. The therapeutic agent and targeting
moiety may also be chemically conjugated. In certain further
embodiments, the antigen-binding fragments may be a full-length
antibody, an Fv fragment, or an scFv fragment.
[0013] In some further aspects, the therapeutic agent comprises a
therapeutic peptide, wherein the therapeutic peptide may be linked
at its carboxy or amino terminus to the first or second
antigen-binding fragment. In still further embodiments of the
invention, the first antigen-binding fragment may be linked at its
carboxy terminus to the therapeutic agent or the second
antigen-binding fragment, or the second antigen-binding fragment
may be linked at its carboxy terminus to the therapeutic agent or
the first antigen-binding fragment.
[0014] The reversed orientation of variable regions may cause the
conjugate to more easily permeate tumor cell or tissue and be more
uniformly distributed to contribute to its greater anti-tumor
activity. Therefore, at least an antigen-binding fragment (e.g.,
sFv) with a V.sub.L-V.sub.H orientation may also have improved
therapeutic efficacy. Based on the general description above, the
conjugate may have at least 18 variations if the first and second
antigen binding fragment are different antigen binding fragments,
such as anti-CD19 and ant-CD22 scFvs. For example, following the
conventional order from N terminus to C terminus, the therapeutic
peptide may be followed by six potential arrangements of two scFvs:
(1) the V.sub.L and V.sub.H regions of anti-CD22 and the V.sub.L
and V.sub.H regions of anti-CD19, (2) the V.sub.L and V.sub.H
regions of anti-CD22 and the V.sub.H and V.sub.L regions of
anti-CD19, (3) the V.sub.H and V.sub.L regions of anti-CD22 and the
V.sub.L and V.sub.H regions of anti-CD19, (4) the V.sub.L and
V.sub.H regions of anti-CD19 and the V.sub.L and V.sub.H regions of
anti-CD22, (5) the V.sub.L and V.sub.H regions of anti-CD19 and the
V.sub.H and V.sub.L regions of anti-CD22, or (6) the V.sub.H and
V.sub.L regions of anti-CD19 and the V.sub.L and V.sub.H regions of
anti-CD22. Alternatively, the therapeutic peptide may be in the C
terminus and preceded by the six possible combinations of V.sub.L
and V.sub.H regions of two scFvs as described above, or may be in
the middle between two scFvs with another six possibilities. In
certain aspects, the conjugate may also be monospecific or
multispecific of recognizing more than two targets with a least a
V.sub.L-V.sub.H structure to improve penetration and efficacy.
[0015] To treat B-cell malignancy, the first or second antigen may
be any B-cell surface marker known in the art, such as CD19, CD22,
CD45, CD10, CD5, CD79a, or polymorphic HLA-DR. Furthermore, in
highly preferred aspects of the invention, the first antigen and
second antigen may be different for bispecificity, for example, the
first antigen is CD19, and the second antigen is CD22. Dual antigen
targeting may be more potent and superior and less toxic compared
with the sum of single targeting.
[0016] Regarding design of the conjugate as a fusion protein,
optimal linkers between different domains may contribute to
improved yield and refolding. In certain aspects of the invention,
the linker connecting the first V.sub.L domain to the first V.sub.H
domain or connecting the second V.sub.L domain to the second
V.sub.H domain may be a peptide linker, preferably comprising at
least three charged resides selected from the group consisting of
lysine, arginine, glutamic acid, aspartic acid, and histidine,
which may improve refolding and help increase protein yield. A
particular example may be an ARL linker (SEQ ID NO:02). The first
antigen-binding fragment may be linked to the second
antigen-binding fragment via a third peptide linker, such as a G45
linker.
[0017] Therapeutic agents are known in the art and may be used in
the methods and compositions of the invention. For example, in some
aspects, the therapeutic agent is a cytotoxic agent, a cytokine, an
anti-angiogenic agent, a chemotherapeutic agent, a pro-apoptosis
agent, an enzyme, a hormone, a growth factor, a peptide, a protein,
an antibiotic, an antibody, a Fab fragment of an antibody, an
antigen, a survival factor, an anti-apoptotic agent, a hormone
antagonist, a virus, a bacteriophage, a bacterium, a liposome, a
cell, a nucleic acid or an expression vector. Preferably, the agent
is a cytotoxic agent, which may comprise a peptide, a polypeptide,
or a small molecule, such as gelonin, ricin, abrin, diphtheria
toxin, Pseudomonas exotoxin, Clostridium perfringens enterotoxin,
dodecandrin, tricosanthin, tricokirin, bryodin, mirabilis antiviral
protein, barley ribosome-inactivating protein (BRIP), pokeweed
antiviral protein (PAPs), saporin, luffin, momordin, colicin,
anthrax toxin, tetanus toxin, botulinum neurotoxin, and fragments
thereof. For example, the cytotoxic agent comprises diphtheria
toxin, the translocation enhancer region of diphtheria toxin, or
the amino terminal 390 amino acids of diphtheria toxin. In another
aspect, the cytotoxic agent may comprise Pseudomonas exotoxin KDEL
(SEQ ID NO:05) or Pseudomonas exotoxin KDEL7 mutant (7mut).
[0018] The skilled artisan will understand that the agent may be an
anti-angiogenic agent which includes, but not is not limited to,
thrombospondin, angiostatin, endostatin or pigment
epithelium-derived factor, angiotensin, laminin peptides,
fibronectin peptides, plasminogen activator inhibitors, tissue
metalloproteinase inhibitors, interferons, interleukin 12, platelet
factor 4, IP-10, Gro-.beta., 2-methoxyoestradiol,
proliferin-related protein, carboxiamidotriazole, CM101,
Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),
interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,
Linomide, thalidomide, pentoxifylline, genistein, TNP-470,
paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470,
platelet factor 4 or minocycline. In a further aspect, the agent
may be a cytokine such as interleukin 1 (IL-1), IL-2, IL-5, IL-10,
IL-11, IL-12, IL-18, interferon-.gamma. (IF-.gamma.), IF-.alpha.,
IF-.beta., tumor necrosis factor-.alpha. (TNF-.alpha.), or GM-CSF
(granulocyte macrophage colony stimulating factor).
[0019] For therapeutic purpose, the conjugate may be further
defined as being comprised in a pharmaceutically acceptable
carrier. There may also be provided a pharmaceutical composition
comprising the conjugate for its superior therapeutic activity, a
nucleic acid molecule comprising a sequence encoding the fusion
protein defining the conjugate and an expression vector comprising
the nucleic acid for various purposes.
[0020] Additional aspects of the invention concern methods of
treating a human patient having a B-cell malignancy, comprising the
step of administering to the patient with the conjugates or the
compositions of the present invention. For example, the B-cell
malignancy may be B-cell subtype of non-Hodgkin's lymphoma,
Burkitt's lymphoma, multiple myeloma, acute lymphoblastic leukemia
(ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous
leukemia (CML), hairy cell leukemia, or prolymphocyte leukemia.
[0021] By targeting tumor cells specifically or preferentially, the
conjugate may exert an anti-tumor activity, such as increasing
tumor-free survival, killing a tumor cell or tissue, inducing
apoptosis of a tumor cell or tissue, inhibiting tumor growth,
inhibiting metastatic spread, reducing tumor burden and inducing
tumor regression. To have a better anti-tumor effect, the conjugate
may be used to treat a patient in combination with chemotherapy,
radiotherapy, surgery, hormone therapy or gene therapy.
[0022] Embodiments discussed in the context of methods and/or
compositions of the invention may be employed with respect to any
other method or composition described herein. Thus, an embodiment
pertaining to one method or composition may be applied to other
methods and compositions of the invention as well.
[0023] As used herein the terms "encode" or "encoding" with
reference to a nucleic acid are used to make the invention readily
understandable by the skilled artisan; however, these terms may be
used interchangeably with "comprise" or "comprising"
respectively.
[0024] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one.
[0025] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0026] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0027] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0029] FIGS. 1A-1B. Construction of DT2219 Variants. FIG. 1A.
Construction of DT2219ARL. (1) The original DT2219EA construct
consisting of the first 389 amino acids of the DT (DT.sub.390), the
V.sub.H and V.sub.L regions of anti-CD22 (sFv) and anti-CD19
(Dorken et al., 1983) linked by a 20 amino acid segment of human
muscle aldolase (hma). (2) To construct DT2219ARL, the
V.sub.H-V.sub.L orientation was reversed and the V.sub.L and
V.sub.H genes of each sFv were conjoined by a fragment encoding the
ARL linker. (3) The final target gene was spliced into pET21d
vector. FIG. 1B. SDS-PAGE gel containing all 3 DT2219 variants used
in these studies. Lane 1 and 8: Molecular weight standards, Lane 2:
95 kDa DT2219ARL, Lane 3: DT2219EA, Lane 4: DT2219EB1, Lane 5: RFB4
monoclonal antibody, Lane 6: HD37 monoclonal antibody, Lane 7:
DT.sub.390 (partially purified). The gel was stained using
Coomassie blue and shows size and purity of the agents.
[0030] FIGS. 2A-2D. The in vitro Effect of the DT2219 Mutant
Proteins. FIG. 2A. Daudi cells were cultured with fusion proteins
and proliferation was measured by uptake of tritiated thymidine.
Data are percentage of control response where control response is
untreated cells. Data are expressed as mean.+-.standard deviation
(SD). The mean values of untreated Daudi cells were
121,001.+-.8,276 cpm/20,000 cells. FIG. 2B. Selectivity was
determined on the CD19.sup.-CD22.sup.- HPBMLT cell line in a
separate experiment. The mean cpm of untreated HPBMLT in this
experiment was 61,993.+-.7,178 cpm/20,000 cells. DT2219ARL differed
significantly from the control Bic3 group at 0.01-100 nM by Student
t-test (p<0.0001). FIG. 2C. In a third experiment, the ability
of the ligands themselves to mediate cytotoxicity was tested by
inactivating the diphtheria toxin with the DT2219GE mutation that
disrupts toxin activity and leaves the ligands intact. The mean cpm
of untreated Daudi in this experiment was 112,164+10,379 cpm/20,000
cells. FIG. 2D. The anti-proliferative effect of DT2219ARL, DT22,
DTIL19, and a mixture of DT22/DT19 on Daudi cells was tested by
measuring .sup.3H-thymidine uptake 72 hours following IT exposure.
Points on each graph represent mean of triplicate samples.+-.SD.
Control counts=61,993.+-.7,178 cpm/20,000 cells.
[0031] FIGS. 3A-3C. The Activity of Mutated DT2219ARL is Mediated
by Both Anti-CD19 sFv and the Anti-CD22 sFv Ligands. Proliferation
studies were performed in which Daudi cells were treated with a
constant concentration of 10 nM DT2219ARL (FIG. 3A), DT2219EA (FIG.
3B), or DT2219EB1 (FIG. 3C) and then blocked with increasing
concentrations of HD37 monoclonal antibody, RFB4 monoclonal
antibody, or non-reactive control Ly5.2 antibody. Thymidine uptake
was then measured. Each line represents the mean of triplicate
determinations.+-.standard deviation (SD). Percent blocking was
calculated in comparison to the unblocked control and then graphed.
Counts for untreated Daudi cells were 59,301.+-.2,804 cpm/20,000
cells.
[0032] FIG. 4. Binding of DT2219ARL-FITC to Monkey PBMC by Direct
Immunofluorescence. Monkey PBMC, normal human PBMC, or normal human
CD22.sup.+ magnetic bead enriched PBMC cells were incubated with
DT2219ARL-FITC, or negative control DTEpCam23. Positive controls
included FITC labeled conventional monoclonal antibodies RFB4-FITC
or HD37-FITC. Flow cytometry was performed and data expressed as a
contour plot showing cells versus increasing fluorescent intensity.
The top 3 panels show human CD22.sup.+ magnetic bead enriched PBMC,
the lowest 3 panels show normal monkey PBMC, while the middle 3
panels show normal human PBMC. The box in each panel outlines the
gated area which shows binding that exceeds the values obtained for
the negative control. The number in the box is the percentage of
positive cells. NE--not evaluated.
[0033] FIGS. 5A-5C. Groups of SCID (Severe Combined
Immunodeficiency) Mice were Given 10.sup.6 Daudi Cells IV to Induce
Systemic Disease. FIG. 5A. Three days following Daudi injection,
mice were given the exact same injection schedule of multiple
intraperitoneal (ip) injections of DT2219ARL and DT2219EB1 in order
to compare them to no treatment controls. Data were graphed as
proportion surviving versus time. Statistical analysis was
performed using the Log-Rank test and the DT2219ARL group
significantly differed from the no treatment group (p<0.001).
FIG. 5B. Three days following Daudi injection, mice were given ip
treatment with DT2219EA and DT2219ARL in comparison to no treatment
controls and to Bic3 immunotoxin control treated mice. The
DT2219ARL group significantly differed from the DT2219EA group, the
Bic3 group, and the no treatment group (p<0.001). FIG. 5C. Three
days following Daudi injection, mice were given a single ip
injection of DT2219EA and DT2219ARL in comparison to no treatment
controls. Only the DT2219ARL group significantly differed from the
no treatment group.
[0034] FIGS. 6A-6C. Effect of IP Administration of DT2219ARL on
Mice Given Systemic B-Cell Cancer by IV Injection of Raji-Luc.
Raji-luc cells stably expressing the luciferase gene were
administered IV to SCID mice. FIG. 6A. Mice were either treated
with DT2219ARL (M1, M2, M3, and M4) on days 3, 5, 11, 16, and 18 or
untreated (M5, M6, M7, and M8). Luciferase bioluminescence was
measured as photons/s/cm.sup.2/sr. FIG. 6B. The same data as shown
in FIG. 6A are graphed in FIG. 6B. Data are expressed as total
activity graphed over time for each individual animal (M1-M8). FIG.
6C. Digital images of illustrating tumor progression in untreated
Raji-luc mice. Bioluminescent imaging is shown for 3 untreated mice
M9-M11 on day 21. Because the Raji-luc line has a GFP reporter gene
as well as a luciferase reporter gene, fluorescent imaging is also
shown for animals M9, M10, and M11. Lymphoma can be seen in lung,
bone marrow, lymph node and compressing the spinal cord which
likely causes hind limb paralysis (HLP). GFP imaging correlates
with luciferase imaging.
[0035] FIGS. 7A-7C. Toxicity of DT2219ARL in Rabbits. Rabbits were
given IV injection of DT2219ARL on days 1, 3, 5, 7. FIG. 7A.
Average weight of two rabbits. FIG. 7B. ALT enzyme levels from the
same rabbits. FIG. 7C. Frozen liver section from a rabbit treated
with 500 .mu.g/kg DT2219ARL. The section was stained with H and E
and is shown at 100.times. magnification.
[0036] FIG. 8. In vitro Effect of 2219KDEL 7mut on Daudi. Daudi
cells were cultured with fusion proteins and proliferation was
measured by uptake of tritiated thymidine. Data are percentage of
control response where control response is untreated cells. Data
are expressed as mean.+-.standard deviation (SD). There is no
difference in the activity of 2219KDEL and 2219KDEL 7mut.
CD3CD3KDEL is an anti-T cell selectivity control immunotoxin not
reactive with Daudi cells.
[0037] FIG. 9. Antibody Response to Immunization with 2219KDEL or
2219KDEL 7mut. 2219KDEL 7mut has reduced immunogenicity. To detect
antitoxin antibodies, immuno-competent BALB/c mice were immunized
with either non-mutated parental 2219KDEL or mutated 2219KDEL.
Serums from individual mice (n=5/group) were analyzed in a modified
ELISA measuring .mu.g/ml anti-toxin IgG. Data were represented as
the average .mu.g IgG/ml. The two groups significantly differed
(p<0.05) and 2219KDEL 7mut was not as immunogenic.
[0038] FIG. 10. Effect of IP Administration of 2219KDEL 7mut on
Mice Given Systemic B-Cell Cancer by IV Injection of 10.sup.6
Raji-Luc. Raji-luc cells stably expressing the luciferase gene were
administered IV to SCID mice. Mice were either treated ip with
2219KDEL 7mut or untreated. Treatment schedule was injection/every
other day (three times/week, MWF). This was called one "course of
treatment." The mice were treated with 4 courses of 20 .mu.g which
began on days 3, 17, 31, 45. Luciferase bio luminescence was
measured as photons/sec/cm.sup.2/sr. Treatment resulted in cancer
inhibition.
DETAILED DESCRIPTION OF THE INVENTION
I. The Present Invention
[0039] Certain aspects of the instant invention provide improved
immuno conjugates and methods for treating B-cell malignancy by
genetic engineering of variable domain orientations. For example, a
bispecific ligand-directed toxin recognizing CD19 and CD22 resulted
in surprisingly long-term tumor-free survival in well-established
animal models. Further embodiments and advantages of the invention
are described below.
II. Conjugates
[0040] Compositions and methods of the present invention involve
genetically engineered targeting conjugates comprising at least a
V.sub.L-V.sub.H structure. The conjugates may comprise a targeting
moiety and a therapeutic agent, which may be chemically conjugated,
crosslinked, or fused at the protein level using conventional
methods.
[0041] Particularly, the conjugate may be an immunotoxin.
Immunotoxins (IT) are synthesized by coupling an antibody or
antigen-binding fragment to a toxin, particularly a potent,
catalytic toxin, such as diphtheria toxin, capable of inhibiting
protein synthesis (Kreitman, 2002). Catalytic toxins are preferable
because one molecule entering the cytosol can kill a cell.
[0042] In certain aspects, bispecific ligand-directed toxins (BLTs)
are contemplated. BLTs are novel single-chain biologicals
synthesized by linking a truncated toxin to two well-established
targeting ligands with the goal of increasing targeting capability.
For successful BLT, the final construct may have better antitumor
activity than its monospecific counterparts or a mixture of the
two, thus indicating an advantage of including both ligands on the
same single-chain molecule (Stish et al., 2007a; Vallera et al.,
2008; Stish et al., 2008; Stish et al., 2007b; Todhunter et al.,
2004). For example, DT2219ARL fulfilled these criteria for a
successful BLT.
[0043] A. Fusion Proteins
[0044] Certain embodiments of the present invention concern fusion
proteins. These molecules generally have all or a substantial
portion of a targeting peptide, linked at the N- or C-terminus, to
all or a portion of a second therapeutic polypeptide or protein.
For example, fusions may employ leader sequences from other species
to permit the recombinant expression of a protein in a heterologous
host. Another useful fusion includes the addition of an
immunologically active domain, such as an antibody epitope, to
facilitate purification of the fusion protein. Inclusion of a
cleavage site at or near the fusion junction will facilitate
removal of the extraneous polypeptide after purification. Other
useful fusions include linking of functional domains, such as
active sites from enzymes, glycosylation domains, cellular
targeting signals or transmembrane regions. In preferred
embodiments, the fusion proteins of the instant invention comprise
a targeting peptide with a V.sub.L-V.sub.H oriented antigen binding
fragment linked to a therapeutic protein or peptide.
[0045] Examples of proteins or peptides that may be incorporated
into a fusion protein include cytostatic proteins, cytocidal
proteins, pro-apoptosis agents, anti-angiogenic agents, hormones,
cytokines, growth factors, peptide drugs, antibodies, Fab fragments
antibodies, antigens, receptor proteins, enzymes, lectins, MHC
proteins, cell adhesion proteins and binding proteins. These
examples are not meant to be limiting and it is contemplated that
within the scope of the present invention virtually any protein or
peptide could be incorporated into a fusion protein comprising a
targeting peptide.
[0046] Methods of generating fusion proteins are well known to
those of skill in the art. Such proteins can be produced, for
example, by chemical attachment using bifunctional cross-linking
reagents, by de novo synthesis of the complete fusion protein, or
by attachment of a DNA sequence encoding the targeting peptide to a
DNA sequence encoding the second peptide or protein, followed by
expression of the intact fusion protein.
[0047] B. Linkers
[0048] Bifunctional cross-linking reagents have been extensively
used for a variety of purposes including preparation of affinity
matrices, modification and stabilization of diverse structures,
identification of ligand and receptor binding sites, and structural
studies. Suitable peptide linkers may be used to link the
therapeutic agent to the targeting moiety in the present invention,
such as an ARL linker used to link V.sub.L to V.sub.H in the
antigen-binding fragments.
[0049] Homobifunctional reagents that carry two identical
functional groups proved to be highly efficient in inducing
cross-linking between identical and different macromolecules or
subunits of a macromolecule, and linking of polypeptide ligands to
their specific binding sites. Heterobifunctional reagents contain
two different functional groups. By taking advantage of the
differential reactivities of the two different functional groups,
cross-linking can be controlled both selectively and sequentially.
The bifunctional cross-linking reagents can be divided according to
the specificity of their functional groups, e.g., amino,
sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,
reagents directed to free amino groups have become especially
popular because of their commercial availability, ease of synthesis
and the mild reaction conditions under which they can be
applied.
[0050] A majority of heterobifunctional cross-linking reagents
contains a primary amine-reactive group and a thiol-reactive group.
In another example, heterobifunctional cross-linking reagents and
methods of using the cross-linking reagents are described (U.S.
Pat. No. 5,889,155, specifically incorporated herein by reference
in its entirety). The cross-linking reagents combine a nucleophilic
hydrazide residue with an electrophilic maleimide residue, allowing
coupling in one example, of aldehydes to free thiols. The
cross-linking reagent can be modified to cross-link various
functional groups.
[0051] If desired, the targeting moiety and the therapeutic agent
may be joined via a biologically-releasable bond, such as a
selectively-cleavable linker or amino acid sequence. For example,
peptide linkers that include a cleavage site for an enzyme
preferentially located or active within a tumor environment are
contemplated. Exemplary forms of such peptide linkers are those
that are cleaved by urokinase, plasmin, thrombin, Factor IXa,
Factor Xa, or a metallaproteinase, such as collagenase, gelatinase,
or stromelysin.
[0052] Amino acids such as selectively-cleavable linkers, synthetic
linkers, or other amino acid sequences may be used to separate a
targeting moiety or peptide from another peptide, adjuvant or a
therapeutic compound.
[0053] Additionally, while numerous types of disulfide-bond
containing linkers are known that can successfully be employed to
conjugate the toxin moiety with the targeting agent, certain
linkers will generally be preferred over other linkers, based on
differing pharmacologic characteristics and capabilities. For
example, linkers that contain a disulfide bond that is sterically
"hindered" are to be preferred, due to their greater stability in
vivo, thus preventing release of the toxin moiety prior to binding
at the site of action. It can be considered as a general guideline
that any biochemical cross-linker that is appropriate for use in an
immunotoxin will also be of use in the present context, and
additional linkers may also be considered.
[0054] Exemplary methods for cross-linking ligands to liposomes are
described in U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511,
each specifically incorporated herein by reference in its
entirety). Various ligands can be covalently bound to liposomal
surfaces through the cross-linking of amine residues. Liposomes, in
particular, multilamellar vesicles (MLV) or unilamellar vesicles
such as micro emulsified liposomes (MEL) and large unilamellar
liposomes (LUVET), each containing phosphatidylethanolamine (PE),
have been prepared by established procedures. The inclusion of PE
in the liposome provides an active functional residue, a primary
amine, on the liposomal surface for cross-linking purposes. Ligands
such as epidermal growth factor (EGF) have been successfully linked
with PE-liposomes. Ligands are bound covalently to discrete sites
on the liposome surfaces. The number and surface density of these
sites are dictated by the liposome formulation and the liposome
type. The liposomal surfaces may also have sites for non-covalent
association. To form covalent conjugates of ligands and liposomes,
cross-linking reagents have been studied for effectiveness and
biocompatibility. Cross-linking reagents include glutaraldehyde
(GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether
(EGDE), and a water soluble carbodiimide, preferably
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Through the
complex chemistry of cross-linking, linkage of the amine residues
of the recognizing substance and liposomes is established.
[0055] Once conjugated, the peptide generally will be purified to
separate the conjugate from unconjugated targeting agents or
coagulants and from other contaminants. A large number of
purification techniques are available for use in providing
conjugates of a sufficient degree of purity to render them
clinically useful.
[0056] Purification methods based upon size separation, such as gel
filtration, gel permeation or high performance liquid
chromatography, will generally be of most use. Other
chromatographic techniques, such as Blue-Sepharose separation, may
also be used. Conventional methods to purify the fusion proteins
from inclusion bodies are particularly useful for the present
invention, such as using weak detergents like sodium
N-lauroyl-sarcosine (SLS).
[0057] In addition to chemical conjugation, a targeting or
therapeutic peptide may be modified at the protein level. Included
within the scope of the invention are protein fragments or other
derivatives or analogs that are differentially modified during or
after translation, for example, by glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, and proteolytic cleavage. Any number of
chemical modifications may be carried out by known techniques,
including but not limited to specific chemical cleavage by cyanogen
bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4,
acetylation, formylation, farnesylation, oxidation, reduction;
metabolic synthesis in the presence of tunicamycin.
[0058] C. Targeting Moiety
[0059] The targeting moiety may comprise at least one antigen
binding fragment, for example, two antigen binding fragments.
Specific examples are anti-CD22 and anti-19 scFvs. Other specific
cell markers may also be used. Non-limiting examples of B-cell
surface markers include CD19, CD22, CD45, CD10, CD5, CD79a, and
polymorphic HLA-DR.
[0060] Examples of antigen binding fragments suitable for the
present invention include, without limitation: (i) the Fab
fragment, consisting of V.sub.L, V.sub.H, CL and CHI domains; (ii)
the "Fv" fragment consisting of the V.sub.L and V.sub.H domains of
a single antibody; (iii) F(ab')2 fragments, a bivalent fragment
comprising two linked Fab fragments; (iv) single chain Fv molecules
("scFv"), wherein a V.sub.H domain and a V.sub.L domain are linked
by a peptide linker which allows the two domains to associate to
form a binding domain; (v) bispecific single chain Fv dimers (see
U.S. Pat. No. 5,091,513) and (vi) diabodies, multivalent or
multispecific fragments constructed by gene fusion (U.S. Patent
Pub. 2005/0214860). Fv, scFv or diabody molecules may be stabilized
by the incorporation of disulphide bridges linking the V.sub.H and
V.sub.L domains. Minibodies comprising a scFv joined to a CH3
domain may also be made (Hu et al., 1996).
[0061] "Single-chain Fv," "scFv" or "sFv" antibody fragments
comprise the V.sub.H and V.sub.L domains of antibody, wherein these
domains are present in a single polypeptide chain. Generally, the
Fv polypeptide further comprises a peptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv see The
Pharmacology of Monoclonal Antibodies (1994).
[0062] CD19, a 95 kDa membrane glycoprotein, is considered by many
to be the most ubiquitous marker expressed on B cells. CD19 is
expressed not only on mature B cells, but also on late pre-B cells.
It is broadly expressed on B-cell leukemia/lymphoma (Anderson et
al., 1984) including B-ALL. For CD19 targeting, investigators using
conventional biochemically linked anti-CD19 IT have reported
anti-cancer effects (Goulet et al., 1997; Stone et al., 1996;
Flavell et al., 1995; Ghetie et al., 1994; Uckun et al., 1986).
However, these have not reached the mainstream because of varied
degrees of effectiveness.
[0063] CD22 or cluster of differentiation-22, is a molecule
belonging to the SIGLEC family of lectins. Generally speaking, CD22
is a regulatory molecule that prevents the overactivation of the
immune system and the development of autoimmune diseases. CD22 is a
sugar binding transmembrane protein, which specifically binds
sialic acid with an immunoglobulin (Ig) domain located at its
N-terminus. The presence of Ig domains makes CD22 a member of the
immunoglobulin superfamily. CD22 functions as an inhibitory
receptor for B cell receptor (BCR) signaling.
[0064] Anti-CD22 IT have proven successful in the treatment of rare
Hairy Cell Leukemia (HCL) (Kreitman et al., 2005). However, HCL
represents a narrow sampling of patients with leukemia and
expanding the use of the drug to the wider population of patients
is critical. In a previous study, the inventors cloned a new
molecule by fusing two repeating sFv subunits recognizing human
CD19 and human CD22 spliced downstream of truncated DT390 to
broaden toxin delivery and anti-leukemia affect (Vallera et al.,
2005). Studies targeting these 2 ligands with monomeric
conventional immunotoxins showed promise and led to clinical trials
(Herrera et al., 2003; Messmann et al., 2000).
[0065] The present invention provides a conjugate that may be
superior to the known anti-CD19, anti-CD22 immunotoxins or existing
bispecific immunotoxins in terms of its novel V.sub.L-V.sub.H
orientation.
III. Therapeutic Agents
[0066] In certain embodiments, it may be desirable to couple
specific bioactive agents to one or more targeting peptides
(particularly CD19 and CD22 dual targeting peptides) for targeted
delivery to an organ, tissue or cell type. Such agents include, but
are not limited to, cytotoxic molecules, cytokines, chemokines,
pro-apoptosis factors and anti-angiogenic factors as well as
imaging agents.
[0067] A. Cytotoxic Agents
[0068] Chemotherapeutic (cytotoxic) agents of potential use
include, but are not limited to, 5-fluorouracil, bleomycin,
busulfan, camptothecin, carboplatin, chlorambucil, cisplatin
(CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin,
estrogen receptor binding agents, etoposide (VP 16),
farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide,
mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea,
plicomycin, procarbazine, raioxifene, tamoxifen, taxol,
temazolomide (an aqueous form of DTIC), transplatinum, vinblastine
and methotrexate, vincristine, or any analog or derivative variant
of the foregoing. Most chemotherapeutic agents fall into the
categories of alkylating agents, antimetabolites, antitumor
antibiotics, corticosteroid hormones, mitotic inhibitors, and
nitrosoureas, hormone agents, miscellaneous agents, and any analog
or derivative variant thereof.
[0069] In addition, there are a variety of protein toxins
(cytotoxic proteins), which include a number of different classes,
such as those that inhibit protein synthesis: ribosome-inactivating
proteins of plant origin, such as ricin, abrin, gelonin, and a
number of others, and bacterial toxins such as pseudomonas exotoxin
and diphtheria toxin.
[0070] Particularly, Diphtheria toxin (DT) was chosen as an example
for construction due to its irreversible catalytic activity and
research demonstrating a single molecule causes cell death
(Yamaizumi et al., 1978). Also, it is desirable to have new
anti-cancer agents that kill by protein synthesis inhibition, a
mechanism entirely different and unrelated to the mechanism of most
conventional chemotherapeutic agents. The truncated form of DT
(DT390; DT390 protein sequence is SEQ ID NO:03, encoded by SEQ ID
NO:04) was used in Examples due to previous research describing a
series of internal frame deletion mutations that established amino
acid 389 as the best location for genetic fusion of DT to targeting
ligands (Williams et al., 1990). DT390 contains the A fragment of
native DT that catalyzes ADP ribosylation of elongation factor 2
(EF-2) leading to irreversible inhibition of protein synthesis and
cell death (Collier, 1975; Oppenheimer and Bodley, 1981).
[0071] Chemotherapeutic agents and methods of administration,
dosages, etc. are well known to those of skill in the art (see for
example, the "Physicians' Desk Reference", Goodman & Gilman's
"The Pharmacological Basis of Therapeutics" and in "Remington's
Pharmaceutical Sciences" 15.sup.th ed., pp 1035-1038 and 1570-1580,
incorporated herein by reference in relevant parts), and may be
combined with the invention in light of the disclosures herein.
Some variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Examples of specific chemotherapeutic
agents and dose regimes are also described herein. Of course, all
of these dosages and agents described herein are exemplary rather
than limiting, and other doses or agents may be used by a skilled
artisan for a specific patient or application. Any dosage
in-between these points, or range derivable therein is also
expected to be of use in the invention.
[0072] B. Cytokines and Chemokines
[0073] The term "cytokine" is a generic term for proteins released
by one cell population that act on another cell as intercellular
mediators.
[0074] Examples of such cytokines are lymphokines, monokines,
growth factors and traditional polypeptide hormones. Included among
the cytokines are growth hormones such as human growth hormone,
N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;
prorelaxin; glycoprotein hormones such as follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone (LH); hepatic growth factor; prostaglandin, fibroblast
growth factor; prolactin; placental lactogen, OB protein; tumor
necrosis factor-.alpha. and -.beta.; mullerian-inhibiting
substance; mouse gonadotropin-associated peptide; inhibin; activin;
vascular endothelial growth factor; integrin; thrombopoietin (TPO);
nerve growth factors such as NGF-.beta.; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-.alpha. and
TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors; interferons such as
interferon-.alpha., -.beta., and -.gamma.; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
IL-15, IL-16, IL-17, IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO,
kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor
necrosis factor and LT. As used herein, the term cytokine includes
proteins from natural sources or from recombinant cell culture and
biologically active equivalents of the native sequence
cytokines.
[0075] Chemokines generally act as chemoattractants to recruit
immune effector cells to the site of chemokine expression. It may
be advantageous to express a particular chemokine gene in
combination with, for example, a cytokine gene, to enhance the
recruitment of other immune system components to the site of
treatment. Chemokines include, but are not limited to, RANTES,
MCAF, MIP1-.alpha., MIP1-.beta., and IP-10. The skilled artisan
will recognize that certain cytokines are also known to have
chemoattractant effects and could also be classified under the term
chemokines.
[0076] C. Regulators of Programmed Cell Death
[0077] Apoptosis, or programmed cell death, is an essential process
for normal embryonic development, maintaining homeostasis in adult
tissues, and suppressing carcinogenesis (Kerr et al., 1972). The
Bcl-2 family of proteins and ICE-like proteases have been
demonstrated to be important regulators and effectors of apoptosis
in other systems. The Bcl-2 protein, discovered in association with
follicular lymphoma, plays a prominent role in controlling
apoptosis and enhancing cell survival in response to diverse
apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985;
Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce,
1986). The evolutionarily conserved Bcl-2 protein now is recognized
to be a member of a family of related proteins, which can be
categorized as death agonists or death antagonists.
[0078] Subsequent to its discovery, it was shown that Bcl-2 acts to
suppress cell death triggered by a variety of stimuli. Also, it now
is apparent that there is a family of Bcl-2 cell death regulatory
proteins that share in common structural and sequence homologies.
These different family members have been shown to either possess
similar functions to Bcl-2 (e.g., Bcl.sub.XL, Bcl.sub.W, Bcl.sub.S,
Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell
death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
[0079] Non-limiting examples of pro-apoptosis agents contemplated
within the scope of the present invention include granzyme B, Bax,
TNF-.alpha., TNF-.beta., TNF-like molecule, TGF-.beta., IL-12,
IL-3, IL-24, IL-18, TRAIL, IFN-.alpha., IFN-.beta., IFN-.gamma.,
Bcl-2, Fas ligand, caspases, gramicidin, magainin, mellitin,
defensin, cecropin, (KLAKLAK).sub.2 (SEQ ID NO:09), (KLAKKLA).sub.2
(SEQ ID NO:010), (KAAKKAA).sub.2 (SEQ ID NO:11) or (KLGKKLG).sub.3
(SEQ ID NO:12).
[0080] D. Angiogenic Inhibitors
[0081] In certain embodiments the present invention may concern
administration of targeting peptides attached to anti-angiogenic
agents, such as angiotensin, laminin peptides, fibronectin
peptides, plasminogen activator inhibitors, tissue
metalloproteinase inhibitors, interferons, interleukin 12, platelet
factor 4, IP-10, Gro-P, thrombospondin, 2-methoxyoestradiol,
proliferin-related protein, carboxiamidotriazole, CM101,
Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),
interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,
Linomide, thalidomide, pentoxifylline, genistein, TNP470,
endostatin, paclitaxel, accutin, angiostatin, cidofovir,
vincristine, bleomycin, AGM-1470, platelet factor 4 or
minocycline.
[0082] Proliferation of tumors cells relies heavily on extensive
tumor vascularization, which accompanies cancer progression. Thus,
inhibition of new blood vessel formation with anti-angiogenic
agents and targeted destruction of existing blood vessels have been
introduced as an effective and relatively non-toxic approach to
tumor treatment. (Arap et al., 1998; Arap et al., 1998; Ellerby et
al., 1999). A variety of anti-angiogenic agents and/or blood vessel
inhibitors are known (e.g., Folkman, 1997; Eliceiri and Cheresh,
2001).
[0083] E. Imaging Agents and Radioisotopes
[0084] In certain embodiments, the claimed targeting peptides or
proteins of the present invention may be attached to imaging agents
of use for imaging and diagnosis of various diseased organs,
tissues or cell types. Many appropriate imaging agents are known in
the art, as are methods for their attachment to proteins or
peptides (see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509, both
incorporated herein by reference). Certain attachment methods
involve the use of a metal chelate complex employing, for example,
an organic chelating agent such a DTP A attached to the protein or
peptide (U.S. Pat. No. 4,472,509). Proteins or peptides also may be
reacted with an enzyme in the presence of a coupling agent such as
glutaraldehyde or periodate. Conjugates with fluorescein markers
are prepared in the presence of these coupling agents or by
reaction with an isothiocyanate.
[0085] Non-limiting examples of paramagnetic ions of potential use
as imaging agents include chromium (III), manganese (II), iron
(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium
(III), samarium (III), ytterbium (III), gadolinium (III), vanadium
(II), terbium (III), dysprosium (III), holmium (III) and erbium
(III), with gadolinium being particularly preferred. Ions useful in
other contexts, such as X-ray imaging, include but are not limited
to lanthanum (III), gold (III), lead (II), and especially bismuth
(III).
[0086] Radioisotopes of potential use as imaging or therapeutic
agents include astatine.sup.211, .sup.14carbon, .sup.51chromium,
.sup.36chlorine, .sup.57cobalt, .sup.58cobalt, copper.sup.67,
.sup.152Eu, gallium.sup.67, .sup.3hydrogen, iodine.sup.123,
iodine.sup.125, iodine.sup.131, indium.sup.111, .sup.59iron,
.sup.32phosphorus, rhenium.sup.186, rhenium.sup.188,
.sup.75selenium, .sup.35sulphur, technicium.sup.99m and
yttrium.sup.90. .sup.125I is often being preferred for use in
certain embodiments, and technicium.sup.99m and indium.sup.111 are
also often preferred due to their low energy and suitability for
long range detection.
[0087] Radioactively labeled proteins or peptides of the present
invention may be produced according to well-known methods in the
art. For instance, they can be iodinated by contact with sodium or
potassium iodide and a chemical oxidizing agent such as sodium hypo
chlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
Proteins or peptides according to the invention may be labeled with
technetium-.sup.99m by ligand exchange process, for example, by
reducing pertechnate with stannous solution, chelating the reduced
technetium onto a Sephadex column and applying the peptide to this
column or by direct labeling techniques, e.g., by incubating
pertechnate, a reducing agent such as SNCl.sub.2, a buffer solution
such as sodium-potassium phthalate solution, and the peptide.
Intermediary functional groups that are often used to bind
radioisotopes that exist as metallic ions to peptides are
diethylenetriaminepenta-acetic acid (DTPA) and ethylene
diaminetetra-acetic acid (EDTA). Also contemplated for use are
fluorescent labels, including rhodamine, fluorescein isothiocyanate
and renographin.
[0088] In certain embodiments, the claimed proteins or peptides may
be linked to a secondary binding ligand or to an enzyme (an enzyme
tag) that will generate a colored product upon contact with a
chromogenic substrate. Examples of suitable enzymes include urease,
alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose
oxidase. Preferred secondary binding ligands are biotin and avidin
or streptavidin compounds. The use of such labels is well known to
those of skill in the art in light and is described, for example,
in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149 and 4,366,241; each incorporated herein by
reference.
[0089] F. Alkylating Agents
[0090] Alkylating agents are drugs that directly interact with
genomic DNA to prevent cells from proliferating. This category of
chemotherapeutic drugs represents agents that affect all phases of
the cell cycle, that is, they are not phase-specific. An alkylating
agent, may include, but is not limited to, a nitrogen mustard, an
ethylenimene, a methylmelamine, an alkyl sulfonate, a nitrosourea
or a triazines. They include but are not limited to: busulfan,
chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine,
ifosfamide, mechlorethamine (mustargen), and melphalan.
[0091] G. Antimetabolites
[0092] Antimetabolites disrupt DNA and RNA synthesis. Unlike
alkylating agents, they specifically influence the cell cycle
during S phase. Antimetabolites can be differentiated into various
categories, such as folic acid analogs, pyrimidinc analogs and
purine analogs and related inhibitory compounds. Antimetabolites
include but are not limited to, 5-fluorouracil (5-FU), cytarabinc
(Ara-C), fludarabine, gemcitabine, and methotrexate.
[0093] H. Natural Products
[0094] Natural products generally refer to compounds originally
isolated from a natural source, and identified as having a
pharmacological activity. Such compounds, analogs and derivatives
thereof may be, isolated from a natural source, chemically
synthesized or recombinantly produced by any technique known to
those of skill in the art. Natural products include such categories
as mitotic inhibitors, antitumor antibiotics, enzymes and
biological response modifiers.
[0095] Mitotic inhibitors include plant alkaloids and other natural
agents that can inhibit either protein synthesis required for cell
division or mitosis. They operate during a specific phase during
the cell cycle. Mitotic inhibitors include, for example, docetaxel,
etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine,
vincristine, and vinorelbine.
[0096] Taxoids are a class of related compounds isolated from the
bark of the ash tree, Taxus brevifolia. Taxoids include but are not
limited to compounds such as docetaxel and paclitaxel. Paclitaxel
binds to tubulin (at a site distinct from that used by the vinca
alkaloids) and promotes the assembly of microtubules.
[0097] Vinca alkaloids are a type of plant alkaloid identified to
have pharmaceutical activity. They include such compounds as
vinblastine (VLB) and vincristine.
[0098] I. Antibiotics
[0099] Certain antibiotics have both antimicrobial and cytotoxic
activity. These drugs also interfere with DNA by chemically
inhibiting enzymes and mitosis or altering cellular membranes.
These agents are not phase specific so they work in all phases of
the cell cycle. Examples of cytotoxic antibiotics include, but are
not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin
(Adriamycin), plicamycin (mithramycin) and idarubicin.
[0100] J. Miscellaneous Agents
[0101] Miscellaneous cytotoxic agents that do not fall into the
previous categories include, but are not limited to, platinum
coordination complexes, anthracenediones, substituted ureas, methyl
hydrazine derivatives, amsacrine, L-asparaginase, and tretinoin.
Platinum coordination complexes include such compounds as
carboplatin and cisplatin (cis-DDP). An exemplary anthracenedione
is mitoxantrone. An exemplary substituted urea is hydroxyurea. An
exemplary methyl hydrazine derivative is procarbazine
(N-methylhydrazine, MIH). These examples are not limiting and it is
contemplated that any known cytotoxic, cytostatic or cytocidal
agent may be attached to targeting peptides and administered to a
targeted organ, tissue or cell type within the scope of the
invention.
[0102] K. Dosages
[0103] The skilled artisan is directed to "Remington's
Pharmaceutical Sciences" 15th Edition, chapter 33, and in
particular to pages 624-652. Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any
event, determine the appropriate dose for the individual subject.
Moreover, for human administration, preparations should meet
sterility, pyrogenicity, and general safety and purity standards as
required by the FDA Office of Biologics Standards.
IV. Proteins and Peptides
[0104] In certain embodiments, the present invention concerns novel
compositions comprising at least one protein or peptide, such as
antigen-binding fragments or therapeutic peptides. These peptides
may be comprised in a fusion protein or conjugated to an agent as
described supra.
[0105] A. Proteins and Peptides
[0106] As used herein, a protein or peptide generally refers, but
is not limited to, a protein of greater than about 200 amino acids,
up to a full length sequence translated from a gene; a polypeptide
of greater than about 100 amino acids; and/or a peptide of from
about 3 to about 100 amino acids. For convenience, the terms
"protein," "polypeptide" and "peptide are used interchangeably
herein.
[0107] In certain embodiments the size of at least one protein or
peptide may comprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, about 130,
about 140, about 150, about 160, about 170, about 180, about 190,
about 200, about 210, about 220, about 230, about 240, about 250,
about 275, about 300, about 325, about 350, about 375, about 400,
about 425, about 450, about 475, about 500, about 525, about 550,
about 575, about 600, about 625, about 650, about 675, about 700,
about 725, about 750, about 775, about 800, about 825, about 850,
about 875, about 900, about 925, about 950, about 975, about 1000,
about 1100, about 1200, about 1300, about 1400, about 1500, about
1750, about 2000, about 2250, about 2500 or greater amino acid
residues.
[0108] As used herein, an "amino acid residue" refers to any
naturally occurring amino acid, any amino acid derivative or any
amino acid mimic known in the art. In certain embodiments, the
residues of the protein or peptide are sequential, without any
non-amino acid interrupting the sequence of amino acid residues. In
other embodiments, the sequence may comprise one or more non-amino
acid moieties. In particular embodiments, the sequence of residues
of the protein or peptide may be interrupted by one or more
non-amino acid moieties.
[0109] Accordingly, the term "protein or peptide" encompasses amino
acid sequences comprising at least one of the 20 common amino acids
found in naturally occurring proteins, or at least one modified or
unusual amino acid, including but not limited to those shown on
Table 1 below.
TABLE-US-00001 TABLE 1 Modified and Unusual Amino Acids Abbr. Amino
Acid Abbr. Amino Acid Aad 2-Aminoadipic acid EtAsn
N-Ethylasparagine Baad 3-Aminoadipic acid Hyl Hydroxylysine Bala
.beta.-alanine, AHyl allo-Hydroxylysine .beta.-Amino-propionic acid
Abu 2-Aminobutyric acid 3 Hyp 3-Hydroxypro line 4Abu 4-Aminobutyric
acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp 6-Aminocaproic
acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid Alle
allo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,
sarcosine Baib 3-Aminoisobutyric acid Melle N-Methylisoleucine Apm
2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric
acid MeYal N-Methylvaline Des Desmosine Nva Norvaline Dpm
2,2'-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionic
acid Orn Ornithine EtGly N-Ethylglycine
[0110] Proteins or peptides may be made by any technique known to
those of skill in the art, including the expression of proteins,
polypeptides or peptides through standard molecular biological
techniques, the isolation of proteins or peptides from natural
sources, or the chemical synthesis of proteins or peptides. The
nucleotide and protein, polypeptide and peptide sequences
corresponding to various genes have been previously disclosed, and
may be found at computerized databases known to those of ordinary
skill in the art. One such database is the National Center for
Biotechnology Information's Genbank and GenPept databases
(http://www.ncbi.nlm.nih.gov/). The coding regions for known genes
may be amplified and/or expressed using the techniques disclosed
herein or as would be known to those of ordinary skill in the art.
Alternatively, various commercial preparations of proteins,
polypeptides and peptides are known to those of skill in the
art.
[0111] B. Protein Purification
[0112] In certain embodiments a protein or peptide may be isolated
or purified. Protein purification techniques are well known to
those of skill in the art. These techniques involve, at one level,
the homogenization and crude fractionation of the cells, tissue or
organ to polypeptide and non-polypeptide fractions. The protein or
polypeptide of interest may be further purified using
chromatographic and electrophoretic techniques to achieve partial
or complete purification (or purification to homogeneity).
Analytical methods particularly suited to the preparation of a pure
peptide are ion-exchange chromatography, gel exclusion
chromatography, polyacrylamide gel electrophoresis, affinity
chromatography, immuno affinity chromatography and isoelectric
focusing. A particularly efficient method of purifying peptides is
fast performance liquid chromatography (FPLC) or even high
performance liquid chromatography (HPLC).
[0113] A purified protein or peptide is intended to refer to a
composition, isolatable from other components, wherein the protein
or peptide is purified to any degree relative to its
naturally-obtainable state. An isolated or purified protein or
peptide, therefore, also refers to a protein or peptide free from
the environment in which it may naturally occur. Generally,
"purified" will refer to a protein or peptide composition that has
been subjected to fractionation to remove various other components,
and which composition substantially retains its expressed
biological activity. Where the term "substantially purified" is
used, this designation will refer to a composition in which the
protein or peptide forms the major component of the composition,
such as constituting about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, or more of the proteins in the
composition.
[0114] Various methods for quantifying the degree of purification
of the protein or peptide are known to those of skill in the art in
light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity therein, assessed by a "-fold
purification number." The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification, and whether or not the
expressed protein or peptide exhibits a detectable activity.
[0115] Various techniques suitable for use in protein purification
are well known to those of skill in the art. These include, for
example, precipitation with ammonium sulphate, PEG, antibodies and
the like, or by heat denaturation, followed by: centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse
phase, hydroxylapatite and affinity chromatography; isoelectric
focusing; gel electrophoresis; and combinations of these and other
techniques. As is generally known in the art, it is believed that
the order of conducting the various purification steps may be
changed, or that certain steps may be omitted, and still result in
a suitable method for the preparation of a substantially purified
protein or peptide.
[0116] There is no general requirement that the protein or peptide
always be provided in their most purified state. Indeed, it is
contemplated that less substantially purified products will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. For example, it is appreciated that a cation-exchange
column chromatography performed utilizing an HPLC apparatus will
generally result in a greater "-fold" purification than the same
technique utilizing a low pressure chromatography system. Methods
exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining
the activity of an expressed protein.
[0117] Affinity chromatography is a chromatographic procedure that
relies on the specific affinity between a substance to be isolated
and a molecule to which it can specifically bind. This is a
receptor-ligand type of interaction. The column material is
synthesized by covalently coupling one of the binding partners to
an insoluble matrix. The column material is then able to
specifically adsorb the substance from the solution. Elution occurs
by changing the conditions to those in which binding will not occur
(e.g., altered pH, ionic strength, temperature, etc.). The matrix
should be a substance that itself does not adsorb molecules to any
significant extent and that has a broad range of chemical, physical
and thermal stability. The ligand should be coupled in such a way
as to not affect its binding properties. The ligand should also
provide relatively tight binding. And it should be possible to
elute the substance without destroying the sample or the
ligand.
V. Nucleic Acids
[0118] Nucleic acids according to the present invention may encode
a targeting peptide, a fusion protein, a therapeutic peptide, or
other protein or peptide. The nucleic acid may be derived from
genomic DNA, complementary DNA (cDNA) or synthetic DNA. Where
incorporation into an expression vector is desired, the nucleic
acid may also comprise a natural intron or an intron derived from
another gene. Such engineered molecules are sometime referred to as
"mini-genes."
[0119] A "nucleic acid" as used herein includes single-stranded and
double-stranded molecules, as well as DNA, RNA, chemically modified
nucleic acids and nucleic acid analogs. It is contemplated that a
nucleic acid within the scope of the present invention may be of
almost any size, determined in part by the length of the encoded
protein or peptide.
[0120] It is contemplated that targeting peptides, fusion proteins
and therapeutic peptides may be encoded by any nucleic acid
sequence that encodes the appropriate amino acid sequence. The
design and production of nucleic acids encoding a desired amino
acid sequence is well known to those of skill in the art, using
standardized codon tables. In preferred embodiments, the codons
selected for encoding each amino acid may be modified to optimize
expression of the nucleic acid in the host cell of interest. Codon
preferences for various species of host cell are well known in the
art.
[0121] In addition to nucleic acids encoding the desired peptide or
protein, the present invention encompasses complementary nucleic
acids that hybridize under high stringency conditions with such
coding nucleic acid sequences. High stringency conditions for
nucleic acid hybridization are well known in the art. For example,
conditions may comprise low salt and/or high temperature
conditions, such as provided by about 0.02 M to about 0.15 M NaCl
at temperatures of about 50 degree to about 70 degree. It is
understood that the temperature and ionic strength of a desired
stringency are determined in part by the length of the particular
nucleic acid(s), the length and nucleotide content of the target
sequence(s), the charge composition of the nucleic acid(s), and to
the presence or concentration of formamide, tetramethylammonium
chloride or other solvent(s) in a hybridization mixture.
VI. B-Cell Malignancy
[0122] In certain embodiments, the invention also provides a method
of treating a subject with a B-cell malignancy, which comprises
administering to the subject an effective amount of the B cell
targeting conjugates or compositions described herein. As used
herein, "subject" means any animal afflicted with a B-cell
malignancy. In preferred embodiments, the subject is a human. As
used herein, "treating" means either slowing, stopping or reversing
the progression of a B-cell malignancy. Other clinical parameters
may also be used to evaluate efficacy of treatment as are known by
the skilled clinician such as increased survival time, inhibition
of metastasis, and the like. In preferred embodiments, "treating"
means reversing the progression to the point of eliminating the
disorder. As used herein, "afflicted with or having a B-cell
malignancy" means that the subject harbors at least one cancerous
cell that expresses B-cell markers, including but not limited to
CD19 and CD22.
[0123] B cells are lymphocytes that play a large role in the
humoral immune response (as opposed to the cell-mediated immune
response, which is governed by T cells). The principal functions of
B cells are to make antibodies against antigens, perform the role
of Antigen Presenting Cells (APCs) and eventually develop into
memory B cells after activation by antigen interaction. B cells are
an essential component of the adaptive immune system.
[0124] The term "B-cell malignancy," and grammatical variants
thereof, are used in the broadest sense to refer to malignancies or
neoplasms of B cells that typically arise in lymphoid tissues, such
as bone marrow or lymph nodes, but may also arise in non-lymphoid
tissues, such as thyroid, gastrointestinal tract, salivary gland
and conjunctiva. The treatment methods of the present invention
specifically concern B-cell malignancies including, without
limitation, B-cell subtype of non-Hodgkin's lymphoma, Burkitt's
lymphoma, multiple myeloma, acute lymphoblastic leukemia (ALL),
chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia
(CML), hairy cell leukemia, and prolymphocytic leukemia.
[0125] B-cell type Non-Hodgkin's Lymphoma is a term that is used to
encompass a large group (over 29 types) of lymphomas caused by
malignant (cancerous) B-cell lymphocytes, and represents a large
subset of the known types of lymphoma. B-cells are known to undergo
many changes in their life cycle dependent on complex intracellular
signaling processes, and apparently different types of B-cell
malignancies can occur at different stages of the life cycle of
B-cells. At the stem cell stage, acute lymphocytic leukemia (ALL)
or lymphoblastic lymphoma/leukemia can typically develop. Precursor
B-cells can develop precursor B lymphoblastic lymphoma/leukemia.
Typical malignancies of immature B-cells include small non-cleaved
cell lymphoma and possibly Burkitt's/non-Burkitt's lymphoma. B
cells before antigen exposure typically develop chronic lymphocytic
leukemia (CLL) or small lymphocytic lymphoma, while after antigen
exposure typically follicular lymphomas, large cell lymphoma and
immunoblastic lymphoma are observed. There are also classification
systems that characterize B-cell lymphomas by the rate of growth
distinguishing aggressive (fast growing) and indolent (slow
growing) lymphomas. For example, Burkitt's/non-Burkitt's lymphoma
and LCL lymphoma belong in the aggressive group, while indolent
lymphomas include follicular center cell lymphomas (FCCL),
follicular large cell lymphomas, and follicular small cleaved cell
lymphomas.
[0126] Non-Hodgkin's Lymphomas are also characterized by the stage
of development. Stage I: cancer is found in only one lymph node
area, or in only one area or organ outside the lymph nodes. Stage
II: (1) Cancer is found in two or more lymph node areas on the same
side of the diaphragm (the thin muscle under the lungs that helps
breathing), or (2) cancer is found in only one area or organ
outside the lymph nodes and in the lymph nodes around it, or (3)
other lymph node areas on the same side of the diaphragm may also
have cancer. Stage III: Cancer is found in lymph node areas on both
sides of the diaphragm. The cancer may also have spread to an area
or organ near the lymph node areas and/or to the spleen. Stage IV:
(1) Cancer has spread to more than one organ or organs outside the
lymph system; cancer cells may or may not be found in the lymph
nodes near these organs, or (2) cancer has spread to only one organ
outside the lymph system, but lymph nodes far away from that organ
are involved.
[0127] B-cell chronic lymphocytic leukemia (also known as "chronic
lymphoid leukemia" or "CLL"), is a type of leukemia, or cancer of
the white blood cells (lymphocytes). CLL affects a particular
lymphocyte, the B cell, which originates in the bone marrow,
develops in the lymph nodes, and normally fights infection. In CLL,
the DNA of a B cell is damaged, so that it can't fight infection,
but it grows out of control and crowds out the healthy blood cells
that can fight infection.
[0128] Acute lymphoblastic leukemia (ALL), is a form of leukemia,
or cancer of the white blood cells characterized by excess
lymphoblasts. Malignant, immature white blood cells continuously
multiply and are overproduced in the bone marrow. ALL causes damage
and death by crowding out normal cells in the bone marrow, and by
spreading (metastasizing) to other organs. ALL is most common in
childhood with a peak incidence at 4-5 years of age, and another
peak in old age. The overall cure rate in children is 85%, and
about 50% of adults have long-term disease-free survival. `Acute`
refers to the undifferentiated, immature state of the circulating
lymphocytes ("blasts"), and to the rapid progression of disease,
which can be fatal in weeks to months if left untreated.
[0129] Current treatment options of B-cell malignancies, including
non-Hodgkin's lymphomas depend on the type and stage of malignancy.
Typical treatment regimens include radiation therapy, also referred
to as external beam therapy, chemotherapy, immunotherapy, and
combinations of these approaches. One promising approach is
radioimmunotherapy (RIT). With external beam therapy, a limited
area of the body is irradiated. With chemotherapy, the treatment is
systemic, and often adversely affects normal cells, causing severe
toxic side-effects. Targeted RIT is an approach in which a B-cell
specific antibody delivers a toxic substance to the site of tumor.
The therapeutic potential of RIT in patients with B-cell NHL has
been shown using different targets, including CD20, CD19, CD22, and
HLA-DR10 (Lym-1). More recently, combined modality therapy (CMT)
has become an increasingly frequent maneuver for the treatment of
solid tumors, and includes radiosensitization of cancer cells by
drugs, and the direct cytotoxic effect of chemotherapy. The most
common chemotherapy regiment for treating NHL is
Cyclophosphamide-Hydroxydoxorubicin-Oncovin
(vincristine)-Prednisone (CHOP) combination therapy. A randomized
study of aggressive, but early stage NHL showed superior results
with CHOP plus involved field radiation over treatment with CHOP
alone. Despite its promise, the disadvantage of treatments
involving external beam radiation is that external beam radiation
can only be delivered in high doses to a limited region of the
body, while NHL is mostly widespread. Accordingly, CMT has proven
clinically useful for locally advanced malignancies.
[0130] Another current approach is combined modality
radioimmunotherapy (CMRIT), which pairs the specific delivery of
systemic radiation (e.g., 90Y-DOTA-peptide-Lym-1) to NHL with the
systemic radiation sensitizing effects of an additional
chemotherapeutic agent. Because in CMRIT radiation is delivered
continuously, cancer cells that are hypoxic may re-oxygenate, or
pass through the radiosensitive G2/M phase of the cell cycle during
the course of treatment, making cure more likely. In addition,
CMRIT provides specificity first, by the specific targeting of NHL
by Lym-1, and second by timing. This allows the radiation
sensitizer to potentially synergize only at the sites targeted by
RIT, thus maximizing efficacy and minimizing toxicity. Several
previous xenograft studies have demonstrated improved synergy when
the radiation synthesizer (Taxol) was given 24-48 hours after
RIT.
[0131] Although CMRIT is currently viewed as the most advanced
therapeutic approach for the treatment of NHL, the engineered
conjugate (e.g., bispecific immunotoxin) of the present invention
alone have been demonstrated to provide superior results in terms
of tumor cell killing and overall survival, when tested in vitro
and in the well accepted Raji and Daudi lymphoma xenograft
models.
VII. Combination Treatments
[0132] In order to increase the effectiveness of a targeted
delivery of therapeutic agents to targeted cells such as B cells in
a subject, it may be desirable to combine these targeting
conjugates or compositions with other agents effective in the
treatment of a cancer or a B-cell malignancy, such as anti-cancer
agents.
[0133] An "anti-cancer" agent is capable of negatively affecting
cancer in a subject, for example, by killing cancer cells, inducing
apoptosis in cancer cells, reducing the growth rate of cancer
cells, reducing the incidence or number of metastases, reducing
tumor size, inhibiting tumor growth, reducing the blood supply to a
tumor or cancer cells, promoting an immune response against cancer
cells or a tumor, preventing or inhibiting the progression of
cancer, or increasing the lifespan of a subject with cancer. More
generally, these other compositions would be provided in a combined
amount effective to kill or inhibit proliferation of the cell. This
process may involve contacting the cells with the expression
construct and the agent(s) or multiple factor(s) at the same time.
This may be achieved by contacting the cell with a single
composition or pharmacological formulation that includes both
agents, or by contacting the cell with two distinct compositions or
formulations, at the same time, wherein one composition includes
the expression construct and the other includes the second
agent(s).
[0134] In the context of the present invention, it is contemplated
that the targeted therapy of the present invention could be used in
conjunction with chemotherapeutic, radiotherapeutic,
immunotherapeutic intervention, or other pro-apoptotic or cell
cycle regulating agents.
[0135] Alternatively, the targeted therapy may precede or follow
the other agent treatment by intervals ranging from minutes to
weeks. In embodiments where the other agent and expression
construct are applied separately to the cell, one would generally
ensure that a significant period of time did not expire between the
time of each delivery, such that the agent and expression construct
would still be able to exert an advantageously combined effect on
the cell. In such instances, it is contemplated that one may
contact the cell with both modalities within about 12-24 h of each
other and, more preferably, within about 6-12 h of each other. In
some situations, it may be desirable to extend the time period for
treatment significantly, however, where several d (2, 3, 4, 5, 6 or
7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations.
[0136] Various combinations may be employed, the targeted therapy
of the present invention is "A" and the secondary agent, such as
radio- or chemotherapy, is "B":
TABLE-US-00002 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/BBB B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0137] Administration of the therapeutic targeting conjugates of
the present invention to a patient will follow general protocols
for the administration of chemotherapeutics, taking into account
the toxicity, if any, of the targeting conjugates. It is expected
that the treatment cycles would be repeated as necessary. It also
is contemplated that various standard therapies, as well as
surgical intervention, may be applied in combination with the
described targeted cell therapy.
[0138] A. Chemotherapy
[0139] Cancer therapies also include a variety of combination
therapies with both chemical and radiation based treatments.
Combination chemotherapies include, for example, cisplatin (CDDP),
carboplatin, procarbazine, mechlorethamine, cyclophosphamide,
camptothecin, ifosfamide, melphalan, chlorambucil, busulfan,
nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,
plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene,
estrogen receptor binding agents, taxol, gemcitabien, navelbine,
farnesyl-protein tansferase inhibitors, transplatinum,
5-fluorouracil, vincristin, vinblastin and methotrexate, or any
analog or derivative variant of the foregoing.
[0140] B. Radiotherapy
[0141] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage on DNA, on the
precursors of DNA, on the replication and repair of DNA, and on the
assembly and maintenance of chromosomes. Dosage ranges for X-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0142] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0143] C. Immunotherapy
[0144] Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent to serve as a second targeting conjugate.
Alternatively, the effector may be a lymphocyte carrying a surface
molecule that interacts, either directly or indirectly, with a
tumor cell target. Various effector cells include cytotoxic T cells
and NK cells.
[0145] In certain aspects, the targeting conjugate may comprise an
antibody or fragment thereof for immunotherapy. Alternatively,
Immunotherapy could be used as part of a combined therapy, in
conjunction with the targeted therapy. The general approach for
combined therapy is discussed below. Generally, the tumor cell must
bear some additional marker that is amenable to targeting, i.e., is
not present on the majority of other cells. Many tumor markers
exist and any of these may be suitable for targeting in the context
of the present invention. Common tumor markers include
carcinoembryonic antigen, prostate specific antigen, urinary tumor
associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,
HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,
laminin receptor, erb B and p155.
[0146] D. Gene Therapy
[0147] In yet another embodiment, the secondary treatment is a gene
therapy in which a therapeutic polynucleotide is administered
before, after, or at the same time the targeting conjugate is
delivered. Delivery of a targeting conjugate in conjunction with a
vector encoding one of the following gene products will have a
combined anti-hyperproliferative effect on target tissues. A
variety of proteins are encompassed within the invention, some of
which are described below.
[0148] The tumor suppressor oncogenes function to inhibit excessive
cellular proliferation. The inactivation of these genes destroys
their inhibitory activity, resulting in unregulated proliferation.
For example, the tumor suppressors p53, p16 and C-CAM may be
used.
[0149] Another inhibitor of cellular proliferation is p16. The
major transitions of the eukaryotic cell cycle are triggered by
cyclin-dependent kinases, or CDKs. p16.sup.INK4 belongs to a newly
described class of CDK-inhibitory proteins that also includes
p16.sup.B, p19, p21 .sup.WAF1, and p27.sup.KIP1. Restoration of
wild-type p16.sup.INK4 function by transfection with a plasmid
expression vector reduced colony formation by some human cancer
cell lines (Okamoto, 1994; Arap, 1995).
[0150] Other genes that may be employed according to the present
invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,
zac1, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16
fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1,
TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fins, trk, ret, gsp,
hst, abl, p300, genes involved in angiogenesis (e.g., VEGF, FGF,
thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.
[0151] Regulators of programmed cell death may also be used in the
present invention for a combined therapy. Apoptosis, or programmed
cell death, is an essential process for normal embryonic
development, maintaining homeostasis in adult tissues, and
suppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family of
proteins and ICE-like proteases have been demonstrated to be
important regulators and effectors of apoptosis in other systems.
Subsequent to its discovery, it was shown that Bcl-2 acts to
suppress cell death triggered by a variety of stimuli. Also, it now
is apparent that there is a family of Bcl-2 cell death regulatory
proteins which share in common structural and sequence homologies.
These different family members have been shown to either possess
similar functions to Bcl-2 (e.g., Bcl.sub.XL, Bcl.sub.W, Bcl.sub.S,
Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell
death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
[0152] E. Surgery
[0153] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0154] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
microscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0155] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0156] F. Other Agents
[0157] It is contemplated that other agents may be used in
combination with the present invention to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adhesion, or agents that
increase the sensitivity of the hyperproliferative cells to
apoptotic inducers. Immunomodulatory agents include tumor necrosis
factor; interferon alpha, beta, and gamma; IL-2 and other
cytokines; F42K and other cytokine analogs; or MIP-1.alpha.,
MIP-1.beta., MCP-1, RANTES, and other chemokines. It is further
contemplated that the upregulation of cell surface receptors or
their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL would
potentiate the apoptotic inducing abilities of the present
invention by establishment of an autocrine or paracrine effect on
hyperproliferative cells. Increases intercellular signaling by
elevating the number of GAP junctions would increase the
anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with the present invention to improve the anti-hyperproliferative
efficacy of the treatments. Inhibitors of cell adhesion are
contemplated to improve the efficacy of the present invention.
Examples of cell adhesion inhibitors are focal adhesion kinase
(FAKs) inhibitors and Lovastatin. It is further contemplated that
other agents that increase the sensitivity of a hyperproliferative
cell to apoptosis, such as the antibody c225, could be used in
combination with the present invention to improve the treatment
efficacy.
[0158] Hormonal therapy may also be used in conjunction with the
present invention or in combination with any other cancer therapy
previously described. The use of hormones may be employed in the
treatment of certain cancers such as breast, prostate, ovarian, or
cervical cancer to lower the level or block the effects of certain
hormones such as testosterone or estrogen. This treatment is often
used in combination with at least one other cancer therapy as a
treatment option or to reduce the risk of metastases.
VIII. Pharmaceutical Compositions
[0159] Where clinical applications are contemplated, it may be
necessary to prepare pharmaceutical compositions--expression
vectors, virus stocks, proteins, antibodies and drugs--in a form
appropriate for the intended application. Generally, this will
entail preparing disclosed targeting conjugate compositions that
are essentially free of impurities that could be harmful to humans
or animals.
[0160] One generally will desire to employ appropriate salts and
buffers to render delivery vectors stable and allow for uptake by
target cells. Buffers also are employed when recombinant cells are
introduced into a patient. Aqueous compositions of the present
invention may comprise an effective amount of a protein, peptide,
fusion protein, recombinant phage and/or expression vector,
dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous medium. Such compositions also are referred to as innocula.
The phrase "pharmaceutically or pharmacologically acceptable"
refers to molecular entities and compositions that do not produce
adverse, allergic, or other untoward reactions when administered to
an animal or a human. As used herein, "pharmaceutically acceptable
carrier" includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
proteins or peptides of the present invention, its use in
therapeutic compositions is contemplated. Supplementary active
ingredients also can be incorporated into the compositions.
[0161] The active compositions of the present invention may include
classic pharmaceutical preparations. Administration of these
compositions according to the present invention are via any common
route so long as the target tissue is available via that route.
This includes oral, nasal, buccal, rectal, vaginal or topical.
Alternatively, administration may be by orthotopic, intradermal,
subcutaneous, intramuscular, intraperitoneal, intraarterial or
intravenous injection. Such compositions normally would be
administered as pharmaceutically acceptable compositions, described
supra.
[0162] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it is preferable to include isotonic
agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0163] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
IX. Kits
[0164] In various aspects of the invention, a kit is envisioned
containing therapeutic agents, diagnostic and/or delivery agents.
In some embodiments, the present invention contemplates a kit for
preparing and/or administering a therapy of the invention. The kit
may comprise one or more sealed vials containing any of the
pharmaceutical compositions of the present invention. In some
embodiments, the lipid is in one vial, and the nucleic acid
component is in a separate vial. The kit may include, for example,
at least one conjugate comprising a targeting moiety with a
V.sub.L-V.sub.H structure and a therapeutic agent, such as a toxin,
one or more lipid component, as well as reagents to prepare,
formulate, and/or administer the components of the invention or
perform one or more steps of the inventive methods. In some
embodiments, the kit may also comprise a suitable container means,
which is a container that will not react with components of the
kit, such as an eppendorf tube, an assay plate, a syringe, a
bottle, or a tube. The container may be made from sterilizable
materials such as plastic or glass.
[0165] The kit may further include an instruction sheet that
outlines the procedural steps of the methods set forth herein, and
will follow substantially the same procedures as described herein
or are known to those of ordinary skill. The instruction
information may be in a computer readable media containing
machine-readable instructions that, when executed using a computer,
cause the display of a real or virtual procedure of delivering a
pharmaceutically effective amount of a therapeutic agent.
X. Examples
[0166] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Construction of DT2219 Variants
[0167] For these studies, three different variations of DT2219 were
synthesized, DT2219EA, DT2219EB1, and DT2219ARL.
[0168] Construction of DT2219EA.
[0169] In 2005, the inventors reported an original DT2219 2760 bp
construct called DT2219EA constructed using DNA shuffling and
assembly PCR (Vallera et al., 2005). DT2219 consisted of an Ncol
restriction site at the N-terminus, followed by a downstream ATG
initiation codon, the first 389 amino acids of the DT (DT390), the
V.sub.H and V.sub.L regions of anti-CD22 (sFv) and anti-CD19
(Kipriyanov et al., 1996) linked by a 20 amino acid segment of
human muscle aldolase (hma), and a Xho1 compatible restriction site
(FIG. 1A). Importantly, Salvadore et al. (2002) reported that
mutating three amino acids Thr-His-Trp (THW) in place of
Ser-Ser-Tyr (SSY) at positions 100, 100A, and 100B in the CDR3
region of the V.sub.H of the anti-CD22 sFv enhanced its affinity,
so these same amino acids were mutated in the assembled plasmid
called pDT2219hmaEA.pET21d, which produced DT2219EA (enhanced
affinity). The synthesis of the DT gene fragment was previously
described (Chan et al., 1995) using diphtheria toxin CRM107 as a
template. The hma fragment was used as a non-immunogenic linker to
connect the two sFvs and was used to enhance the level of protein
production and ultimately the level of purity of the molecule. HMA
is 363 amino acids in length and the inventors used the final 20
amino acids (PSGQAGAAASESLFVSNHAY (SEQ ID NO:13)). DNA sequencing
analysis (University of Minnesota, Advanced Genetic Analysis
Center) was used to verify that the gene had been cloned in frame
and was correct in sequence.
[0170] Construction of DT2219EB1.
[0171] DT2219EB1 was created by mutating DT2219EA by modifying two
hot spot amino acids (S30G and N31R) in the anti-CD22 V.sub.L
region as previously reported by Ho et al. (2005). The sequence
change was verified.
[0172] Construction of DT2219ARL.
[0173] The hybrid gene encoding DT2219ARL was synthesized using
assembly PCR. The major differences between DT2219ARL and DT2219EA
were (1) reversal of the orientation of the V.sub.H and V.sub.L
chains. In DT2219ARL, the V.sub.L preceded the V.sub.H (FIG. 1A).
(2) The V.sub.L and V.sub.H genes of each sFv were conjoined by a
fragment encoding the ARL linker (GSTSGSGKPGSGEGSTKG (SEQ ID
NO:14)) and the two sFv genes were linked by a fragment encoding
G4S linker. In its final configuration, the DT2219ARL Nco1/Xho1
gene fragment encoded a start codon followed first by 389 aa of DT,
and then a 7 aa linker EASPEEA, followed by the anti-CD22 sFv, and
then the CD19 sFv. The final target gene was spliced into pET21d
vector expression vector and inclusion bodies expressed. A Food and
Drug Administration (FDA) Investigational New Drug (IND)
Application is now approved for the clinical phase I evaluation of
DT2219ARL.
[0174] As specificity controls for these studies, the inventors
constructed a bivalent fusion protein consisting of DT390 fused to
two repeating sFvs recognizing human CD3epsilon called Bic3
(Vallera et al., 2005). Anti-CD3epsilon recognizes a domain of the
T cell receptor (Vallera et al., 1996). An additional control
included DT390EpCam23, DT390 spliced to anti-EpCam sFv and
anti-ErbB2sFv sFv. Anti-EpCam and anti-ErbB2sFv have been used by
others to synthesize recombinant IT (Di Paolo et al., 2003; Batra
et al., 1991). ErbB2 is a tumor-associated antigen belonging to the
epidermal growth factor receptor family and implicated in poor
prognosis and more aggressive course in many human cancers
including breast, lung, ovary and stomach (Menard et al.,
2003).
Example 2
Expression and Purification of DT2219 Variants
[0175] To test activity of DT2219 variants, these recombinant
immunotoxins were expressed and purified. Plasmid was transformed
into the Escherichia coli strain BL21(DE3)(EMD, Madison Wis.).
Bacteria were grown in 600 ml Luria Broth supplemented with 100
.mu.g/ml carbenicillin in a 21 flask at 37.degree. C. with shaking.
Expression of the hybrid gene was induced by the addition of
isopropyl-b-D-thiogalactopyranoside (IPTG, FisherBiotech Fair Lawn,
N.J.). Two hours after induction, the bacteria were harvested by
centrifugation. The cell pellets were suspended and homogenized
using a polytron homogenizer. After sonication and centrifugation,
the pellets were extracted with 0.3% sodium deoxycholate, 5% Triton
X-100, 10% Glycerin, 50 mM Tris, 50 mM NaCl, 5 mM EDTA, pH 8.0 and
washed.
[0176] The proteins were refolded using a sodium
N-lauroyl-sarcosine (SLS) air oxidation method modified from a
previously reported procedure for isolating sFv (Vallera et al.,
2005). Refolded DT2219 variants were purified by FPLC ion exchange
chromatography (Q Sepharose Fast Flow, Sigma, St. Louis, Mo.) using
a continuous gradient from 0.2 M to 0.5 M NaCl in 20 mM Tris-HCl,
pH 9.0 over 4 column volumes.
[0177] Following ion exchange chromatography, 95 kDa DT2219ARL,
DT2219EA, and DT2219EB1 were greater than 95% pure as determined by
Coomassie blue staining (FIG. 1B).
Example 3
Cytotoxicity of Various Immunotoxins (IT) on the Daudi Cancer Cell
Line
[0178] To test cytotoxicity of various immunotoxins (IT), Daudi was
selected as a target cell line in these studies because flow
cytometry studies showed greater than 95% positivity for both CD19
and CD22. To determine the ability of DT2219ARL, DT2219EA, or
DT2219EB1 to kill Daudi, these IT were tested in a proliferation
assay and a representative experiment is shown (FIG. 2A). DT2219ARL
showed an IC.sub.50 of 0.2 nM. DT2219EA showed an IC.sub.50 of 0.4
nM. DT2219EB1 showed an IC.sub.50 of 0.1 nM. None of these curves
statistically differed. FIG. 2B shows a different experiment in
which DT2219ARL had no effect on CD22.sup.-CD19.sup.-HPBMLT T
leukemia cells. In contrast, HPBMLT were readily killed with an
anti-T cell IT called Bic3. To create the mutant DT2219GE gene, the
DT2219 gene was disrupted by a single glycine to aspartic acid
mutation at position 53 of the DT390 molecule known to inactivate
the catalytic activity of the DT A chain. Whereas parental
DT2219ARL showed an IC.sub.50 of 0.06 nM, the mutated DT2219GE
protein minimally inhibited Daudi proliferation. Together, these
data showed that DT2219 variants were potent and selective in their
ability to inhibit CD22.sup.+CD19.sup.+ target cells and that the
killing of DT2219 IT is caused by the DT moiety, not the 2219
moiety (FIG. 2C). Trypan blue viability assays were performed in
addition to proliferation assays and as an additional check to
verify that DT2219ARL was indeed killing and not simply inhibiting
cell proliferation/protein synthesis.
[0179] Furthermore, increased activity of DT2219ARL is due to the
presence of the anti-CD22 and anti-CD19 sFv ligands on a single
molecule. Proliferation assays were conducted in order to determine
if the increased activity of DT2219ARL was a result of the
increased number of binding molecules present on a bispecific IT.
FIG. 2D shows the data comparing the activity DT2219ARL to the
monospecific DT22 and DT19, as well as a combination of both
monospecific IT against Daudi cells. A mixture of DT22 and DT19
resulted in an identical number of ligands as are present in the
same concentration of DT2219ARL. Against Daudi, the monospecific
DT22 was able to kill with an IC.sub.50 of 3.05 nM. Monospecific
DT19 was less effective. However, DT2219ARL showed an IC.sub.50 of
0.15 nM, representing about a 1000-fold increase in activity as
compared to DT19 and a 20-fold increase in activity as compared to
DT22. Interestingly, a mixture of DT22 and DTIL19 showed no
increase in activity over DT22 alone. These data demonstrate the
superior activity of DT2219ARL is due to the presence of both
ligands on a single-chain molecule.
[0180] Antibodies and Cell Lines.
[0181] The anti-CD19 monoclonal antibody hybridoma HD37 that
secretes mouse IgG1 kappa has been previously described by Dorken
et al. (1983) and has been studied as a targeted toxin conjugated
to ricin toxin A chain (Stone et al., 1996). RFB4 (anti-CD22) was
provided by Dr. Ellen Vitetta, University of Texas Southwestern
Medical Center, Dallas, Tex. Anti-Ly5.2, a rat IgG2a from clone
A20-1.7, generously provided by Dr. Uli Hammerling, Sloan Kettering
Cancer Research Center, New York, N.Y. Anti-Ly5.2 was used as a
control since it recognized mouse CD45.1, a hematopoietic cell
surface marker not expressed on human cells.
[0182] Human cell lines included the CD19.sup.-CD22.sup.- T cell
leukemia HPBMLT Morikawa et al., 1978) and the CD22.sup.+19.sup.+
Burkitt's lymphomas Daudi (Klein et al., 1968) and Raji Pulbertaft,
1964). Raji was genetically altered by transfection with dual
reporter genes encoding both firefly luciferase and GFP creating
the Raji-luc cell line for imaging. Raji-luc was subcloned using
flow cytometric cell sorting in order to obtain stable
transfectants that were highly bio luminescent.
[0183] Measuring DT2219ARL Activity In Vitro.
[0184] To determine the effect of DT2219 on normal B and malignant
B cell function, the Daudi CD19.sup.+CD22.sup.+ Burkitt's lymphoma
cell line was used. Flow cytometry shows that Daudi is >98%
positive for both CD19 expression and CD22 expression. Cells
(10.sup.5) were plated in a 96-well flat-bottom plate in RPMI
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100
U/ml penicillin, 100 .mu.m/ml streptomycin. Immunotoxin in varying
concentrations was added to triplicate wells containing cells. The
plates were incubated at 37.degree. C., 5% CO.sub.2 for 72 h. Cells
were then incubated with 1 .mu.Ci [methyl-.sup.3H]-thymidine (GE
Healthcare, UK) per well for 8 h and harvested onto glass fiber
filters, washed, dried and counted for 10 min in a standard
scintillation counter. Data were analyzed using Prism 4 (GraphPad
Software, Inc.) and were presented as "percent control response"
calculated by dividing the cpm of untreated cells by the cpm of the
immunotoxin-treated cells (.times.100).
[0185] Statistical Analyses.
[0186] Groupwise comparisons of continuous data were made by
Student's t-test. A computer program for compiling life table and
statistical analysis by the Log-Rank test was used to analyze
survival data. Probability (p) values <0.05 were considered
significant
Example 4
Blocking DT2219 Activity
[0187] To confirm that the anti-CD19 sFv and anti-CD22 sFv ligands
were both still active in DT2219, blocking experiments were
performed with the parental RFB4 and HD37 monoclonal antibodies.
Proliferation experiments were performed in which increasing
amounts of blocking antibody were added to a constant inhibitory
concentration of 10 nM DT2219 immunotoxin which inhibited about 90%
of Daudi cell proliferation (IC.sub.90). FIG. 3A shows that
increasing concentrations of RFB4 or HD37 inhibit the proliferation
of 0 nM DT2219ARL in a dose-dependent manner. Saturation is reached
around 50 nM. The addition of an irrelevant control antibody
anti-Ly5.2 had no effect. Neither antibody blocked 100% of the
activity because blocking one ligand would not necessarily fully
block the other. Similar results were observed when DT2219EA or
DT2219EB1 were blocked in an identical fashion (FIGS. 3B-C). None
of the antibodies alone were stimulatory to Daudi cells at these
concentrations. Together, the similarity of these curves indicated
that that the anti-CD19 and anti-CD22 ligands on the DT2219
variants appeared to bind with similar monovalent affinity. Also,
both sFvs were active on the DT2219 molecules which were highly
specific.
[0188] Blocking studies were conducted to test the specificity of
DT2219ARL. Briefly, 0.5, 5, 50, or 500 nM RFB4 or HD37 were added
to media containing 10 nM DT2219EA, DT2219EB1, or DT2219ARL.
Resulting mixtures were added to wells containing Daudi cells and
proliferation was measured by H-thymidine uptake as described. The
mouse specific antibody Ly 5.2 was studied as a negative control.
Data were presented as "percent control response" as described
above.
Example 5
Target Cell Binding Flow Cytometry Studies
[0189] To determine whether the cytotoxicity data related to the
ability of the various immunotoxins to bind their target, the
recombinant immunotoxins were labeled with FITC and tested for
target cell binding with flow cytometry. Briefly, Table 2 shows
that DT2219ARL-FITC was highly reactive with normal human B cells
in the form of PBMC with a K.sub.d of 28 nM. For these studies, the
inventors gated the 7% B cells found in normal human peripheral
blood. These findings were confirmed by testing DT2219ARL against
magnetic bead enriched CD22.sup.+CD19.sup.+ B cells (enriched to
90%). The K.sub.d of the enriched cells was similar at 20 nM. When
DT2219ARL was tested against human malignant B cell lines in the
form of the Daudi and Raji cell lines, the K.sub.d of Daudi was 133
nM and Raji was 39 nM. These findings indicate that different
K.sub.ds can be anticipated on different cell lines, some lower
than others.
[0190] Finally, the inventors tested DT2219ARL binding against
human malignant B cells in the form of peripheral blood B-CLL from
two different patients. Patient 1 had a K.sub.d of 36, while
patient 2 had a K.sub.d of 181. Together, these data indicate that
DT2219ARL will bind malignant B cells from patients, but
patient-to-patient variation may be anticipated, perhaps due to
variances in CD22 and CD19 expression levels. As a negative control
the inventors tested the binding of irrelevant immunotoxins that do
not bind to human B cells, DTEpCam23-FITC and Bic3-FITC. They
showed a K.sub.d of 1879 and 1032 nM, respectively indicating that
the binding of DT2219ARL is specific. These data suggest that there
is no major difference in the binding of DT2219ARL to normal and
malignant B cells and that binding is specific.
[0191] DT2219ARL was labeled with FITC using the standard labeling
procedure and verified at 2-3 FITC molecules/DT2219ARL molecule.
Cells were incubated with DT2219ARL-FITC in the dark, washed, and
then run assayed on a Becton-Dickinson FACSCaliber. K.sub.d values
were determined using PRISM software. R2 values indicate how well
the regression plots fit data points.
TABLE-US-00003 TABLE 2 K.sub.d Values of DT2219ARL-FITC on Various
Cells on Malignant and Normal B Cells Kd (nM) R.sup.2 Normal B
Cells Human PBMC 28 0.90 Enriched CD22.sup.+ Cells 20 0.95
Malignant B Cells (Cell lines) Daudi 133 0.97 Raji 39 0.99
Malignant B Cells (Patient B-CLL cells) Patient 1 36 0.97 Patient 2
181 0.99 Negative Control IT Binding to Raji DTe23EpCAM-FITC 1879
0.99
[0192] To study the specific binding of DT2219ARL to cells, the
reactivity of DT2219ARL-FITC with human PBMC, monkey PBMC, and
magnetic bead enriched CD22.sup.+ human PBMC was compared (FIG. 4).
The top 3 panels show that CD22 enriched human PBMC were highly
reactive (79.1%) with a saturating concentration of DT2219ARL-FITC
(100 nM). A negative control DTEpCam23-FITC was not as reactive
with human CD22.sup.+ enriched PBMC. A control conventional
anti-CD22-FITC antibody (RFB4) was highly reactive. In the same
experiment, the lowest 3 panels show that DT2219ARL-FITC did not
recognize monkey PBMC (2.28%), even though the positive control
anti-CD22 antibody did recognize them (31.3%). The parental
anti-CD19 antibody HD37 did not recognize monkey cells. The middle
3 panels showed that DT2219ARL-FITC did recognize human PBMC. The
parental anti-CD22 and anti-CD19 monoclonal antibodies also
recognized B cells in the peripheral blood. Note that there are
considerably less positive B cells because these are PBMC and not a
CD22-enriched population.
[0193] Flow Cytometry Studies.
[0194] To determine comparative K.sub.ds, DT2219ARL-FITC and
control Bic3 (DT390 fused to two anti-CD3 sFv)-FITC and
DTe23EpCAM-FITC were reacted with Daudi cells, Raji cells, normal
human peripheral blood mononuclear cells (PBMC), normal human
CD22.sup.+, magnetic bead enriched PBMC, and patient CLL cells.
Cells were incubated with recombinant FITC-labeled proteins at
saturating concentrations for 45 minutes at 4.degree. C. Positive
cells were quantitated using a Becton Dickinson FACS Calibur.
K.sub.ds were calculated using PRISM software. DT2219ARL-FITC
reactivity was also determined with non-human primate cells. Rhesus
monkey PBMC cells were obtained through RAR, University of
Minnesota. As controls, binding was simultaneously assessed against
human PBMC and human CD22.sup.+, magnetic bead enriched PBMC
enriched using a CD22 isolation kit and a MACS system (Miltenyi
Biotec, Auburn, Calif.).
Example 6
Effects of DT2219 IT in SCID Mice with Systemic Cancer
[0195] Injection of the Daudi cells intravenously into SCID mice
results in a systemic tumor that infiltrates all major organs and
is reminiscent of human leukemia. To determine if DT2219ARL was
effective against established systemic leukemia and whether it
differed in its effectiveness from DT2219EB1, systemic cancer was
initiated in mice and ip treatments were started on day 3. FIG. 5A
shows that mice given 6 ip injections of DT2219ARL survived
significantly longer than mice given treatment with DT2219EB1 or
untreated mice (p<0.001). All of the DT2219ARL treated mice
survived to day 90 when the experiments were terminated. All of the
untreated control mice and the DT2219EB1 treated mice were dead by
day 90. Three of five mice given DT2219EB1 died early by day 10
with weight loss indicating that DT2219EB1 is more toxic than
DT2219ARL. FIG. 5B shows a different experiment in which mice were
given 11 injections of DT2219ARL. Again, all of these mice survived
150 days compared to groups of mice treated with control Bic3 or
untreated controls (p<0.001). DT2219EA also showed enhanced
survival, but over 60% of these mice were dead by day 80. No early
toxic deaths resulted from DT2219EA or DT2219ARL treatment. FIG. 5C
shows that when mice are given a single dose of DT2219ARL survival
is significantly better than untreated controls (p<0.01).
DT2219EA also shows a protective effect that is not as pronounced
as the effect with DT2219ARL.
[0196] To study a second human B-cell malignancy in a model which
could be imaged in real time, Raji-luc was injected intravenously
into SCIDs to induce systemic cancer. FIG. 6A shows that tumor
progressed quickly since it was detected in all untreated mice
(n=4/group) by day 12-18 and all untreated mice developed hind limb
paralysis on days 18 through 46. Animals injected with Raji-luc
develop this CNS complication with a 100% incidence. Three of four
(75%) of the DT2219ARL treated mice were completely tumor-free on
day 87. Tumor progressed in one of the treated mice on day 18. FIG.
6B shows the total photon activity graphed over time for each
mouse. In FIG. 6C, three additional mice were injected with
Raji-luc and not treated in order to study the aggressive nature of
the tumor with a dual reporter gene cell line. Luciferase
bioluminescent imaging showed these animals all developed tumor by
day 14. GFP imaging of the same mice on day 21 confirmed tumor
presence in lung and immune system and revealed the tumor had a
propensity for the bone marrow and spinal cord.
[0197] Together, these data indicate that DT2219ARL was able to
prevent the onset of established fatal systemic cancer in two
highly aggressive human B-cell malignancy models in SCID mice.
[0198] Mouse Efficacy Studies.
[0199] Female SCID/hu mice were purchased from NCI, Frederick
Cancer Research and Development Center, Animal Production Area and
housed in an AAALAC-accredited specific pathogen-free facility
under the care of the Department of Research Animal Resources,
University of Minnesota. Animal research protocols were approved by
the University of Minnesota Institutional Animal Care and Use
Committee (IACUC). Animals were housed in microisolator cages to
minimize the possibility of transmission of any contaminating
virus.
[0200] In Experiment 1 (FIG. 5A), 10.sup.6 Daudi cells in 200 .mu.l
sterile, endotoxin-free PBS were injected intravenously into SCID
mice via caudal vein. After Daudi injection on day 0, one 20 .mu.g
IV injection of DT2219ARL or DT2219EB1 was given on day 3 with five
subsequent 10 .mu.g/200 .mu.l ip injections on days 5, 10, 24, 26
and 31. Body weights were documented three times per week. Since
this Daudi substrain always metastasizes to the central nervous
system resulting in hind-limb paralysis (HLP), paralyzed mice were
deemed pre-terminal and euthanized by University approved IACUC
procedures.
[0201] In Experiment 2 (FIG. 5B), mice were given 10.sup.6 Daudi
cells IV on day 0 and divided into groups. Groups of 7 mice (no
treatment group, n=5) were given ip injections of 20 .mu.g/200
.mu.l DT2219ARL, DT2219EA, or Bic3 on days 3 and 6; 10 .mu.g/200
.mu.l ip injections on days 21, 24, 26, 31, 38, 47, 54, 56 and
59.
[0202] In Experiment 3 (FIG. 5C), mice were given 10.sup.6 Daudi
cells IV on day 0. A single 20 .mu.s ip injection of DT2219ARL or
DT2219EA was given on day 3. Body weights were determined. Mice
exhibiting hind limb paralysis were euthanized.
[0203] In Experiment 4 (FIG. 6), to test the efficacy of DT2219ARL
against a different CD22.sup.+CD19.sup.+ human B-cell malignancy,
mice were given 10.sup.6 Raji-luc cells IV on day 0 and then were
treated with a single 20 .mu.g ip injection of drug on days 3, 5,
11, 16, and 18. Mice were imaged on day 5, 12, 18, 25, 32, 39, 46,
60, 74, and 87. Images were captured using Xenogen Ivis imaging
system (Xenogen Corporation, Hopkington Mass.) and analyzed with
IGOR Pro 4.09a software (WaveMetrics, Inc., Portaland, Oreg.).
Prior to imaging, mice were anesthetized using isoflorane gas. All
mice received 100 .mu.l of a 30 mg/ml D-luciferin aqueous solution
(Gold Biotechnology, St. Louis, Mo.) as a substrate for luciferase
10 minutes before imaging. All images represent a 5 minute exposure
time and all regions of interest (ROI) are expressed in units of
photons/sec/cm.sup.2/sr.
Example 7
Effects of DT2219ARL in Rabbits
[0204] To determine the maximum tolerated dose (MTD) of DT2219ARL,
rabbits were injected with a course of 4 IV injections given every
other day. Rabbits were given either 50, 100, 200, or 500 .mu.g/kg.
Two animals were treated at each dosage. FIG. 7A shows the average
weight loss and reveals a minimal effect at 50 or 100 .mu.g/kg
treatment as compared to the untreated controls. At 200 .mu.g/kg
there was a more pronounced weight loss amounting to less than 20%
of starting weight that is not considered life threatening by the
IACUC. Weight loss is likely due to non-specific toxicities of the
agent, becoming obvious at higher doses. These are likely due to
non-specific uptake of DT390, primarily causing liver damage.
[0205] BUN (Blood-Urea-Nitrogen) levels correlated with increasing
concentrations of DT2219ARL with only the 500 .mu.g/kg dosage
causing elevations. This increase only reached 100 mg/dL and was
considered minimal and histology confirmed mild effects in the
kidneys. In contrast, DT2219ARL was highly damaging to liver at 500
.mu.g/kg, but not at 200 .mu.g/kg. Not only did these animals lose
weight, but FIG. 7B shows a precipitous rise in ALT levels
indicating dose-dependent hepatic damage. Damage was confirmed by
histology studies which showed necrosis and fatty degeneration
consistent with grade 4 liver damage (FIG. 7C). Together, the ALT,
histology, and weight data confirmed that the dose-dependent
toxicity of DT2219ARL was likely due to non-specific uptake of
DT390, primarily causing liver damage. The 50,000 .mu.g/kg dosage
damaged the liver and the MTD was 200 .mu.g/kg. Histology studies
of the heart, lung, and spleen did not show any evidence of
cellular damage or toxicity.
[0206] In the rabbit studies, female New Zealand White rabbits (2.2
kg) were purchased from Bakkom Rabbitry (Viroqua, Wis.) and housed
under the care of Resource Animal Resources as described above.
Catheters were implanted in the ears for IV drug administration.
Animals were weighed to determine the effect of drug on weight.
Blood samples were obtained centrifuged immediately at 5000 rpm.
Individual serum samples were analyzed on a Kodak ETA-CHEM 950 by
the Clinical Chemistry Laboratory, University of Minnesota
Hospital, Fairview (Minneapolis, Minn.). The BUN assays were read
spectrophotometrically at 670 nm. In the ALT assay, the oxidation
of NADH was used to measure ALT activity at 340 nm.
Example 8
Design and Cytotoxity of 2219KDEL and 2219KDEL 7 Mutants
[0207] This design of bispecific ligand-directed toxin (BLT) also
works when the inventors used truncated pseudomonas exotoxin fused
to 2219 scFvs instead of DT390. The PE has been genetically
engineered to reduce its immunogenicity as reported by the Ira
Pastan Lab (Alderson et al., 2009).
[0208] In order to determine if other toxins could be used, the
inventors bioengineered a nucleotide sequence to express 2219KDEL
(protein sequence is SEQ ID NO:5; encoded by SEQ ID NO:6). In this
instance, they fused the same 2219 scFvs from DT2219 to truncated
PE. The amino acids KDEL were added to replace REDLK at the
C-terminus of PE since this has been previously shown to enhance ER
retention and enhance toxicity. Another mutant was created called
2219KDEL 7mut (protein sequence is SEQ ID NO:7; encoded by SEQ ID
NO:8) in which 8 hydrophilic amino acids on PE were mutated to
reduce its immunogenicity (Onda et al., 2008; Pastan et al., 2009).
Both 2219KDEL and 2219KDEL 7mut were tested for their ability to
inhibit Daudi cell proliferation in vitro (FIG. 8) and showed high
cytotoxicity on Daudi cells.
Example 9
Clinical Trial of Phase I Study of DT2219ARL
[0209] Phase I clinical study was designed to determine the MTD
(maximum tolerated dose) of DT2219ARL for treatment of chemotherapy
refractory or relapsed and bone marrow transplant ineligible or
bone marrow transplant relapsed B-cell lineage leukemia or relapsed
B-cell lineage lymphoma. It is anticipated that approximately 36
patients will be needed for this phase I evaluation and that this
study should be completed within 3 years.
[0210] Immunophenotypic analysis of lymphoblasts from children and
adults with B-lineage ALL demonstrate that virtually all (>95%)
have expression of CD19 and >80% express CD22 (Frankel et al.,
2002; Bene, 2005). The marked prevalence of both markers on the
tumor cells should allow for effective targeting of DT2219ARL.
[0211] DT2219ARL protein is supplied frozen in sterile 1 mL
colorless, type I glass vials with an 11 mm rubber stopper and an
aluminum seal ring and is formulated at 1 mg drug in 1 mL of 0.15 M
NaCl/10 mM sodium phosphate+0.5% Polysorbate 80, pH 7.4. Lot
#120706 will be used for this trial. Vials used in drug preparation
were sterile pyrogen-free, 1 mL (Hollister Stier, Miles Inc.
#280090-M01).
[0212] Patients will be screened for eligibility and after
eligibility is confirmed and informed consent obtained,
pretreatment labs will be done and patients will be admitted.
Patients will begin on prophylactic allopurinol 300 mg po qd. Each
day prior to treatment, patients will receive acetaminophen 325 mg
po, diphenhydramine 25 mg IV (12.5 mg IV for weight <25 kg),
hydrocortisone 100 mg IV (50 mg IV for weight <25 kg), rantidine
50 mg IV (1 mg/kg for weight <50 kg), and normal saline 1 L IV
over five hours (20 mL/kg for weight <50 kg).
[0213] DT2219ARL will be administered into a free-flowing IV over a
period of 4 hour QOD.times.4. Vital signs including blood pressure,
pulse, temperature, respirations, and pulse oximetry will be
measured every 15-30 minutes for one hour and then hourly for 5
hours and then q 4-8 hours while hospitalized. Patients will be
closely monitored for toxicities. Careful I/O will be measured.
Additional blood will be collected pre-treatment and every 2-3 days
for 1 week and then on days 15 and 28 for anti-DT2219ARL and
DT2219ARL levels. Blood counts and chemistries will be measured
every 2-3 days for 1 week and at days 15 and 28. Supportive
measures will include acetaminophen for fevers, meperidine for
chills, anti-emetics for nausea and vomiting, normal saline or
furosemide to maintain fluid balance/blood pressure/pulmonary
function, electrolyte replacement, albumin to maintain serum
albumin at 3 g/dL or greater. Anaphylactoid reactions will be
treated with 100 mg methylprednisolone IV, diphenhydramine 25 mg
IV, or 0.3 cc epinephrine (1:1000) IV and transfer to an ICU
setting for monitoring. One cycle of treatment will be given.
[0214] The selection of the starting dose for this trial is based
on the two species toxicology and prior Phase I trials using
diphtheria toxins and recombinant chain ITs described in an earlier
section (Vallera et al., 2005). The DT2219ARL starting dose is 0.5
.mu.g/kg/d ( 1/400th the MTD in rats and rabbits) for patient #1.
Dose will be escalated to 1.25 .mu.g/kg/d for patient #2 and 2.5
.mu.g/kg/d for patient #3. The lower doses in single patient
cohorts are to identify the risk of capillary leak syndrome
toxicities prior to expansion into the higher dose cohorts. These
patients are to be treated sequentially, where dosing of the next
higher cohort may proceed after completion of the first dosing
cycle. A clinical monitoring plan to identify capillary leak
syndrome will include evidence of orthostatic hypotension
unresponsive to two normal saline boluses or serum albumin <3
g/dL unresponsive to a single 0.5 g/kg albumin infusion. In
addition, any drug-related grade 2 toxicity will necessitate
expansion to a 3 patient cohort and subsequent dose escalation by
33%. If no drug-related grade 2 toxicity is observed in the first
three patients, subsequent dose escalation will be by 100% until
evidence of biological activity (grade 2 drug related toxicity) is
observed. At that point, dose escalation will be decreased to about
35% increments. If signs of marrow recovery are observed prior to
the second week of observation and/or signs of drug-related
toxicity have resolved to less than grade 2, additional patients
could be added to the cohorts at an earlier time point. No patient
will be entered on an escalating dosage level until at least 3
patients have been treated at the previous level and observed for
toxicity for at least 3 weeks after the last dose of treatment.
[0215] Dose-limiting toxicity (DLT) is defined as any drug-related
grade 3 or higher toxicity. Dose levels will be escalated in
cohorts of three patients as long as no drug-related
non-hematologic toxicity >grade 3 is observed and marrow
recovery is sufficiently rapid. If one patient is observed to
suffer >grade 3 drug-related toxicity, the cohort will be
expanded. If not more than one patient in the expanded cohort of
six patients experience drug-related DLT, dose escalation will
resume. If two patients enrolled at the same dose level in a cohort
of up to six patients experience drug-related DLT, the MTD has been
exceeded, and dose escalations will cease. The next lower dose
level will be considered the MTD and three additional patients
treated at the newly defined MTD level to determine an accurate
toxicity profile. If no patients in the expanded cohort at the
lower MTD experience DLT, a one-half step escalation (about 17%)
will be added to more carefully define the MTD. Responses will be
based on response criteria for therapeutic trials of leukemia with
clearance of marrow and peripheral blasts and recovery of normal
hematopoiesis (Pui and Evans, 2006) or by RECIST criteria for
lymphomas (Cheson, 2008).
[0216] Correlative studies include the evaluation of response and
toxicity in relation to pre-treatment leukemia burden, prior
treatments, patient age, sex, leukemia cytogenetics, leukemia and
lymphoma CD19 and CD22 antigen densities, PK and antibody
levels.
[0217] Post-treatment assessment will include bone marrow as
appropriate for ALL leukemia and lymphoma assessment, PET CT for
lymphoma assessment, cardiac ejection fraction if appropriate,
blood counts and chemistries.
[0218] Table 3 is description of the patients that have been
treated with FDA IND-Approved DT2219ARL. Patients were all in
cancer relapse and treated intravenously every-other-day for a
total of 4 treatments (QOD.times.4). Study is incomplete and still
accruing patients. Patients according to the approved protocol were
ALL or CLL (Ages 20-71) and positive for either CD19 or CD22. Four
dose levels have now been completed: 0.5 .mu.g/kg, 1.25 .mu.g/kg,
2.5 .mu.g/kg, and 5 .mu.g/kg. Generally, the findings are the same
at all of these low dose levels in all 7 patients. The drug is safe
at these low doses since bloodwork and enzymes (not shown) showed
no evidence of toxicity. There have been no responses, a finding
that correlates with the pk data showing that no drug was in the
serum (not shown). It is safe to continue with the phase 1 dose
escalation.
TABLE-US-00004 TABLE 3 Completion Status of Phase I Study of
DT2219ARL (IND# 100780) Pt. Disease Status Dosage # of Doses
Completion Pre- CD CD # Facility Age Gen. Race Diagnosis prior to
Therapy mcg/kg Received Status Response TreatBlast % 19 22 01
S&W 64 M White CLL Fludarabine 0.5 4 OffStudy- Disease 1 58 1
Refractory (Day 28) Progression 02 S&W 71 M White CLL
Fludarabine 1.25 4 OffStudy- Disease -- 99 1 Refractory (Day 15)
Progression 03 MDACC 23 M Hisp ALL Relapsed 2.5 4 Off Study-
Disease 91 93 97 pay 9) Progression 04 MDACC 20 M Hisp ALL Relapsed
5.0 4 Off study No 97 100 64 (Day 8) response 05 MDACC 32 F Hisp
ALL Relapsed 5.0 4 Off study No 76 -- 79 (Day 8) response 06 MDACC
42 F White ALL Relapsed 5.0 4 Off study No 95 95 -- (Day 15)
response 07 S&W 11 F White ALL Relapsed/ 5.0 2 Off study No 97
86 5 Refractory (Day 5) response
[0219] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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Sequence CWU 1
1
141904PRTArtificial SequenceSynthetic peptide 1Met Gly Ala Asp Asp
Val Val Asp Ser Ser Lys Ser Phe Val Met Glu 1 5 10 15 Asn Phe Ser
Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile 20 25 30 Gln
Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp 35 40
45 Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala
50 55 60 Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala
Gly Gly 65 70 75 80 Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val
Leu Ala Leu Lys 85 90 95 Val Asp Asn Ala Glu Thr Ile Lys Lys Glu
Leu Gly Leu Ser Leu Thr 100 105 110 Glu Pro Leu Met Glu Gln Val Gly
Thr Glu Glu Phe Ile Lys Arg Phe 115 120 125 Gly Asp Gly Ala Ser Arg
Val Val Leu Ser Leu Pro Phe Ala Glu Gly 130 135 140 Ser Ser Ser Val
Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu 145 150 155 160 Ser
Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln 165 170
175 Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val
180 185 190 Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp
Trp Asp 195 200 205 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser
Leu Lys Glu His 210 215 220 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser
Pro Asn Lys Thr Val Ser 225 230 235 240 Glu Glu Lys Ala Lys Gln Tyr
Leu Glu Glu Phe His Gln Thr Ala Leu 245 250 255 Glu His Pro Glu Leu
Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 260 265 270 Val Phe Ala
Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 275 280 285 Val
Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 290 295
300 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly
305 310 315 320 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser
Ile Ala Leu 325 330 335 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu
Val Gly Glu Leu Val 340 345 350 Asp Ile Gly Phe Ala Ala Tyr Asn Phe
Val Glu Ser Ile Ile Asn Leu 355 360 365 Phe Gln Val Val His Asn Ser
Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 370 375 380 His Lys Thr Gln Pro
Phe Glu Ala Ser Gly Gly Pro Glu Asp Ile Gln 385 390 395 400 Met Thr
Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val 405 410 415
Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp 420
425 430 Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr
Thr 435 440 445 Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser 450 455 460 Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
Glu Gln Glu Asp Phe 465 470 475 480 Ala Thr Tyr Phe Cys Gln Gln Gly
Asn Thr Leu Pro Trp Thr Phe Gly 485 490 495 Gly Gly Thr Lys Leu Glu
Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys 500 505 510 Pro Gly Ser Gly
Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val Glu 515 520 525 Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys 530 535 540
Ala Ala Ser Gly Phe Ala Phe Ser Ile Tyr Asp Met Ser Trp Val Arg 545
550 555 560 Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Tyr Ile Ser
Ser Gly 565 570 575 Gly Gly Thr Thr Tyr Tyr Pro Asp Thr Val Lys Gly
Arg Phe Thr Ile 580 585 590 Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
Leu Gln Met Ser Ser Leu 595 600 605 Lys Ser Glu Asp Thr Ala Met Tyr
Tyr Cys Ala Arg His Ser Gly Tyr 610 615 620 Gly Thr His Trp Gly Val
Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu 625 630 635 640 Val Thr Val
Ser Ala Gly Gly Gly Gly Ser Asp Ile Leu Leu Thr Gln 645 650 655 Thr
Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser 660 665
670 Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn
675 680 685 Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile
Tyr Asp 690 695 700 Ala Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe
Ser Gly Ser Gly 705 710 715 720 Ser Gly Thr Asp Phe Thr Leu Asn Ile
His Pro Val Glu Lys Val Asp 725 730 735 Ala Ala Thr Tyr His Cys Gln
Gln Ser Thr Glu Asp Pro Trp Thr Phe 740 745 750 Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg Gly Ser Thr Ser Gly Ser 755 760 765 Gly Lys Pro
Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu 770 775 780 Gln
Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val Lys Ile 785 790
795 800 Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn
Trp 805 810 815 Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly
Gln Ile Trp 820 825 830 Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys
Phe Lys Gly Lys Ala 835 840 845 Thr Leu Thr Ala Asp Glu Ser Ser Ser
Thr Ala Tyr Met Gln Leu Ser 850 855 860 Ser Leu Ala Ser Glu Asp Ser
Ala Val Tyr Phe Cys Ala Arg Arg Glu 865 870 875 880 Thr Thr Thr Val
Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 885 890 895 Gly Thr
Ser Val Thr Val Ser Ser 900 22715DNAArtificial SequenceSynthetic
primer 2atgggcgctg atgatgttgt tgattcttct aaatcttttg tgatggaaaa
cttttcttcg 60taccacggga ctaaacctgg ttatgtagat tccattcaaa aaggtataca
aaagccaaaa 120tctggtacac aaggaaatta tgacgatgat tggaaagggt
tttatagtac cgacaataaa 180tacgacgctg cgggatactc tgtagataat
gaaaacccgc tctctggaaa agctggaggc 240gtggtcaaag tgacgtatcc
aggactgacg aaggttctcg cactaaaagt ggataatgcc 300gaaactatta
agaaagagtt aggtttaagt ctcactgaac cgttgatgga gcaagtcgga
360acggaagagt ttatcaaaag gttcggtgat ggtgcttcgc gtgtagtgct
cagccttccc 420ttcgctgagg ggagttctag cgttgaatat attaataact
gggaacaggc gaaagcgtta 480agcgtagaac ttgagattaa ttttgaaacc
cgtggaaaac gtggccaaga tgcgatgtat 540gagtatatgg ctcaagcctg
tgcaggaaat cgtgtcaggc gatcagtagg tagctcattg 600tcatgcataa
atcttgattg ggatgtcata agggataaaa ctaagacaaa gatagagtct
660ttgaaagagc atggccctat caaaaataaa atgagcgaaa gtcccaataa
aacagtatct 720gaggaaaaag ctaaacaata cctagaagaa tttcatcaaa
cggcattaga gcatcctgaa 780ttgtcagaac ttaaaaccgt tactgggacc
aatcctgtat tcgctggggc taactatgcg 840gcgtgggcag taaacgttgc
gcaagttatc gatagcgaaa cagctgataa tttggaaaag 900acaactgctg
ctctttcgat acttcctggt atcggtagcg taatgggcat tgcagacggt
960gccgttcacc acaatacaga agagatagtg gcacaatcaa tagctttatc
gtctttaatg 1020gttgctcaag ctattccatt ggtaggagag ctagttgata
ttggtttcgc tgcatataat 1080tttgtagaga gtattatcaa tttatttcaa
gtagttcata attcgtataa tcgtcccgcg 1140tattctccgg ggcataaaac
gcaaccattt gaagcttccg gaggtcccga ggatattcaa 1200atgactcaaa
ctacttcttc tttgtctgct tctttgggtg atagagttac tatttcttgt
1260agagcttctc aagatatttc taactacttg aactggtacc aacaaaagcc
agatggtact 1320gttaagttgt tgatttacta cacttccatt ttgcattctg
gtgttccatc tagattctct 1380ggttctggtt ctggtactga ttactctttg
actatttcta acttggaaca agaagatttc 1440gctacttact tctgtcaaca
aggtaatact ttgccatgga ctttcggtgg tggtactaag 1500ttggaaatta
agggtagcac ctctggctcc ggaaaaccgg gaagcggtga agggtccacc
1560aagggtgaag ttcaattggt tgaatctggt ggtggtttgg ttaagccagg
tggttctttg 1620aagttgtctt gtgctgcttc tggtttcgct ttctctattt
acgatatgtc ttgggttaga 1680caaactccag aaaagagatt ggaatgggtt
gcttacattt cttctggtgg tggtactact 1740tactacccag atactgttaa
gggtagattc actatttcta gagataacgc taagaacact 1800ttgtacctgc
aaatgtcttc tctgaagtct gaagataccg ctatgtacta ctgtgctaga
1860cattccggtt acggtaccca ctggggtgtt ttgttcgctt actggggtca
aggtactttg 1920gttactgttt ctgctggtgg cggtggatcc gatatcttgc
tcacccaaac tccagcttct 1980ttggctgtgt ctctagggca gagggccacc
atctcctgca aggccagcca aagtgttgat 2040tatgatggtg atagttattt
gaactggtac caacagattc caggacagcc acccaaactc 2100ctcatctatg
atgcatccaa tctagtttct gggattccac ccaggtttag tggcagtggg
2160tctgggacag acttcaccct caacatccat cctgtggaga aggtggatgc
tgcaacctat 2220cactgtcagc aaagtactga agatccgtgg acgttcggtg
gaggcaccaa gctggaaatc 2280aaacggggta gcacctctgg ctccggaaaa
ccgggaagcg gtgaagggtc caccaagggt 2340caggtgcagc tgcagcagtc
tggggctgag ctggtgaggc ctgggtcctc agtgaagatt 2400tcctgcaagg
cttctggcta tgcattcagt agctactgga tgaactgggt gaagcagagg
2460cctggacagg gtcttgagtg gattggacag atttggcctg gagatggtga
tactaactac 2520aatggaaagt tcaagggtaa agccactctg actgcagacg
aatcctccag cacagcctac 2580atgcaactca gcagcctagc atctgaggac
tctgcggtct atttctgtgc aagacgggag 2640actacgacgg taggccgtta
ttactatgct atggactact ggggtcaagg aacctcagtc 2700accgtctcct catag
27153390PRTArtificial SequenceSynthetic peptide 3Met Gly Ala Asp
Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu 1 5 10 15 Asn Phe
Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile 20 25 30
Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp 35
40 45 Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala
Ala 50 55 60 Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys
Ala Gly Gly 65 70 75 80 Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys
Val Leu Ala Leu Lys 85 90 95 Val Asp Asn Ala Glu Thr Ile Lys Lys
Glu Leu Gly Leu Ser Leu Thr 100 105 110 Glu Pro Leu Met Glu Gln Val
Gly Thr Glu Glu Phe Ile Lys Arg Phe 115 120 125 Gly Asp Gly Ala Ser
Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly 130 135 140 Ser Ser Ser
Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu 145 150 155 160
Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln 165
170 175 Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg
Val 180 185 190 Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu
Asp Trp Asp 195 200 205 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu
Ser Leu Lys Glu His 210 215 220 Gly Pro Ile Lys Asn Lys Met Ser Glu
Ser Pro Asn Lys Thr Val Ser 225 230 235 240 Glu Glu Lys Ala Lys Gln
Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 245 250 255 Glu His Pro Glu
Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 260 265 270 Val Phe
Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 275 280 285
Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 290
295 300 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp
Gly 305 310 315 320 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln
Ser Ile Ala Leu 325 330 335 Ser Ser Leu Met Val Ala Gln Ala Ile Pro
Leu Val Gly Glu Leu Val 340 345 350 Asp Ile Gly Phe Ala Ala Tyr Asn
Phe Val Glu Ser Ile Ile Asn Leu 355 360 365 Phe Gln Val Val His Asn
Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 370 375 380 His Lys Thr Gln
Pro Phe 385 390 41170DNAArtificial SequenceSynthetic primer
4atgggcgctg atgatgttgt tgattcttct aaatcttttg tgatggaaaa cttttcttcg
60taccacggga ctaaacctgg ttatgtagat tccattcaaa aaggtataca aaagccaaaa
120tctggtacac aaggaaatta tgacgatgat tggaaagggt tttatagtac
cgacaataaa 180tacgacgctg cgggatactc tgtagataat gaaaacccgc
tctctggaaa agctggaggc 240gtggtcaaag tgacgtatcc aggactgacg
aaggttctcg cactaaaagt ggataatgcc 300gaaactatta agaaagagtt
aggtttaagt ctcactgaac cgttgatgga gcaagtcgga 360acggaagagt
ttatcaaaag gttcggtgat ggtgcttcgc gtgtagtgct cagccttccc
420ttcgctgagg ggagttctag cgttgaatat attaataact gggaacaggc
gaaagcgtta 480agcgtagaac ttgagattaa ttttgaaacc cgtggaaaac
gtggccaaga tgcgatgtat 540gagtatatgg ctcaagcctg tgcaggaaat
cgtgtcaggc gatcagtagg tagctcattg 600tcatgcataa atcttgattg
ggatgtcata agggataaaa ctaagacaaa gatagagtct 660ttgaaagagc
atggccctat caaaaataaa atgagcgaaa gtcccaataa aacagtatct
720gaggaaaaag ctaaacaata cctagaagaa tttcatcaaa cggcattaga
gcatcctgaa 780ttgtcagaac ttaaaaccgt tactgggacc aatcctgtat
tcgctggggc taactatgcg 840gcgtgggcag taaacgttgc gcaagttatc
gatagcgaaa cagctgataa tttggaaaag 900acaactgctg ctctttcgat
acttcctggt atcggtagcg taatgggcat tgcagacggt 960gccgttcacc
acaatacaga agagatagtg gcacaatcaa tagctttatc gtctttaatg
1020gttgctcaag ctattccatt ggtaggagag ctagttgata ttggtttcgc
tgcatataat 1080tttgtagaga gtattatcaa tttatttcaa gtagttcata
attcgtataa tcgtcccgcg 1140tattctccgg ggcataaaac gcaaccattt
11705362PRTArtificial SequenceSynthetic peptide 5Pro Glu Gly Gly
Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His 1 5 10 15 Leu Pro
Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu 20 25 30
Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr 35
40 45 Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
Asn 50 55 60 Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu
Ala Ile Arg 65 70 75 80 Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr
Leu Ala Ala Ala Glu 85 90 95 Ser Glu Arg Phe Val Arg Gln Gly Thr
Gly Asn Asp Glu Ala Gly Ala 100 105 110 Ala Asn Ala Asp Val Val Ser
Leu Thr Cys Pro Val Ala Ala Gly Glu 115 120 125 Cys Ala Gly Pro Ala
Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr 130 135 140 Pro Thr Gly
Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser 145 150 155 160
Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His 165
170 175 Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly
Thr 180 185 190 Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val
Arg Ala Arg 195 200 205 Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe
Tyr Ile Ala Gly Asp 210 215 220 Pro Ala Leu Ala Tyr Gly Tyr Ala Gln
Asp Gln Glu Pro Asp Ala Arg 225 230 235 240 Gly Arg Ile Arg Asn Gly
Ala Leu Leu Arg Val Tyr Val Pro Arg Ser 245 250 255 Ser Leu Pro Gly
Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu 260 265 270 Ala Ala
Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg 275 280 285
Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr 290
295 300 Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser
Ala 305 310 315 320 Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu
Asp Pro Ser Ser 325 330 335 Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala
Leu Pro Asp Tyr Ala Ser 340 345 350 Gln Pro Gly Lys Pro Pro Lys Asp
Glu Leu 355 360 6 1089DNAArtificial SequenceSynthetic primer
6cccgagggcg gcagcctggc cgcgctgacc gcgcaccagg cttgccacct
gccgctggag
60actttcaccc gtcatcgcca gccgcgcggc tgggaacaac tggagcagtg cggctatccg
120gtgcagcggc tggtcgccct ctacctggcg gcgcggctgt cgtggaacca
ggtcgaccag 180gtgatccgca acgccctggc cagccccggc agcggcggcg
acctgggcga agcgatccgc 240gagcagccgg agcaggcccg tctggccctg
accctggccg ccgccgagag cgagcgcttc 300gtccggcagg gcaccggcaa
cgacgaggcc ggcgcggcca acgccgacgt ggtgagcctg 360acctgcccgg
tcgccgccgg tgaatgcgcg ggcccggcgg acagcggcga cgccctgctg
420gagcgcaact atcccactgg cgcggagttc ctcggcgacg gcggcgacgt
cagcttcagc 480acccgcggca cgcagaactg gacggtggag cggctgctcc
aggcgcaccg ccaactggag 540gagcgcggct atgtgttcgt cggctaccac
ggcaccttcc tcgaagcggc gcaaagcatc 600gtcttcggcg gggtgcgcgc
gcgcagccag gacctcgacg cgatctggcg cggtttctat 660atcgccggcg
atccggcgct ggcctacggc tacgcccagg accaggaacc cgacgcacgc
720ggccggatcc gcaacggtgc cctgctgcgg gtctatgtgc cgcgctcgag
cctgccgggc 780ttctaccgca ccagcctgac cctggccgcg ccggaggcgg
cgggcgaggt cgaacggctg 840atcggccatc cgctgccgct gcgcctggac
gccatcaccg gccccgagga ggaaggcggg 900cgcctggaga ccattctcgg
ctggccgctg gccgagcgca ccgtggtgat tccctcggcg 960atccccaccg
acccgcgcaa cgtcggcggc gacctcgacc cgtccagcat ccccgacaag
1020gaacaggcga tcagcgccct gccggactac gccagccagc ccggcaaacc
gccgaaggac 1080gagctatga 10897877PRTArtificial SequenceSynthetic
peptide 7Met Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala
Ser Leu 1 5 10 15 Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
Asp Ile Ser Asn 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
Gly Thr Val Lys Leu Leu 35 40 45 Ile Tyr Tyr Thr Ser Ile Leu His
Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu 65 70 75 80 Gln Glu Asp Phe
Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro 85 90 95 Trp Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser 100 105 110
Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val 115
120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser
Leu 130 135 140 Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile
Tyr Asp Met 145 150 155 160 Ser Trp Val Arg Gln Thr Pro Glu Lys Arg
Leu Glu Trp Val Ala Tyr 165 170 175 Ile Ser Ser Gly Gly Gly Thr Thr
Tyr Tyr Pro Asp Thr Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 195 200 205 Met Ser Ser Leu
Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg 210 215 220 His Ser
Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala Tyr Trp Gly 225 230 235
240 Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Asp Ile
245 250 255 Leu Leu Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly
Gln Arg 260 265 270 Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp
Tyr Asp Gly Asp 275 280 285 Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro
Gly Gln Pro Pro Lys Leu 290 295 300 Leu Ile Tyr Asp Ala Ser Asn Leu
Val Ser Gly Ile Pro Pro Arg Phe 305 310 315 320 Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val 325 330 335 Glu Lys Val
Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu Asp 340 345 350 Pro
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser 355 360
365 Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
370 375 380 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro
Gly Ser 385 390 395 400 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Ala Phe Ser Ser Tyr 405 410 415 Trp Met Asn Trp Val Lys Gln Arg Pro
Gly Gln Gly Leu Glu Trp Ile 420 425 430 Gly Gln Ile Trp Pro Gly Asp
Gly Asp Thr Asn Tyr Asn Gly Lys Phe 435 440 445 Lys Gly Lys Ala Thr
Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 450 455 460 Met Gln Leu
Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 465 470 475 480
Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 485
490 495 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ala Ser
Gly 500 505 510 Gly Pro Glu Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr
Ala His Gln 515 520 525 Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg
His Arg Gln Pro Arg 530 535 540 Gly Trp Glu Gln Leu Glu Gln Cys Gly
Tyr Pro Val Gln Arg Leu Val 545 550 555 560 Ala Leu Tyr Leu Ala Ala
Arg Leu Ser Trp Asn Gln Val Asp Gln Val 565 570 575 Ile Ala Asn Ala
Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu 580 585 590 Ala Ile
Arg Glu Ser Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala 595 600 605
Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu 610
615 620 Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val
Ala 625 630 635 640 Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp
Ala Leu Leu Glu 645 650 655 Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu
Gly Asp Gly Gly Asp Val 660 665 670 Ser Phe Ser Thr Arg Gly Thr Gln
Asn Trp Thr Val Glu Arg Leu Leu 675 680 685 Gln Ala His Arg Gln Leu
Glu Glu Gly Gly Tyr Val Phe Val Gly Tyr 690 695 700 His Gly Thr Phe
Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val 705 710 715 720 Arg
Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Ala Gly Phe Tyr Ile 725 730
735 Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro
740 745 750 Asp Ala Ala Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val
Tyr Val 755 760 765 Pro Arg Ser Ser Leu Pro Gly Phe Tyr Ala Thr Ser
Leu Thr Leu Ala 770 775 780 Ala Pro Glu Ala Ala Gly Glu Val Glu Arg
Leu Ile Gly His Pro Leu 785 790 795 800 Pro Leu Arg Leu Asp Ala Ile
Thr Gly Pro Glu Glu Ser Gly Gly Arg 805 810 815 Leu Glu Thr Ile Leu
Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile 820 825 830 Pro Ser Ala
Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp 835 840 845 Pro
Ser Ser Ile Pro Asp Ser Glu Gln Ala Ile Ser Ala Leu Pro Asp 850 855
860 Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys Asp Glu Leu 865 870 875
82634DNAArtificial SequenceSynthetic primer 8atggatattc aaatgactca
aactacttct tctttgtctg cttctttggg tgatagagtt 60actatttctt gtagagcttc
tcaagatatt tctaactact tgaactggta ccaacaaaag 120ccagatggta
ctgttaagtt gttgatttac tacacttcca ttttgcattc tggtgttcca
180tctagattct ctggttctgg ttctggtact gattactctt tgactatttc
taacttggaa 240caagaagatt tcgctactta cttctgtcaa caaggtaata
ctttgccttg gactttcggt 300ggtggtacta agttggaaat taagggtagc
acctctggct ccggaaaacc gggaagcggt 360gaagggtcca ccaagggtga
agttcaattg gttgaatctg gtggtggttt ggttaagcca 420ggtggttctt
tgaagttgtc ttgtgctgct tctggtttcg ctttctctat ttacgatatg
480tcttgggtta gacaaactcc agaaaagaga ttggaatggg ttgcttacat
ttcttctggt 540ggtggtacta cttactaccc agatactgtt aagggtagat
tcactatttc tagagataac 600gctaagaaca ctttgtacct gcaaatgtct
tctctgaagt ctgaagatac cgctatgtac 660tactgtgcta gacattccgg
ttacggtacc cactggggtg ttttgttcgc ttactggggt 720caaggtactt
tggttactgt ttctgctggt ggcggtggat ccgatatctt gctcacccaa
780actccagctt ctttggctgt gtctctaggg cagagggcca ccatctcctg
caaggccagc 840caaagtgttg attatgatgg tgatagttat ttgaactggt
accaacagat tccaggacag 900ccacccaaac tcctcatcta tgatgcatcc
aatctagttt ctgggattcc acccaggttt 960agtggcagtg ggtctgggac
agacttcacc ctcaacatcc atcctgtgga gaaggtggat 1020gctgcaacct
atcactgtca gcaaagtact gaagatccgt ggacgttcgg tggaggcacc
1080aagctggaaa tcaaacgggg tagcacctct ggctccggaa aaccgggaag
cggtgaaggg 1140tccaccaagg gtcaggtgca gctgcagcag tctggggctg
agctggtgag gcctgggtcc 1200tcagtgaaga tttcctgcaa ggcttctggc
tatgcattca gtagctactg gatgaactgg 1260gtgaagcaga ggcctggaca
gggtcttgag tggattggac agatttggcc tggagatggt 1320gatactaact
acaatggaaa gttcaagggt aaagccactc tgactgcaga cgaatcctcc
1380agcacagcct acatgcaact cagcagccta gcatctgagg actctgcggt
ctatttctgt 1440gcaagacggg agactacgac ggtaggccgt tattactatg
ctatggacta ctggggtcaa 1500ggaacctcag tcaccgtctc ctcagaagct
tccggaggtc ccgagcccga gggcggcagc 1560ctggccgcgc tgaccgcgca
ccaggcttgc cacctgccgc tggagacttt cacccgtcat 1620cgccagccgc
gcggctggga acaactggag cagtgcggct atccggtgca gcggctggtc
1680gccctctacc tggcggcgcg gctgtcgtgg aaccaggtcg accaggtgat
cgccaacgcc 1740ctggccagcc ccggcagcgg cggcgacctg ggcgaagcga
tccgcgagtc gccggagcag 1800gcccgtctgg ccctgaccct ggccgccgcc
gagagcgagc gcttcgtccg gcagggcacc 1860ggcaacgacg aggccggcgc
ggccaacgcc gacgtggtga gcctgacctg cccggtcgcc 1920gccggtgaat
gcgcgggccc ggcggacagc ggcgacgccc tgctggagcg caactatccc
1980actggcgcgg agttcctcgg cgacggcggc gacgtcagct tcagcacccg
cggcacgcag 2040aactggacgg tggagcggct gctccaggcg caccgccaac
tggaggaggg aggctatgtg 2100ttcgtcggct accacggcac cttcctcgaa
gcggcgcaaa gcatcgtctt cggcggggtg 2160cgcgcgcgca gccaggacct
cgacgcgatc tgggccggtt tctatatcgc cggcgatccg 2220gcgctggcct
acggctacgc ccaggaccag gaacccgacg cagccggccg gatccgcaac
2280ggtgccctgc tgcgggtcta tgtgccgcgc tcgagcctgc cgggcttcta
cgccaccagc 2340ctgaccctgg ccgcgccgga ggcggcgggc gaggtcgaac
ggctgatcgg ccatccgctg 2400ccgctgcgcc tggacgccat caccggcccc
gaggagtcag gcgggcgcct ggagaccatt 2460ctcggctggc cgctggccga
gcgcaccgtg gtgattccct cggcgatccc caccgacccg 2520cgcaacgtcg
gcggcgacct cgacccgtcc agcatccccg actcggaaca ggcgatcagc
2580gccctgccgg actacgccag ccagcccggc aaaccgccga aggacgagct atga
2634914PRTArtificial SequenceSynthetic peptide 9Lys Leu Ala Lys Leu
Ala Lys Lys Leu Ala Lys Leu Ala Lys 1 5 10 1014PRTArtificial
SequenceSynthetic peptide 10Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala
Lys Lys Leu Ala 1 5 10 1114PRTArtificial SequenceSynthetic peptide
11Lys Ala Ala Lys Lys Ala Ala Lys Ala Ala Lys Lys Ala Ala 1 5 10
1221PRTArtificial SequenceSynthetic peptide 12Lys Leu Gly Lys Lys
Leu Gly Lys Leu Gly Lys Lys Leu Gly Lys Leu 1 5 10 15 Gly Lys Lys
Leu Gly 20 1320PRTArtificial SequenceSynthetic peptide 13Pro Ser
Gly Gln Ala Gly Ala Ala Ala Ser Glu Ser Leu Phe Val Ser 1 5 10 15
Asn His Ala Tyr 20 1418PRTArtificial SequenceSynthetic peptide
14Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1
5 10 15 Lys Gly
* * * * *
References