U.S. patent application number 14/051816 was filed with the patent office on 2014-05-29 for nucleic acids encoding biologically active polypeptides derived from a novel early stage pregnancy factor designated maternin (ma).
This patent application is currently assigned to NOBEL BIOSCIENCES LLC. The applicant listed for this patent is NOBEL BIOSCIENCES LLC. Invention is credited to Joseph Bryant, Robert C. Gallo, Yanto Lunardi-Iskandar.
Application Number | 20140148582 14/051816 |
Document ID | / |
Family ID | 26845258 |
Filed Date | 2014-05-29 |
United States Patent
Application |
20140148582 |
Kind Code |
A1 |
Gallo; Robert C. ; et
al. |
May 29, 2014 |
NUCLEIC ACIDS ENCODING BIOLOGICALLY ACTIVE POLYPEPTIDES DERIVED
FROM A NOVEL EARLY STAGE PREGNANCY FACTOR DESIGNATED MATERNIN
(MA)
Abstract
The invention relates to nucleotides encoding therapeutic
polypeptides and fragments thereof isolated from beta-human
chorionic gonadotropin (.beta.-hCG) found in early pregnancy urine,
now synthetically produced and designated Maternin. The therapeutic
polypeptides and their functional equivalents are useful in
treating and/or preventing various medical conditions. Examples of
therapeutic effects of the therapeutic polypeptides include
anti-HIV, anti-cancer, anti-wasting, pro-hematopoietic (e.g.,
anemias, radiation-mediated bone marrow damage, and trauma-mediated
blood loss), and anti-angiogenic effects. The invention also
provides pharmaceutical compositions comprising the therapeutic
polypeptides, as well as methods for using the therapeutic
polypeptides, functional equivalents and/or pharmaceutical
compositions in the treatment and/or prevention of such medical
conditions.
Inventors: |
Gallo; Robert C.; (Bethesda,
MD) ; Bryant; Joseph; (Baltimore, MD) ;
Lunardi-Iskandar; Yanto; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOBEL BIOSCIENCES LLC |
Gaithersburg |
MD |
US |
|
|
Assignee: |
NOBEL BIOSCIENCES LLC
Gaithersburg
MD
|
Family ID: |
26845258 |
Appl. No.: |
14/051816 |
Filed: |
October 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13159285 |
Jun 13, 2011 |
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14051816 |
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09632831 |
Aug 4, 2000 |
7994278 |
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13159285 |
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60188777 |
Mar 13, 2000 |
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60147825 |
Aug 6, 1999 |
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Current U.S.
Class: |
530/387.3 ;
435/331; 530/387.9 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/59 20130101; A61K 48/00 20130101; C07K 2319/00 20130101;
C07K 16/26 20130101 |
Class at
Publication: |
530/387.3 ;
435/331; 530/387.9 |
International
Class: |
C07K 16/26 20060101
C07K016/26 |
Goverment Interests
1. FEDERAL FUNDING
[0002] The research leading to at least one aspect of the invention
described herein was made with the support of the U.S. government,
under National Institute of Health Grant No. PO1CA78817. The U.S.
government may have certain rights to aspects of the invention
described herein.
Claims
1-115. (canceled)
116. An isolated antibody, or a fragment of an antibody, which
specifically binds to a peptide selected from the group consisting
of: (a) MA (SEQ ID NO: 2); (b) pMA (SEQ ID NO: 3); (c) MA.sub.S1
(SEQ ID NO: 4); (d) MA.sub.S2 (SEQ ID NO: 5); (e) MA.sub.S3 (SEQ ID
NO: 6); (f) MA.sub.S5 (SEQ ID NO: 7); (g) MA.sub.S9 (SEQ ID NO: 8);
(h) MA.sub.S10 (SEQ ID NO: 9); (i) MA.sub.S11 (SEQ ID NO: 10); (j)
.beta.-hCG 55-88 (SEQ ID NO: 11); (k) .beta.-hCG 55-90 (SEQ ID NO:
12); (l) .beta.-hCG 55-91 (SEQ ID NO: 13); (m) .beta.-hCG 55-74
(SEQ ID NO: 14); (n) .beta.-hCG 6-37 (SEQ ID NO: 15); (o)
.beta.-hCG 6-38 (SEQ ID NO: 16); (p) .beta.-hCG 6-39 (SEQ ID NO:
17); and (q) .beta.-hCG 6-40 (SEQ ID NO: 18).
117. The antibody of claim 116, wherein the antibody is a
monoclonal antibody.
118. The antibody of claim 116, wherein the antibody is a humanized
antibody.
119. A cell producing the antibody of claim 116, produced by the
fusion of an immortal cell line to an immunoglobulin-producing
plasma cell producing said monoclonal antibody.
120-123. (canceled)
Description
2. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional U.S. patent application
Ser. No. 13/159,285, filed Jun. 13, 2011, which is a divisional of
U.S. patent application Ser. No. 09/632,831, filed Aug. 4, 2000,
now U.S. Pat. No. 7,994,278, which claims priority from U.S.
Provisional Patent Application No. 60/147,825, filed Aug. 6, 1999,
and U.S. Provisional Patent Application No. 60/188,777, filed Mar.
13, 2000; the entire disclosure of these applications is
incorporated herein by reference.
2.1. SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 4, 2014, is named 9272-102DIV2_SL.txt and is 22,073 bytes
in size.
3. FIELD OF THE INVENTION
[0004] The invention relates to therapeutic polypeptides useful in
treating and/or preventing various medical conditions. Examples of
therapeutic effects of the therapeutic polypeptides include
anti-HIV, anti-cancer, anti-wasting, pro-hematopoietic (e.g.,
anemias, radiation or chemotherapy-mediated bone marrow damage, and
trauma-mediated blood loss), anti-angiogenic and anti-inflammatory
effects. The invention also provides pharmaceutical compositions
comprising the therapeutic polypeptides, as well as methods for
using the therapeutic polypeptides, functional equivalents and/or
pharmaceutical compositions in the treatment and/or prevention of
such medical conditions.
4. BACKGROUND OF THE INVENTION
[0005] This invention is the culmination of a lengthy research
endeavor that began with an observation that a particular group of
immunodeficient mice, inoculated with Kaposi's sarcoma (KS) cells,
did not develop KS as expected. The researchers extracted KS cells
from AIDS patients, grew the cells in vitro and then injected the
cells into the immunodeficient mice, expecting the mice to develop
KS. While most mice injected with the cells did develop KS, the
researchers surprisingly identified a group of ten mice in which KS
failed to develop.
[0006] In attempting to explain the failure of these mice to
develop KS, the researchers first considered whether the anomaly
was the result of a laboratory error, such as a failure to properly
inject the mice. Closer observation revealed that all ten
KS-negative mice were pregnant. A group of females and males had
been mistakenly housed together. While the male mice had grown
large malignant tumors, the females had only small tumors or no
tumors at all. Further observation indicated that females with
small tumors were in later stages of pregnancy, and those with no
tumors were in early stages of pregnancy. Based on these collective
observations, the researchers surmised that one or more factors
produced in the early stages of pregnancy was responsible for
impeding the development of KS lesions.
[0007] In the ensuing months, the group designed and executed a
controlled study of the effects of pregnancy on KS. The results of
the study were published in the May 4, 1995, issue of Nature..sup.1
In this study, the researchers injected KS cells into 24 female
mice and 21 male mice. Four of the female mice were then caged with
2 males. All 4 females became pregnant. One month later, the males
had developed large malignant tumors; and as expected, females in
late stages of their pregnancy had developed smaller tumors, while
newly pregnant females were tumor free.
[0008] These results confirmed the group's suspicions that a
pregnancy-related factor was responsible for the inhibition of KS.
The group noted that the pattern of no KS lesions in early stages
of pregnancy and increasingly larger lesions at later stages of
pregnancy inversely corresponds to the production of human
chorionic gonadotropin (hCG). High levels of hCG are produced
during early pregnancy, while lower levels are produced in later
stages of pregnancy. The researchers investigated the anti-KS
activity of crude commercial hCG preparations. These investigations
revealed that some lots of some commercially available crude hCG
preparations exhibited the same anti-KS effect seen in the pregnant
mice.
[0009] In a related study published in the Oct. 24, 1996 issue of
The New England Journal of Medicine,.sup.2 the investigators tested
hCG preparations on 30 human subjects. The results were compared
with 6 others who were given a placebo. Twenty-four subjects
received intralesional injections of preparations of hCG at doses
of 250, 500, 1000, or 2000 international units (IU) three times a
week for 2 weeks. In 5 of the 6 subjects who received the 2000 IU
hCG dose, KS lesions shrank significantly or completely
disappeared. Lesions in at least one subject in each of the 250,
500, and 1000 dose ranges also shrank or disappeared. This study
provided further evidence that some lots of some commercial hCG
preparations exhibit anti-KS effects. The results also demonstrated
that certain crude commercial preparations of hCG can reduce or
reverse symptoms of KS in humans.
[0010] In another related experiment, the researchers administered
a 2000 IU hCG dose preparation to 6 subjects. These subjects were
compared to six others who received a placebo. KS lesions shrank or
completely disappeared in all 6 subjects receiving hCG, while none
of the controls showed any change.
[0011] These results were further confirmed in a study published in
the January/February 1998 issue of the Journal of Human
Virology..sup.3 In this study, 13 subjects with advanced AIDS-KS
and visceral KS were treated with a commercial hCG preparation
known to have anti-KS effects. Of 12 subjects treated with the hCG
preparation, 5 had a dramatic response to the therapy and overall
tolerance to the treatment was excellent.
[0012] In addition to the surprising anti-KS properties revealed by
this series of experiments, the group also observed that the hCG
preparations had no toxic side effects, common in other anti-cancer
therapeutics. In fact, some subjects reported increased energy,
enhanced appetite and even weight gain, while the control group
showed no such changes.
[0013] The variability of results among lots and sources of the hCG
preparations led the group to conclude that the active factor,
which was clearly present only in some lots from some sources,
could not be hCG itself. And although the crude preparations
produced some positive results, the variability in the crude
preparations was not conducive to the development of a standard
therapeutic protocol. The variability of results prevented an
accurate determination of ideal dose, route or frequency of
administration.
[0014] The group had made following observations: [0015] even
though the commercial preparations of hCG had a standardized amount
of hCG, the observed activity varied from manufacturer to
manufacturer and within a particular manufacturer's hCG
preparations; [0016] some of the commercial hCG preparations had no
activity; [0017] pure hCG (native purified and recombinant) had
none of the observed activities; [0018] when some commercial
preparations of hCG were intentionally depleted of hCG activity,
they maintained the previously observed activities; and [0019] mice
do not have the genes for chorionic gonadotropin, but the observed
activities were found in sera and the urine of mice in early
pregnancy.
[0020] These observations led to the hypothesis that the activity
of the hCG preparations is mediated by one or more molecules which
co-purify with hCG in only some of the commercial preparations.
[0021] Next, the researchers tested fragments of hCG for
therapeutic activity. A group of fragments from the .beta.-chain of
hCG (SEQ ID NO: 1) were identified which exhibited various
anti-HIV, anti-cancer and pro-hematopoietic effects..sup.4 Among
the peptides exhibiting such effects were the Satellin A1 peptide
[.beta.-hCG 45-57:
Leu-Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys (SEQ ID NO:
19); the Satellin A1 branched peptide: .beta.-hCG 45-57
[Leu-Gln-Dab(Pro)-Val-Leu-Pro-Dab(Pro)-Leu-Pro-Gln-Val-Val-Cys
((SEQ ID NO: 52) see SEQ ID NO: 19, for primary sequence), where
"Dab" represents diaminobutyric acid, and Dab(Pro) indicates a
proline peptide-bonded to the amino side chain of Dab], the
Satellin A2 circularized peptide [.beta.-hCG 45-57 with a cysteine
residue added to the N-terminus, circularized via a disulfide bond
between the cysteine residues:
Cys-Leu-Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys (SEQ ID NO:
20)], and the Satellin B peptide [.beta.-hCG 109-119:
Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser (SEQ ID NO: 23). Other
.beta.-hCG (SEQ ID NO: 1) peptide fragments demonstrating some
degree of efficacy included the following: 109-145, 47-55, 48-56,
45-57 fused to 109-119, and 45-57 in combination with 109-119.
[0022] Although the already-discovered group of .beta.-hCG
fragments clearly were efficacious in treating KS and other
conditions, it appeared that one or more factors remained to be
discovered, i.e., the factor or factors which accounted for the
higher degree of efficacy exhibited by the commercial hCG
preparations. As discussed more fully in the detailed description
of the invention, the present inventors have isolated two such
highly active factors. The inventors have named these peptides
"Maternin.TM." for the purpose of future commercialization, and the
peptides are generically referred to herein as the MA peptides: MA
(SEQ ID NO: 2) and pMA (SEQ ID NO: 3), which are the subject of the
instant application.
[0023] Other researchers have investigated the use of preparations
of heterodimeric hCG in the treatment of HIV. Such preparations
have been shown to reduce the reverse transcriptase activity in
HIV-1 infected lymphocytes and monocytes in culture.sup.5 and to
prevent transmission of HIV from lymphocytes to trophoblasts in
vitro..sup.6 One researcher has proposed the use of doses of hCG
below those necessary to induce a humoral immune response for
treating of HIV infection..sup.7 Researchers have also reported
that treatment with hCG improves T cell counts and physical
symptoms in certain HIV infected subjects..sup.8 However, none of
these researchers have successfully isolated novel, highly active
peptides from commercial preparations of hCG or urine.
[0024] Additionally, the .beta.-hCG (SEQ ID NO: 1) has been
reported to reduce HIV production in lymphocytes at doses from 100
pg/ml to 100 .mu.g/ml and in monocytes at doses up to approximately
10 .mu.g/ml, with higher doses actually increasing the level of
viral production in monocytes..sup.9 Lunardi-Iskandar et al..sup.10
reported that hCG, .beta.-hCG (SEQ ID NO: 1), as well as a
.beta.-hCG carboxy-terminal peptides of amino acids 109-145 and
109-119 are efficacious in the treatment of Kaposi's Sarcoma.
However, this work did not contemplate the presence of additional
specific .beta.-hCG-derived products with highly active anti-KS
properties, or any of the other therapeutic effects for such
products as described herein.
[0025] The inventors also previously identified fractions of early
pregnancy urine with consistently potent therapeutic effects, such
as anti-HIV, anti cancer, anti-wasting and pro-hematopoietic
effects..sup.11 These effects were shown to be present in fractions
of a source of native hCG or .beta.-hCG. Particularly active
fractions included material eluting from a gel filtration column
with an apparent molecular weight of approximately 40 kD, 15 kD or
2-3 kD. The same peak fractions not only killed KS tumor cells but
also inhibited HIV-1 replication in vitro, suppressed HIV-1
expression in HIV-1 transgenic mice, and inhibited the development
of AIDS in SIV.sub.MAC 251 infected macaques..sup.12 The instant
invention is an extension of this work, and is the result of a
lengthy and extensive program of experimentation directed towards
identifying the one or more pharmacologically active constituents
of early pregnancy urine.
[0026] Following our original report, several groups attempted to
identify one or another of the molecules responsible for these
activities. These reports were limited to in vitro studies showing
some inhibition of KS cell growth in culture or of decreased HIV
replication with some already well-known urinary products such as
EDN RNase,.sup.13 other RNases,.sup.14 lysozyme,.sup.15 and the
native gylcosylated .beta.-core of .beta.-hCG.sup.16 or with a
putative purified hCG..sup.17 One group described some in vitro
anti-KS activity with a low molecular weight fraction obtained from
a crude hCG preparation, the chemical nature of which was not
defined or further characterized..sup.18 Importantly, none of these
reports showed in vivo results, and none showed the multiple
effects described here. Moreover, with the exception of the hCG
related products, none of these molecules are known to be
relatively high during early pregnancy, and the report of positive
results with native glycosylated .beta.-core emphasized selective
inhibition only of the growth of KS cells..sup.19
[0027] In our earlier work.sup.20 and confirmed in this report, we
found no activity in vitro or in vivo in any of the various
experimental systems described here with purified native
glycosylated .beta.-core, .beta.-hCG, .alpha.-hCG, and/or hCG.
Earlier and distinct from these in vitro studies, Russo and
co-workers reported that a crude commercial hCG preparation
(ProFasi, Organon) inhibited carcinogenesis induced by DMBA in
rats..sup.21 As indicated, we have demonstrated that pure hCG has
no effect on any tumor cell we have studied, and the crude ProFasi
hCG was inactive.
[0028] 4.1 Human Immunodeficiency Virus Therapy
[0029] The human immunodeficiency virus (HIV) is a member of the
lentivirus family of retroviruses..sup.22 Retroviruses are small
enveloped viruses containing a single-stranded RNA genome.
[0030] HIV has been implicated as the primary cause of acquired
immune deficiency syndrome (AIDS),.sup.23 a slowly progressing
degenerative immunological disease. There are at least two distinct
types of HIV: HIV-1.sup.24 and HIV-2,.sup.25 and a large amount of
genetic heterogeneity exists within populations of each type.
[0031] In humans, HIV replication occurs predominantly in CD4.sup.+
T lymphocyte populations. HIV infection leads to depletion of this
cell type and eventually to immune incompetence, opportunistic
infections, neurological dysfunctions, neoplastic growth, wasting
and ultimately death.
[0032] The HIV viral particle comprises a viral core, composed in
part of capsid proteins, together with the viral RNA genome and
enzymes required for early replicative events. Myristylated Gag
protein forms an outer shell around the viral core. This outer
shell is, in turn, surrounded by a lipid membrane envelope derived
from the infected cell membrane.
[0033] The HIV envelope surface glycoproteins are synthesized as a
single 160 kilodalton precursor protein. A cellular protease
cleaves the precursor during viral budding into two glycoproteins,
gp41 and gp120. The gp41 component is a transmembrane glycoprotein.
The gp120 component is an extracellular glycoprotein which remains
non-covalently associated with gp41, possibly in a trimeric or
multimeric form..sup.26
[0034] HIV-associated diseases represent a major world health
problem. Researchers attempting to develop anti-HIV drugs have
focused various specific stages of the HIV life cycle as targets
for therapeutic intervention..sup.27 For example, research efforts
have targeted virally encoded reverse transcriptase (RT) as a point
of attack for anti-HIV therapeutics. These efforts have resulted in
a number of reverse-transcriptase-targeted drugs, including
2',3'-dideoxynucleoside analogs such as AZT, ddI, ddC, and
d4T..sup.28
[0035] The capacity of HIV to mutate has been a major obstacle in
the search for a cure for HIV infection. Mutations can render
effective drugs less effective, or even ineffective.
[0036] Moreover, it has become clear that the ability of
drug-resistant strains of HIV to emerge is greatly diminished by
the use of multi-drug treatment regimens. Multi-drug regimens are
currently the preferred treatment for HIV-infected subjects. For
example, combining anti-HIV compounds which target reverse
transcriptase (e.g., azidothymidine (AZT), lamivudine (3TC),
dideoxyinosine (ddI), dideoxycytidine (ddC)) with an HIV-1 protease
inhibitor produces a far more potent effect (2 to 3 logs reduction)
on viral load than monotherapy using a reverse transcriptase
inhibitor alone (about 1 log reduction) Impressive results have
been obtained using a combination of AZT, ddI, 3TC and
ritonavir..sup.29
[0037] Other researchers have sought to develop therapies which
prevent HIV from entering its target cells. The focus of these
efforts has been on CD4, the cell surface receptor for HIV. For
example, researchers have found that recombinant soluble CD4
inhibits infection of CD4.sup.+ T cells by some HIV-1
strains..sup.30 However, certain primary HIV-1 isolates are
relatively less sensitive to inhibition by recombinant CD4..sup.31
In addition, clinical trials using recombinant soluble CD4 have
produced inconclusive results..sup.32
[0038] Researchers have also targeted late stages of HIV
replication in the development of anti-HIV therapies. Late stages
involve crucial virus-specific processing of certain viral
proteins. This processing depends on the activity of a viral
protease, and drugs are being developed which inhibit this
protease..sup.33
[0039] Chemokines produced by CD8.sup.+ T cells have also been
investigated as anti-HIV agents..sup.34 In particular, RANTES,
MIP-1.alpha. and MIP-1.beta., are known to suppress HIV-1 p24
antigen production in cells infected with HIV-1 or HIV-2 isolates
in vitro..sup.35
[0040] Attention is also being given to the development of vaccines
for the treatment of HIV infection. The HIV-1 envelope proteins
(gp160, gp120, gp41) appear to be the major antigens for anti-HIV
antibodies produced by AIDS patients,.sup.36 and they may be the
most promising antigen candidates for anti-HIV vaccine development.
Various portions of gp160, gp120, and/or gp41 are also being tested
for use as immunogenic targets for the host immune system..sup.37
Vaccines directed against HIV proteins are problematic because
rapid viral mutation commonly renders such vaccines ineffective.
Moreover, vaccines may be ineffective for individuals with active
HIV infections.
[0041] 4.2 Therapies for Hematopoietic Deficiencies
[0042] The morphologically recognizable and functionally capable
cells circulating in blood include erythrocytes, neutrophilic,
eosinophilic, and basophilic granulocytes, B-, T-, non B-, non
T-lymphocytes, and platelets. These mature hematopoietic cells
derive from and develop into morphologically recognizable dividing
precursor cells for the respective lineages, such as erythroblasts
for the erythrocyte series, myeloblasts, promyelocytes and
myelocytes for the granulocyte series, and megakaryocytes for
platelets. The precursor cells derive from more primitive cells
that can simplistically be divided into two major subgroups: stem
cells and progenitor cells..sup.38
[0043] The definitions of stem and progenitor cells are operational
and depend on functional, rather than on morphological, criteria.
Stem cells have extensive self-renewal or self-maintenance capacity
(a necessity since absence or depletion of these cells could result
in the complete depletion of one or more cell lineages)..sup.39
Pluripotential stem cells differentiate into several sub-lines of
progenitor cells. These sub-lines typically have a more limited or
completely eliminated self-renewal capacity. Progenitor cells
ultimately produce morphologically recognizable precursor
cells..sup.40
[0044] A variety of infectious agents, genetic abnormalities and
environmental factors can cause a deficiency in one or more
hematopoietic cell types. For example, hematological deficiencies
have been observed in HIV-1 infected individuals, including a
reduction in CD4.sup.+ T cells and cytopenias of one or more
hematopoietic lineages.
[0045] HIV-related cytopenias are often associated with bone marrow
morphologic abnormalities and deficient progenitor cell
growth..sup.41 For example, idiopathic thrombocytopenic purpura
(ITP), characterized by significant reduction in platelet numbers,
often afflicts subjects infected with HIV..sup.42 The destruction
of platelets appears to be mediated by platelet associated
autoantibodies..sup.43 Management of ITP generally involves
immunosuppression; consequently, treatment of ITP in HIV infected
patients is complicated, since administration of immunosuppressive
drugs is extremely detrimental to HIV-infected individuals.
[0046] Chemotherapy and radiation therapy are still the frontline
methods used in the treatment of cancer and certain immunological
disorders. It is well-known that these therapies cause a number of
detrimental side-effects, such as pancytopenias or combinations of
anemia, neutropenia and thrombocytopenia. The increase or
replacement of hematopoietic cells is often crucial to the success
of such treatments..sup.44
[0047] Aplastic anemia is another serious condition characterized
by hematopoietic deficiency. In the absence of stem cell therapy,
aplastic anemias are commonly fatal. Without new stem cells,
approximately 60-75% of individuals presenting with this disorder
die within 12 months. The overall incidence of these diseases is
approximately 25 new cases per million persons per year. No single
pathogenic mechanism can account for all aplastic anemias; however,
in most cases, provision of new hematopoietic stem cells is
sufficient to allow permanent recovery..sup.45 Standard treatment
involves bone marrow transplants; however, some subjects with
aplastic anemia reject the transplanted marrow. This complication
is particularly common among subjects who have been immunologically
sensitized as a result of multiple therapeutic blood
transfusions.
[0048] A common therapy for many hematological disorders, as well
as the destruction of the endogenous hematopoietic cells caused by
chemotherapy or radiotherapy, is bone marrow transplantation.
However, the availability of bone marrow transplantation is
restricted, since it is extremely rare to have perfectly matched
(genetically identical) donors. Moreover, the complications of bone
marrow incompatibility (e.g., host versus graft reaction and graft
versus host disease) are often lethal. Even with closely matched
family donors, the complications of partial mismatching cause
substantial mortality and morbidity.
[0049] Researchers have also investigated the use of peripheral
blood as a source of stem cells for hematopoietic
reconstitution..sup.46 Promising results have been obtained for
subjects with various leukemias.sup.47 and subjects with
lymphoma..sup.48 However, some studies using peripheral blood have
failed to effect reconstitution..sup.49
[0050] There is a need for methods which enable in vitro expansion
of blood cells and for therapies which increase the production of
hematopoietic cells in vivo and which reduce the need for reliance
on bone marrow transplantation and/or blood transfusions.
[0051] 4.3 Therapies for Wasting Syndromes
[0052] Wasting syndrome is generally characterized by a decrease in
body mass of more than 10% from baseline body weight and a
disproportionate loss of body mass with respect to body fat..sup.50
Wasting is distinguished from starvation, in which higher levels of
body fat than body cell mass are depleted..sup.51 Wasting is
associated with a variety of conditions, including HIV infection,
other infectious diseases, sepsis, cancer, chronic cardiovascular
disease and diarrhea..sup.52 Wasting is a significant factor in the
mortality of subjects presenting with infections or cancer.
[0053] Current and potential therapies for wasting syndromes
include nutritional support, appetite enhancers such as dronabinol
and megestrol acetate, anabolic therapies, such as growth hormone,
and cytokine inhibitors. However, nutritional support and appetite
enhancers have the disadvantages that subjects tend to gain only
fat and not overall body mass. Administration of growth hormone,
and cytokine inhibitors are still being tested and may pose a risk
of side effects..sup.53
[0054] Treatment of wasting is critical to the survival and
well-being of patients presenting with serious diseases such as
cancer, chronic diarrhea and AIDS; accordingly, there is a need in
the art for safe and effective therapies for wasting syndromes.
[0055] 4.4 Cancer Therapy
[0056] A tumor (i.e., a neoplasm) is a mass resulting from
abnormal, uncontrolled cell growth. Tumors can be benign or
malignant. Benign tumors generally remain localized. The term
"malignant" generally means that the tumor can invade and destroy
neighboring body structures and spread to distant sites to cause
death..sup.54
[0057] Treatment options for cancer include, for example, surgery,
chemotherapy and radiation treatment. Such options are commonly
either ineffective or present serious side effects. Accordingly,
there is a need for new drugs for use in the treatment of
cancer.
[0058] Kaposi's Sarcoma (KS) is a rare type of cancer, the
incidence of which is greatly increased in HIV infected
subjects..sup.55 KS tumors are characterized by the presence of
hyperplastic cells derived from vascular endothelial cells..sup.56
In some cases, neoplastic cells with chromosomal abnormalities are
also present in the tumors..sup.57
[0059] Currently available therapies for KS include radiotherapy,
.alpha.-interferon and systemic chemotherapy..sup.58 However,
hematological and non-hematological toxicities limit the prolonged
use of chemotherapy and .alpha.-interferon in conjunction with
anti-retroviral agents commonly used in the treatment of
AIDS..sup.59 Thus, new anti-KS drugs are needed, preferably drugs
which are also compatible with other AIDS therapeutics.
[0060] 4.5 Anti-Angiogenesis
[0061] A variety of disease conditions, such as cancer, are
currently treated using therapeutics which control angiogenesis.
Angiogenesis is the formation of new blood vessels from existing
capillaries. Angiogenesis is a pathological component of a number
of diseases. For example, angiogenesis is necessary to enable
tumors to grow beyond a few mm in size. Researchers have
investigated a number of endogenous compounds thought to
negatively-regulate angiogenesis, including angiostatin (a fragment
of plasminogen), endostatin (a fragment of collagen), vasostatin (a
fragment of kallikrein), and interferons .alpha. and .beta., and
interleukin 12..sup.60 Other agents currently being considered
include: SU5416 (a small-molecule angiogenesis inhibitor); IM862 (a
naturally-occurring bipeptide and antiangiogenic agent); EMD121974
(a cyclic pentapeptide and a potent and selective inhibitor of
alpha VB3); interferon-.alpha. 2B; ZD4190; SU6668 and PD
0173073.
5. SUMMARY OF THE INVENTION
[0062] The invention provides isolated MA peptides. The isolated MA
peptides exhibit a variety of therapeutic effects, as more fully
described in Sections 8 and 9. Examples of such therapeutic effects
include anti-HIV effects, anti-cancer effects, anti-wasting
effects, and pro-hematopoietic effects. Preferred MA peptides
include: [0063] MA (SEQ ID NO: 2); and [0064] pMA (SEQ ID NO:
3).
[0065] Functional equivalents of the MA peptides are also provided
by the invention. Preferred functional equivalents include: [0066]
MA.sub.S1 (SEQ ID NO: 4); [0067] MA.sub.S2 (SEQ ID NO: 5); [0068]
MA.sub.S3 (SEQ ID NO: 6); [0069] MA.sub.S5 (SEQ ID NO: 7); [0070]
MA.sub.S9 (SEQ ID NO: 8); [0071] MA.sub.S10 (SEQ ID NO: 9); [0072]
MA.sub.S11 (SEQ ID NO: 10); [0073] .beta.-hCG 55-88 (SEQ ID NO:
11); [0074] .beta.-hCG 55-90 (SEQ ID NO: 12); [0075] .beta.-hCG
55-91 (SEQ ID NO: 13); [0076] .beta.-hCG 55-74 (SEQ ID NO: 14);
[0077] .beta.-hCG 6-37 (SEQ ID NO: 15); [0078] .beta.-hCG 6-38 (SEQ
ID NO: 16); .beta.-hCG 6-39 (SEQ ID NO: 17); and [0079] .beta.-hCG
6-40 (SEQ ID NO: 18).
[0080] In one aspect, the functional equivalents consist of
polypeptides which comprise within their amino acid sequences, the
sequence of one or more MA peptides, and preferably exclude at
least some .beta.-hCG (SEQ ID NO: 1) amino acid residues contiguous
to the MA peptide sequence.
[0081] In one embodiment of the invention, the functional
equivalents are 5 to 38 amino acids in length and are truncations
of any of the following peptides: MA (SEQ ID NO: 2); pMA (SEQ ID
NO: 3); MA.sub.S1 (SEQ ID NO: 4); MA.sub.S2 (SEQ ID NO: 5);
MA.sub.S3 (SEQ ID NO: 6); MA.sub.S5 (SEQ ID NO: 7); MA.sub.S9 (SEQ
ID NO: 8); MA.sub.S10 (SEQ ID NO: 9); MA.sub.S11 (SEQ ID NO: 10);
.beta.-hCG 55-88 (SEQ ID NO: 11); .beta.-hCG 55-90 (SEQ ID NO: 12);
.beta.-hCG 55-91 (SEQ ID NO: 13); .beta.-hCG 55-74 (SEQ ID NO: 14);
.beta.-hCG 6-37 (SEQ ID NO: 15); .beta.-hCG 6-38 (SEQ ID NO: 16);
.beta.-hCG 6-39 (SEQ ID NO: 17); and .beta.-hCG 6-40 (SEQ ID NO:
18). The truncations preferably result in peptides that are from 5
to 30 amino acid residues in length, more preferably from 5 to 20
amino acid residues in length, still more preferably from 5 to 15
amino acid residues in length, and ideally from 5 to 10 amino acid
residues in length. In a related embodiment, the truncated
polypeptides include peptides from the group consisting of MA (SEQ
ID NO: 2); pMA (SEQ ID NO: 3); MA.sub.S1 (SEQ ID NO: 4); MA.sub.S2
(SEQ ID NO: 5); MA.sub.S3 (SEQ ID NO: 6); MA.sub.S5 (SEQ ID NO: 7);
MA.sub.S9 (SEQ ID NO: 8); MA.sub.S10 (SEQ ID NO: 9); MA.sub.S11
(SEQ ID NO: 10); .beta.-hCG 55-88 (SEQ ID NO: 11); .beta.-hCG 55-90
(SEQ ID NO: 12); .beta.-hCG 55-91 (SEQ ID NO: 13); .beta.-hCG 55-74
(SEQ ID NO: 14); .beta.-hCG 6-37 (SEQ ID NO: 15); .beta.-hCG 6-38
(SEQ ID NO: 16); .beta.-hCG 6-39 (SEQ ID NO: 17); and .beta.-hCG
6-40 (SEQ ID NO: 18) having 1, 2, 3, 4, 5 or 6 amino acid residues
deleted from an amino terminus or carboxy terminus thereof.
Moreover, the functional equivalents include polypeptides which
comprise one or more of these truncated MA peptides.
[0082] The functional equivalents of the invention exclude full
length human .beta.-hCG (SEQ ID NO: 1).
[0083] The invention also provides pharmaceutical compositions
comprising one or more therapeutic polypeptides and/or one or more
functional equivalents of the invention. The pharmaceutical
compositions are useful in the treatment of a variety of conditions
as described more fully in Sections 8 and 9. Examples include HIV
infection, cancer, wasting, and hematopoietic deficiencies.
[0084] Furthermore, the present invention provides an MA peptide
isolated from early pregnancy urine consisting of the amino acid
sequence of SEQ ID NO: 2 (MA). The invention also provides an MA
peptide isolated from early pregnancy urine consisting of the amino
acid sequence of SEQ ID NO: 3 (pMA).
[0085] In addition to the foregoing compositions and formulations,
the invention provides methods for treating and/or preventing
various medical conditions. Examples of conditions suitably treated
and/or prevented according to the methods of the invention include
HIV infection, cancer, wasting, hematopoietic deficiency, and
pathological angiogenesis. The subject of the methods of the
invention may be a human or other animal, preferably a mammal, such
as a dog, cat, horse, sheep, cow or rat. The methods generally
comprise administering to a subject in need thereof a
pharmaceutically effective amount of one or more therapeutic
polypeptides of the invention.
[0086] In one aspect of the invention, the condition treated is
cancer. Examples of cancers which may be suitably treated according
to the methods of the invention include brain cancer, breast
cancer, lung cancer, pancreatic cancer, prostate cancer, renal
cancer, and hematopoietic malignancies. The MA peptides may be used
to treat any cancer for which the MA peptides have anti-cancer
activity; cancers in addition to those described herein may readily
be identified by those of skill in the art by screening the novel
compounds of the invention using readily available assays. In a
preferred aspect, the cancer is Kaposi's sarcoma. The therapeutic
polypeptide(s) and/or functional equivalent(s) may also be
administered in conjunction with another anti-cancer therapy, such
as radiation therapy, anti-cancer chemotherapy, and/or anti-cancer
vaccines.
[0087] In another aspect of the invention, the condition treated is
a hematopoietic deficiency caused by radiation and/or chemical
exposure. The radiation and/or chemical exposure may be associated
with a medical therapy, such as anti-cancer therapy. The
therapeutic polypeptide may be administered before, during or after
the radiation and/or chemical exposure.
[0088] The invention also provides a rationally designed peptide
exhibiting a therapeutic effect selected from the group consisting
of anti-HIV effects, anti-cancer effects, anti-wasting effects,
radioprotective effects, anti-angiogenic effects, anti-inflammatory
effects and pro-hematopoietic effects, comprising at least one
motif from each of the following groups: (a) SH3 and PDZ domain
motifs; and (b) phosphorylation and myristoylation domain
motifs.
[0089] In a preferred embodiment, the motifs are selected from SH3
domain motifs, PDZ domain motifs, phosphorylation domain motifs and
myristoylation domain motifs as found in mammalian luteinizing
hormone and/or mammalian chorionic gonadotropin.
[0090] In a more preferred embodiment, the motifs are selected from
SH3 domain motifs, PDZ domain motifs, phosphorylation domain motifs
and myristoylation domain motifs as found in a source selected from
the group consisting of .beta.-hCG (SEQ ID NO: 1), horse .beta. CG
(SEQ ID NO: 24), sheep .beta. LH (SEQ ID NO: 25), pig .beta. LH
(SEQ ID NO: 26), dog .beta. LH (SEQ ID NO: 27), bovine .beta. LH
(SEQ ID NO: 28), rat .beta. LH (SEQ ID NO: 29), cat .beta. LH (SEQ
ID NO: 30), and human .beta. LH (SEQ ID NO: 31).
[0091] Preferably no more than 20 amino acid residues separate the
(a) motif from the (b) motif.
[0092] Preferably the (a) motif comprises an SH3 motif and the (b)
motif comprises a phosphorylation motif. Moreover, it is also
preferred that no more than 10 amino acid residues separate the SH3
motif from the phosphorylation motif, more preferably no more than
5 amino acid residues separate the SH3 motif from the
phosphorylation motif, and ideally, the SH3 motif and the
phosphorylation motif are immediately contiguous.
[0093] In a related embodiment, the (a) motif comprises an SH3
motif and the (b) motif comprises a myristoylation motif.
Preferably no more than 15 amino acid residues separate the SH3
motif from the myristoylation motif, more preferably no more than
10 amino acid residues separate the SH3 motif from the
myristoylation motif, and still more preferably no more than 5
amino acid residues separate the SH3 motif from the myristoylation
motif. Ideally, the SH3 motif and the myristoylation motif are
immediately contiguous.
[0094] The rationally designed peptide of the invention may
suitably comprise more than one (a) motif and/or more than one (b)
motif.
[0095] Motifs for inclusion in the rationally designed polypeptides
of the invention are preferably selected from the following: (1)
.beta.-hCG 4-7, 50-53, 70-73, 124-129, 141-144, 30-33, 41-44, and
139-142; and (2) .beta.-hCG 22-27, 47-52, 71-76, 66-68, 94-99,
109-112, and 120-122.
[0096] The (a) and (b) motifs may also be provided together in a
continuous segment of a chorionic gonadotropin or leutenizing
hormone amino acid sequence, e.g., .beta.-hCG 6-37, 6-38, 6-39 and
6-40 (SEQ ID NOS: 13, 14, 15 and 16).
[0097] Preferred rationally designed peptides include .beta.-hCG
1-39, 1-29, 1-35, 41-54, 66-76, 93-130, 93-131, 93-132, 93-135,
93-144, 93-145, 41-54 linked to 55-92, 41-54 linked to 59-89, or
41-54 linked to 55-76.
[0098] Preferred sources for motifs include HUSI-II polypeptide,
.beta.-hCG (SEQ ID NO: 1), horse .beta. CG (SEQ ID NO: 24), sheep
.beta. LH (SEQ ID NO: 25), pig .beta. LH (SEQ ID NO: 26), dog
.beta. LH (SEQ ID NO: 27), bovine .beta. LH (SEQ ID NO: 28), rat
.beta. LH (SEQ ID NO: 29), cat .beta. LH (SEQ ID NO: 30), and human
.beta. LH (SEQ ID NO: 31).
[0099] The rationally designed peptides are typically at least 5
and less than 100 amino acid residues in length, preferably at
least 5 and less than 50 amino acid residues in length, more
preferably at least 5 and less than 25 amino acid residues in
length, still more preferably, at least 5 and less then 20 amino
acid residues in length, and ideally at least 5 and less than 15
amino acid residues in length.
[0100] The invention also provides fragments of HUSI-II comprising
an SH3 motif and flanking residues, preferably HUSI-II fragments
40-46, 40-60, 40-59, 40-58, 40-66, 40-67, 40-68.
[0101] In a related aspect, the invention provides a method for
producing a peptide library for screening for a therapeutic effect
(e.g., anti-HIV effects, anti-cancer effects, anti-wasting effects,
radioprotective effects, anti-angiogenic effects, anti-inflammatory
effects and pro-hematopoietic effects). The method generally
comprises producing a set of rationally designed peptides, each
peptide comprising at least one motif from each of the following
groups (a) SH3 and PDZ domain motifs; and (b) phosphorylation and
myristoylation domain motifs. The peptide library may be screened
to identify peptides exhibiting the target therapeutic
effect(s).
[0102] 5.1 Definitions
[0103] A "therapeutically effective" amount or dose is an amount or
dose which prevents or delays the onset or progression of an
indicated disease or other adverse medical condition. The term also
includes an amount sufficient to arrest or reduce the severity of
an ongoing disease or other adverse medical condition, and also
includes an amount necessary to enhance normal physiological
functioning.
[0104] As used herein, "treatment" of a disease or other adverse
medical condition, should be broadly interpreted based on the
therapeutic effects described herein as variously including
palliative, active, causal, conservative, medical, palliative,
prophylactic, and/or symptomatic treatment, treatment designed to
delay the onset or progression of the disease or other adverse
medical condition, as well as treatment designed to arrest or
reducing the severity of an ongoing disease or other adverse
medical condition.
[0105] As used herein, a "pharmaceutically acceptable" component
(such as a salt, carrier, excipient or diluent) of a formulation
according to the present invention is a component which (1) is
compatible with the other ingredients of the formulation in that it
can be combined with the therapeutic polypeptides of the invention
without eliminating the biological activity of the therapeutic
polypeptides; and (2) is suitable for use in non-human animals or
humans without undue adverse side effects (e.g., toxicity,
irritation, and allergic response). Side effects are "undue" when
their risk outweighs the benefit provided by the pharmaceutical
composition.
[0106] As used herein, a "pharmaceutically acceptable" with
reference to the degree of purity of a therapeutic polypeptide or
nucleic acid indicates that the therapeutic polypeptide or nucleic
acid (1) is free of contaminating materials that would eliminate
the biological activity of the therapeutic polypeptide or nucleic
acid; and (2) is free of contaminating materials that would render
the therapeutic polypeptide or nucleic acid unsuitable for
administration to non-human animals or humans by causing undue
adverse side effects (e.g., toxicity, irritation, and allergic
response). Side effects are "undue" when their risk outweighs the
benefit provided by the therapeutic polypeptide or nucleic
acid.
[0107] The term "substantially pure" when used in reference to a
therapeutic polypeptide or nucleic acid is defined herein to mean a
therapeutic polypeptide or nucleic acid that is substantially free
from other contaminating proteins, nucleic acids, and other
biologicals derived from an original source organism, recombinant
DNA expression system, or from a synthetic procedure employed in
the synthesis or purification of the therapeutic polypeptide of
nucleic acid (e.g., chromatography reagents and polymers, such as
acrylamide or agarose). Purity may be assayed by standard methods.
Purity evaluation may be made on a mass or molar basis.
[0108] The term "functional equivalent" as used herein refers to a
polypeptide sequence comprising a full-length MA peptide sequence,
or comprising a fragment, analogue, derivative or truncation
isoform of a full-length MA peptide. Functional equivalents also
include, for example, an MA peptide in salt, complex, analogue, or
derivative form, as well as a fragment, derivative or analogue of a
native MA peptide. Functional equivalents retain some or all of the
biological activity of the corresponding MA peptide. Functional
equivalents exclude full-length .beta.-hCG.
[0109] The word "transform" is broadly used herein to refer to
introduction of an exogenous polynucleotide sequence into a
prokaryotic or eukaryotic cell by any means known in the art
(including for example, direct transmission of a polynucleotide
sequence from a cell or virus particle, transmission by infective
virus particles, and transmission by any known
polynucleotide-bearing substance) resulting in a permanent or
temporary alteration of genotype and in an immortal or non-immortal
cell line.
[0110] The term "polypeptide" as used herein is intended to refer
to amino acid sequences of any length, for example, the term
specifically includes both peptides and proteins.
[0111] The terms "administer," "administration" and the like, as
used herein with reference to a polypeptide, are intended to be
inclusive of any means for delivering the polypeptide to a subject.
For example, these terms are broadly inclusive of direct delivery
of a polypeptide to a subject by conventional routes, as well as
delivery of a prodrug which metabolizes in vivo to provide the
polypeptide to a subject.
6. BRIEF DESCRIPTION OF THE FIGURES
[0112] FIG. 1. Purification and identification of MA.
Panel A: shows an HPLC profile utilizing a SUPERDEX.TM. column (HR
10/30, cut-off 100-7000 Da) of the Pall-Filtron Microsep purified
fraction. Panel B: shows the results of SDS-PAGE elucidating
peptides that generally ran with markers of about 4-6 kDa and 3-4
kDa. Panel C: shows the results of mass spectroscopy using
MALDI-TOF and an SA matrix. Panel D: shows the sequence identity as
confirmed by MA.sub.S2 peptide synthesis with demonstrable
reproducibility of bioactivities.
[0113] FIG. 2. Immunoblots of MA and pMA with monoclonal antibodies
(MoAbs).
Panel A: Western blot analysis of MoAb to MA Panel B: Western blot
analysis of MoAb to pMA Panel C: Western blot analysis of MoAB
(B-210) to .beta.-core
[0114] FIG. 3. Anti-tumor effects of native and synthetic MA on
human tumor cells in vitro.
Panel A: Dose dependent inhibition of Kaposi sarcoma (KSY-1) growth
by MA as measured by colony formation assays. The assays were on
day 7, with the indicated amount of test material (MA, MA.sub.S1,
MA.sub.S2, MA.sub.S3, purified native (CR127) rhCG, control
peptides AA 21-52, or native glycosylated .beta.-core) added at
time 0 (40). Panel B: Dose dependent inhibition of prostate
carcinoma cell line (PC-3) growth by MA as measured by colony
formation assays. The assays were each on day 7, with the indicated
amount of test material (MA, MA.sub.S1, MA.sub.S2, MA.sub.S3,
purified native (CR127) rhCG, control peptides AA 21-52, or native
glycosylated .beta.-core) added at time 0. Panel C: Dose dependent
inhibition of breast carcinoma cell line (HTB-123) growth by MA as
measured by colony formation assay on day 7, with the indicated
amount of HTB 123 added at time 0. Panel D: Dose dependent
inhibition of primary breast cancer cells from biopsy of an
intraductal breast carcinoma and cultured for 3 days as measured by
colony formation assay on day 7, with the indicated amount of
cultured primary breast cancer cells added at time 0. Panel E: Dose
dependent cell killing (apoptosis) of KSY-1 cells by MA as measured
by trypan blue exclusion and Annexin V staining (41). In assays,
the PC3 cells were treated with test materials (MA, MA.sub.S1,
MA.sub.S2, MA.sub.S3, purified native (CR127) rhCG, control
peptides AA 21-52, or native glycosylated .beta.-core) in the
indicated amounts. Panel F: Dose dependent apoptosis of prostate
carcinoma (PC-4) cells by MA as measured by trypan blue exclusion
and Annexin V staining. In assays, the PC-4 cells were treated with
test materials (MA, MA.sub.S1, MA.sub.S2, MA.sub.S3, purified
native (CR127) rhCG, control peptides AA 21-52, or native
glycosylated .beta.-core) in the indicated amounts. Panel G: Dose
dependent apoptosis of breast carcinoma (HTB 124) cells by MA as
measured by trypan blue exclusion and Annexin V staining. In
assays, the HTB 124 cells were treated with test materials (MA,
MA.sub.S1, MA.sub.S2, MA.sub.S3, purified native (CR127) rhCG,
control peptides AA 21-52, or native glycosylated .beta.-core) in
the indicated amounts. Panel H: Dose dependent apoptosis of breast
carcinoma (primary intraductal breast carcinoma biopsied tissue)
cells by MA as measured by trypan blue exclusion and Annexin V
staining. In assays, the breast carcinoma cells were treated with
test materials (MA, MA.sub.S1, MA.sub.S2, MA.sub.S3, purified
native (CR127) rhCG, control peptides AA 21-52, or native
glycosylated .beta.-core) in the indicated amounts.
[0115] FIG. 4. Three-dimensional confocal microscopic analysis
showing selective induction of apoptosis of human neoplastic cells
by MA.
Panel A: Confocal microscopy results showing normal nuclear
morphology in human normal cells following high doses of MA. Panel
B: Induction of apoptosis in KS Y-1 cells. Panels C and F:
Induction of apoptosis in two prostate carcinoma cell lines. Panel
D: Induction of apoptosis in gliobastoma cells (HTB 16). Panel E:
Induction of apoptosis in cell lines from carcinomas of the breast
(HBT124). Panel G: Induction of apoptosis in pancreatic cancer
cells (CRL 1682). Panel H: Induction of apoptosis in lung cancer
cells (HTB-184). Panel I: Induction of apoptosis in renal cancer
cells (HTB-46). Panels J and K: Representative examples using the
prostate and breast carcinomas (PCANJ and BCAJR, respectively).
[0116] FIG. 5. Inhibition of development of tumors in
immunodeficient mice transplanted with human cancer cells.
Panel A: Top left shows gross appearance of untreated prostate
carcinoma tumor (PC3); top middle shows histological appearance of
untreated tumor; top right shows MA histology of MA-treated tumor,
showing hypocellularity and degenerative changes of remaining
cells. Second row left shows gross appearance of MA-treated
prostate carcinoma showing marked tumor regression; middle and
right show histology of MA- and MA.sub.S2-treated tumor showing
hypocellularity and degenerative changes of the cells. Panel B: Top
left shows gross appearance of untreated KS tumor; top middle shows
histology of untreated KS tumor; top right shows histology of
MA-treated tumor showing dying cells. Bottom left shows mouse with
KS tumor abolished after MA treatment; histology of MA-treated
tumor showing dying cells; bottom right shows KS cells undergoing
apoptosis after treatment with MA.sub.S2. Brown color in cells of
bottom right box is APO-TAG indicating apoptosis. Panel C:
Inhibition of tumor size by suboptimal doses (about 60 pMples) of
MA (SEQ ID NO: 2); pMA (SEQ ID NO: 3); MA.sub.S1 (SEQ ID NO: 4) and
MA.sub.S2 (SEQ ID NO: 5) in comparison with CR127, pure recombinant
.beta.-hCG, ahCG, and pure native glycosylated .beta.-core, crude
preparations of hCG (APL, Wyeth-Ayrest) and .beta.-hCG (CG10,
Sigma).
[0117] FIG. 6. Anti-tumor effects of MA peptides on spontaneous
tumor growth in immunocompetent mice and rats.
Panel A: Results of treatment of mouse fibrosarcomas with MA for 2
weeks; left panel shows results with untreated cells; left center
shows hemotoxin and eosin (H&E) staining in MA-treated cells
showing hypocellularity; right center and right show TUNEL assays,
indicating apoptosis. Panel B: Results of treatment of one rat with
a tumor of approximately 3.times.3 cm leading to complete tumor
regression. Left top shows gross external appearance of massive
carcinoma of the breast. Left bottom shows treated animal with
tumor resolution. Center-left shows H&E staining in untreated
mouse (top) and MA-treated mouse (below). Right and center-right
show histological sections from MA- and MA.sub.S1-treated rat,
stained with APO-TAG to show apoptosis. Panel C: Confocal
microscopy of Lewis lung carcinoma cells untreated and after
treatment with MA; from left-to-right shows untreated tumor cells,
treatment with r.beta.-hCG (5 .mu.g/ml), native MA (5 .mu.g/ml),
and MA.sub.S1 (5 .mu.g/ml). Panel D: Confocal microscopy of B16
melanoma carcinoma cells untreated and after treatment with MA,
under same conditions as Lewis lung carcinoma cells (Panel C).
[0118] FIG. 7. Inhibition of HIV-1 and SIV replication by MA.
Panel A: Dose dependent inhibition of HIV-1 (IIIB) infection of
human PBMCs by MA, MA.sub.S1, MA.sub.S2, and MA.sub.S3 at
approximately 30 to 100 nM is shown, compared to lack of inhibition
by similar concentrations of hCG (CR127), control peptides AA
21-52, native glycosylated .beta.-core, r.beta.-hCG, and
r.alpha.-hCG, and untreated control. Panel B: Dose dependent
inhibition of HIV-1 (IIIB) infection of primary CD4+ T-cells by MA,
MA.sub.S1, MA.sub.S2, and MA.sub.S3 at approximately 30 to 100 nM
is shown, compared to lack of inhibition by similar concentrations
of hCG (CR127), control peptides AA 21-52, native glycosylated
.beta.-core, r.beta.-hCG, and r.alpha.-hCG, and untreated control.
Panel C: Results of dose dependent inhibition of HIV-1 (IIIB)
infection of primary CD4+ T-cells is shown by MA, MA.sub.S1,
MA.sub.S2, and MA.sub.S3 at approximately 30 to 100 nM, compared to
lack of inhibition by similar concentrations of hCG (CR127),
control peptides AA 21-52, native glycosylated .beta.-core,
r.beta.-hCG, and r.alpha.-hCG, and untreated control. Panel C: Dose
dependent inhibition of HIV-1 (Ba-L) infection of primary
macrophages by MA, MA.sub.S1, MA.sub.S2, and MA.sub.S3 at
approximately 30 to 100 nM is shown, compared to lack of inhibition
by similar concentrations of hCG (CR127), control peptides AA
21-52, native glycosylated .beta.-core, r.beta.-hCG, and
r.alpha.-hCG, and untreated control. Panel D: Dose dependent
inhibition of SIV.sub.MAC 251 infection of rhesus macaque PBMCs by
MA, MA.sub.S1, MA.sub.S2, and MA.sub.S3 at approximately 30 to 100
nM is shown, compared to lack of inhibition by similar
concentrations of hCG (CR127), control peptides AA 21-52, native
glycosylated .beta.-core, r.beta.-hCG, and r.alpha.-hCG, and
untreated control.
[0119] FIG. 8. MA represses HIV-1 gene expression in HIV-1
transgenic mice.
Panel A: Two untreated mice show hyperkeratosis and diminished size
(red arrows). In contrast, transgenics breast feeding from mothers
treated with native MA (200 ng) for 7 days show normal development.
Panel B: Shows % survival of MA peptide-treated mice as compared to
controls. Panel C: Shows weight gain of MA peptide-treated mice as
compared to controls.
[0120] FIG. 9. Pro-hematopoietic effects of MA peptides in vitro
using a human bone marrow derived culture.
Panel A: MA promotion of CFU-GEMM, which includes precursors of
granulocytes, red blood cells, and macrophages. Panel B: promotion
of BFU-e, which are red blood cell precursors. Panel C: shows
promotion of CFU-GM, which include myeloid (granulocyte) and
macrophage precursors. Panel D: Promotion of T-CFC, which are cells
capable of forming colonies of T-cells.
[0121] FIG. 10 shows the results of treatment of mice and rats with
MA before or after lethal irradiation.
Panel A: Hematopoietic recovery phase in mice and rats for total
lymphocytes. Panel B: Hematopoietic recovery phase in mice and rats
for hemoglobin (HBG). Panel C: Hematopoietic recovery phase in mice
and rats for red blood cells (RBC). Panel D: Survival of mice and
rats treated prior to irradiation. Panel E: Survival of mice and
rats treated during irradiation, with treatment maintained for 4
weeks following irradiation.
[0122] FIG. 11. Hematopoietic recovery of rats and monkeys.
Panel A: In a study showing the rescue Rescue of rats from the
ultimate lethal effects of high dose paclitaxel, the graph shows
survival of animals treated prior to administration of paclitaxel.
Panel B: In the study showing the rescue of rats from the ultimate
lethal effects of high dose paclitaxel, the graph shows survival of
animals undergoing treatment within 24 hours of paclitaxel
administration and maintained on 3 weekly injections of 100 ng for
four weeks. Panel C: Hematopoietic recovery phase in animals for
total lymphocytes. Panel D: Hematopoietic recovery phase in animals
for platelets (PLT). Panel E: Hematopoietic recovery phase in
animals for hemoglobin (HGB). Panel F: Hematopoietic recovery phase
in animcals for red blood cells (RBCs).
[0123] FIG. 12. MA restores hematopoiesis in rats with 40% blood
loss.
Panels A-D: Recovery in rats untreated vs. those given i.p. MA or
MA.sub.S1 24 and 48 hrs after the blood loss (each point represents
a mean of 10 animals). MA and MA.sub.S1 treated animals show a
prompt recovery of all peripheral blood cells. Panels E-H: Blood
counts in three monkeys. Each panel illustrates the mean values for
the three animals treated with MA.sub.S1. Panel I: Shows SIVmac251
viral load in Macaca mutata.
[0124] FIG. 13. Silver stained 4-12% Bis-Tris NuPage SDS-PAGE
gel.
Column fractions were derived from non-pregnant female urine
plus/minus a spike with MA.sub.S2 (100 .mu.g/L).
[0125] FIG. 14. Western blot of SDS-PAGE showing MA-reactive
species.
MA-reactive species only in acid peak (lane 4) and MA.sub.S2
control (lane 6).
[0126] FIG. 15. Anti-MA antibodies remove the anti-viral effect of
MA.sub.S2, resulting in elevated HIV-I p24 production.
[0127] FIG. 16. Coomassie stained gel and Western blot of test
expressions showing .alpha.-MA reactive material of the expected
size (58 kDa).
Panel A: Coomassie stained gel. Panel B: Western blot.
[0128] FIG. 17. Silver stained SDS-PAGE and Western blot showing
column purification of clone 517.3 ex ressed Bact:MA.sub.S2 and
immunoreactivit with .alpha.-MA antibodies.
Panel A: Silver stained SDS-PAGE. Panel B: Western blot.
7. BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0129] The sequences used herein are summarized in Table 1:
TABLE-US-00001 SEQ ID NO: Name .beta.hCG AA Sequence 1 .beta.-hCG
1-145 Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr
Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr
Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val Leu
Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe Glu Ser
Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asp Pro Val Val Ser Tyr Ala
Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser Thr Thr Asp Cys
Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp Pro Arg Phe Gln Asp
Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu
Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 2 MA 55-89 Val Val Cys
Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg
Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser Cys Gln 3 pMA
55-92 Val Val Cys Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu
Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu
Ser Cys Gln Cys Ala Leu 4 MA.sub.S1 62-76 Val Arg Phe Glu Ser Ile
Arg Leu Pro Gly Cys Pro Arg Gly Val 5 MA.sub.S2 58-87 Asn Tyr Arg
Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn
Pro Val Val Ser Tyr Ala Val Ala Leu Ser 6 MA.sub.S3 55-89 Val Val
Cys Asn Tyr Arg Asp Val Arg Phe Gln Ser Ile Arg Leu Pro Gly Cys Pro
Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser Cys Gln 7
MA.sub.S5 68-74 Arg Leu Pro Gly Cys Pro Arg 8 MA.sub.S9 A::70-73::G
Ala Pro Gly Cys Pro Gly 9 MA.sub.S10 69-74 Leu Pro Gly Cys Pro Arg
10 MA.sub.S11 69-73::Q Leu Pro Gly Cys Pro Gln 11 .beta.-hCG 55-88
Val Val Cys Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg 55-88 Leu
Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu
Ser Cys 12 .beta.-hCG 55-90 Val Val Cys Asn Tyr Arg Asp Val Arg Phe
Glu Ser Ile Arg 55-90 Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val
Val Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys 13 .beta.-hCG 55-91 Val
Val Cys Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg 55-91 Leu Pro
Gly Cys Pro Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser
Cys Gln Cys Ala 14 .beta.-hCG 55-74 Val Val Cys Asn Tyr Arg Asp Val
Arg Phe Glu Ser Ile Arg 55-74 Leu Pro Gly Cys Pro Arg 15 .beta.-hCG
6-37 Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu Ala Val Glu 6-37
Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr Ile Cys Ala Gly
Tyr 16 .beta.-hCG 6-38 Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu
Ala Val Glu 6-38 Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr
Thr Ile Cys Ala Gly Tyr Cys 17 .beta.-hCG 6-39 Arg Pro Arg Cys Arg
Pro Ile Asn Ala Thr Leu Ala Val Glu 6-39 Lys Glu Gly Cys Pro Val
Cys Ile Thr Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro 18
.beta.-hCG 6-40 Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu Ala Val
Glu 6-40 Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr Ile
Cys Ala Gly Tyr Cys Pro Thr 19 SAT.sub.A1 45-57 Leu Gln Gly Val Leu
Pro Ala Leu Pro Gln Val Val Cys 20 SAT.sub.A2 C::45-57 Cys Leu Gln
Gly Val Leu Pro Ala Leu Pro Gln Val Val Cys (circularized) 21
SAT.sub.A3 C::45-47::R:: Cys Leu Gln Gly Arg Leu Pro Ala Leu Pro
Arg Val Val Cys 49-57 22 SAT.sub.A4 N/A Cys Arg Leu Pro Gly Leu Pro
Arg Cys 23 SAT 109-119 Thr Crs Asp Asp Pro Arg Phe Gln Asp Ser Ser
indicates data missing or illegible when filed
Peptides Other than .beta.-hCG:
TABLE-US-00002 SEQ ID NO: Name Sequence 24 choriogonadotropin
.beta.- Met Glu Thr Leu Gln Gly Leu Leu Leu Trp Met Leu Leu Ser
chain precursor - horse Val Gly Gly Val Trp Ala Ser Arg Gly Pro Leu
Arg Pro Leu Cys Arg Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala
Cys Pro Ile Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro
Ser Met Val Arg Val Met Pro Ala Ala Leu Pro Ala Ile Pro Gln Pro Val
Cys Thr Tyr Arg Glu Leu Arg Phe Ala Ser Ile Arg Leu Pro Gly Cys Pro
Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala Leu Ser Cys His Cys
Gly Pro Cys Gln Ile Lys Thr Thr Asp Cys Gly Val Phe Arg Asp Gln Pro
Leu Ala Cys Ala Pro Glu Ala Ser Ser Ser Ser Lys Asp Pro Pro Ser Gln
Pro Leu Thr Ser Thr Ser Thr Pro Thr Pro Gly Ala Ser Arg Arg Ser Ser
His Pro Leu Pro Ile Lys Thr Ser 25 lutropin .beta.-chain Met Glu
Met Leu Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly precursor - sheep
Val Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln Pro
Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val Cys Ile Thr
Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Leu Ser Met Lys Gln Val Leu
Pro Val Ile Leu Pro Pro Met Pro Gln Arg Val Cys Thr Tyr His Glu Leu
Arg Phe Ala Ser Val Arg Leu Pro Gly Cys Pro Pro Gly Val Asp Pro Met
Val Ser Phe Pro Val Ala Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser
Ser Thr Asp Cys Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro
Pro Leu Pro Asp Ile Leu Phe Leu 26 lutropin .beta.-chain Met Glu
Met Leu Gln Gly Leu Leu Leu Trp Leu Leu Leu Ser precursor - pig Val
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro Ile
Asn Ala Thr Leu Ala Ala Glu Asn Glu Ala Cys Pro Val Cys Ile Thr Phe
Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg Val Leu Pro
Ala Ala Leu Pro Pro Val Pro Gln Pro Val Cys Thr Tyr Arg Glu Leu Ser
Phe Ala Ser Ile Arg Leu Pro Gly Cys Pro Pro Gly Val Asp Pro Thr Val
Ser Phe Pro Val Ala Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser
Ser Asp Cys Gly Gly Pro Arg Ala Gln Pro Leu Ala Cys Asp Arg Pro Leu
Leu Pro Gly Leu Leu Phe Leu 27 lutropin .beta.-chain Leu Gln Gly
Leu Leu Leu Trp Leu Leu Leu Ser Val Gly Gly precursor -- dog Val
Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro Ile Leu Ala Thr
Leu Ala Ala Glu Asn Glu Ala Cys Pro Val Cys Ile Thr Phe Thr Thr Thr
Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg Val Leu Pro Ala Ala Leu
Pro Pro Val Pro Gln Pro Val Cys Thr Tyr His Glu Leu His Phe Ala Ser
Ile Arg Leu Pro Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro
Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Leu Ser Asn Ser Asp Cys
Gly Gly Pro Arg Ala Gln Ser Leu Ala Cys Asp Arg Pro Leu Leu Pro Gly
Leu Leu Phe Leu 28 lutropin .beta.-chain Met Glu Met Phe Gln Gly
Leu Leu Leu Trp Leu Leu Leu Gly precursor - bovine Val Ala Gly Val
Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln Pro Ile Asn Ala Thr
Leu Ala Ala Glu Lys Glu Ala Cys Pro Val Cys Ile Thr Phe Thr Thr Ser
Ile Cys Ala Gly Tyr Cys Pro Ser Met Lys Arg Val Leu Pro Val Ile Leu
Pro Pro Met Pro Gln Arg Val Cys Thr Tyr His Glu Leu Arg Phe Ala Ser
Val Arg Leu Pro Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro
Val Ala Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu Pro Asp
Ile Leu Phe Leu 29 luteinizing hormone .beta.- Met Glu Arg Leu Gln
Gly Leu Leu Leu Trp Leu Leu Leu Ser subunit - Rattus Pro Ser Val
Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu norvegicus Cys Arg Pro
Val Asn Ala Thr Leu Ala Ala Glu Asn Glu Phe Cys Pro Val Cys Ile Thr
Phe Thx Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg Val Leu
Pro Ala Ala Leu Pro Pro Val Pro Gln Pro Val Cys Thr Tyr Arg Glu Leu
Arg Phe Ala Ser Val Arg Leu Pro Gly Cys Pro Pro Gly Val Asp Pro Ile
Val Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Leu Ser
Ser Ser Asp Cys Gly Gly Pro Arg Thr Gln Pro Met Thr Cys Asp Leu Pro
His Leu Pro Gly Leu Leu Leu Phe 30 luteinizing hormone .beta.- Met
Glu Met Leu Gln Gly Leu Leu Leu Leu Trp Leu Leu Leu subunit
precursor - Leu Asn Val Gly Gly Val Trp Thr Ser Arg Glu Pro Leu Arg
Pro Leu Cys Arg Pro Ile Asn Ala Thr Leu Ala Ala Glu Asn Glu Ala Cys
Pro Val Cys Val Thr Phe Thr Thr Thr Ile Cys Ala Gly Tyr Cys Pro Ser
Met Met Arg Val Leu Pro Ala Ala Leu Pro Pro Val Pro Gln Pro Val Cys
Thr Tyr Arg Glu Leu Arg Phe Ala Ser Val Arg Leu Pro Gly Cys Pro Pro
Gly Val Asp Pro Val Val Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly
Pro Cys Arg Leu Ser Ser Ser Asp Cys Gly Gly Pro Arg Ala Gln Pro Leu
Ala Cys Asp Arg Pro Pro Leu Pro Gly Leu Leu Phe Leu 31 .beta.-chain
of human LH Met Glu Met Leu Gln Gly Leu Leu Leu Leu Leu Leu Leu Ser
Met Gly Gly Ala Trp Ala Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro
Ile Asn Ala Ile Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr
Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val Leu
Gln Ala Val Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg Asp Val
Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asp Pro Val
Val Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Arg Ser
Thr Ser Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp His Pro
Gln Leu Ser Gly Leu Leu Phe Leu
8. DETAILED DESCRIPTION OF THE INVENTION
[0130] The inventors have identified specific therapeutic
polypeptides with a molecular mass of about 3.8 kDa, generally
referred to herein as MA peptides. The inventors have also designed
and made smaller forms (truncated at C-terminal and N-terminal
ends), which mimic the therapeutic activities of the MA peptides.
The inventors have confirmed the identity of the native MA peptides
by preparing synthetic peptides encompassing the same sequences.
Previous work by the inventors had indicated activities in the 3-5
kDa range but also in a higher molecular weight range approximating
17 kDa..sup.61 However, with the purification schemes employed
here, no activity was found at 17 kDa. This may be explained by the
presence in the earlier samples of MA that was to bound to other
structures or multimers of MA.
[0131] 8.1 MA Peptides
[0132] The inventors have isolated MA (SEQ ID NO: 2) and pMA (SEQ
ID NO: 3) from native sources of hCG. MA was previously referred to
in an earlier U.S. Patent Application No. 60/147,825 as a
"pregnancy peptide" or "PP." MA (SEQ ID NO: 2) and pMA (SEQ ID NO:
3) exhibit various advantageous pharmacological activities as more
fully discussed below. MA (SEQ ID NO: 2) corresponds to amino acids
55-89 of .beta.-hCG (SEQ ID NO: 1), and pMA corresponds to amino
acids 55-92 of .beta.-hCG (SEQ ID NO: 1).
[0133] The inventors have also designed the following synthetic
mimetics of the MA peptides: MA.sub.S1 (SEQ ID NO: 4); MA.sub.S2
(SEQ ID NO: 5); MA.sub.S3 (SEQ ID NO: 6); MA.sub.S5 (SEQ ID NO: 7);
MA.sub.S9 (SEQ ID NO: 8); MA.sub.S10 (SEQ ID NO: 9); and MA.sub.S11
(SEQ ID NO: 10). A similar set of peptides with utility according
to the practice of the invention are .beta.-hCG 55-90 and
.beta.-hCG 55-91 (SEQ ID NOS: 11 and 12), i.e., MA with one or two
C-terminal extensions, respectively, from the 3-hCG amino acid
sequence.
[0134] MA (SEQ ID NO: 2); pMA (SEQ ID NO: 3); MA.sub.S1 (SEQ ID NO:
4); MA.sub.S2 (SEQ ID NO: 5); MA.sub.S3 (SEQ ID NO: 6); MA.sub.S5
(SEQ ID NO: 7); MA.sub.S9 (SEQ ID NO: 8); MA.sub.S10 (SEQ ID NO:
9); MA.sub.S11 (SEQ ID NO: 10); .beta.-hCG 55-88 (SEQ ID NO: 11);
.beta.-hCG 55-90 (SEQ ID NO: 12) and .beta.-hCG 55-91 (SEQ ID NO:
13) are collectively referred to herein as the "MA peptides."
[0135] The invention also provides functional equivalents of the MA
peptides, as described more fully in Section 8.2. The group
consisting of MA peptides and functional equivalents, but excluding
full-length .beta.-hCG, is referred to herein as the "therapeutic
polypeptides." The therapeutic polypeptides also preferably exclude
the native form of the full length native glycosylated .beta.-core
of .beta.-hCG.
[0136] In one aspect of the invention, a therapeutic polypeptide is
provided in a substantially pure form. The therapeutic polypeptide
is preferably greater than about 90% free of contaminating
polypeptides, more preferably greater than about 99% free of
contaminating polypeptides, still more preferably, greater than
about 99.9% free of contaminating polypeptides. In a preferred
embodiment, the purified therapeutic polypeptide lacks all
contaminating proteins. The therapeutic polypeptide is preferably
provided at a pharmaceutically acceptable level of purity.
[0137] 8.2 Functional Equivalents
[0138] The functional equivalents of the invention exclude the
complete .beta.-chain of hCG (SEQ ID NO: 1) and preferably exclude
the .beta.-hCG (SEQ ID NO: 1) 57-93 peptide. In one aspect of the
invention, the inventors' previously discovered polypeptides and
polypeptides comprising such peptides are excluded from the
functional equivalents of the invention, e.g., Satellin A1 (SEQ ID
NO: 19); the Satellin A1 branched peptide: .beta.-hCG 45-57
[Leu-Gln-Dab(Pro)-Val-Leu-Pro-Dab(Pro)-Leu-Pro-Gln-Val-Val-Cys
((SEQ ID NO: 52) see SEQ ID NO: 19, for primary sequence), where
"Dab" represents diaminobutyric acid, and Dab(Pro) indicates a
proline peptide-bonded to the amino side chain of Dab], the
Satellin A2 circularized peptide [.beta.-hCG 45-57 (SEQ ID NO: 20)
with a cysteine residue added to the N-terminus, circularized via a
disulfide bond between the cysteine residues], and the Satellin B
peptide (SEQ ID NO: 23), as well as .beta.-hCG (SEQ ID NO: 1)
109-145, 47-55, 48-56, 45-57 fused to 109-119, and 45-57 in
combination with 109-119. The following peptides are also
preferably excluded from the functional equivalents of the
invention: .beta.-hCG (SEQ ID NO: 1) 41-53, 41-54, 42-53, 42-53,
43-53, 44-53, 44-57, 45-53, 45-54, 45-55, 45-56, 45-57, 45-58,
46-53, 47-53, 47-54, 47-56, 47-58, 48-145, 58-145, and 109-119.
However, these previously discovered peptides may be included in
various method and formulation aspects of the invention, as
discussed in more detail below.
[0139] The functional equivalents of the invention retain some or
all of the corresponding biological activity of the corresponding
peptide fragments with native amino acid sequences, and preferably
exhibit enhanced activity.
[0140] 8.2.1 Rationally Designed Polypeptides
[0141] The functional equivalents include rationally designed
peptides. The inventors have identified several repeated patterns
within the .beta.-hCG sequence and its active fragments that have
permitted successful prediction by the inventors of smaller active
fragments (i.e., fragments comprising some or all of the
therapeutic activity of MA) of MA and other peptides derived from
.beta.-hCG (SEQ ID NO: 1), horse .beta. CG (SEQ ID NO: 24), sheep
.beta. LH (SEQ ID NO: 25), pig .beta. LH (SEQ ID NO: 26), dog
.beta. LH (SEQ ID NO: 27), bovine .beta. LH (SEQ ID NO: 28), rat
.beta. LH (SEQ ID NO: 29), cat .beta. LH (SEQ ID NO: 30), and human
.beta. LH (SEQ ID NO: 31) that possess some or all of the
therapeutic activities of MA. The repeated patterns include SH3
motifs, PDZ motifs, myristoylation sites and phosphorylation
sites.
[0142] Src-homology 3 (SH3) domains generally consist of sequences
of approximately 60 amino acid residues generally forming two small
.beta. sheets that bind to each other at right angles. Ligands for
SH3 domains include peptides with short proline-rich motifs,
especially the PxxP motif, where P stands for proline and x stands
for any other amino acid. Activation of SH3 domains mediates the
formation of signaling complexes. SH3 binding motifs are typically
7-9 amino acids in length at their core structure. The SH3 domain
is involved in cell-to-cell communication and in signal
transduction from the cell surface to the nucleus. An SH3 binding
interaction is responsible for the complexing of the proto-oncogene
Vav to Grb-2, found only in hematopoietic cells. An SH3 interaction
is also responsible for activation of Ras in abnormal cells
resulting in apoptosis; in normal cells, the same interaction
induces proliferation. This multi-action pathway has also been
reported to play a role in embryogenesis.
[0143] PDZ domains are modular protein-protein interaction domains
that are specialized for binding to specific C-terminal peptide
sequences and to other PDZ domains. Many proteins contain multiple
PDZ domains, thereby allowing them to function as multivalent
scaffolds for organizing large protein complexes. PDZ proteins
recognize the consensus sequence X-S/T-X-V/I, where x is any amino
acid and S is serine, T is threonine, V is valine and I is
isoleucine.
[0144] Various proteins in signal transduction pathways are
myristoylated, and myristoylation is involved in the membrane
interactions of various proteins. The intermediate hydrophobic
nature of the modification plays an important role in the
reversible membrane anchoring of these proteins. Protein
myristoylation is also involved in protein-protein interactions,
which are regulated by the interplay between ptotein
phosphorylation and membrane phospholipds.
[0145] Phosphorylation is the addition of a phosphate to an organic
compound, such as glucose to produce glucose monophosphate,
typically through the action of phosphorylase or kinase.
Phosphorylation at particular sites on proteins can trigger an SH3
mediated activity or activate and/or inhibit the activity of a
cyclin-CDK complex.
[0146] The following motifs are present in the beta chain of hCG:
[0147] SH3 domain motifs: .beta.-hCG 4-7, 50-53, 70-73, 124-129,
141-144 [0148] PDZ domain motifs: .beta.-hCG 30-33, 41-44, 139-142
[0149] myristoylation sites: .beta.-hCG 22-27, 47-52, 71-76 [0150]
phosphorylation sites: .beta.-hCG 66-68, 94-99, 109-112,
120-122
[0151] The following motifs are present in the beta chain precursor
of horse coriogonadotropin: [0152] SH3 domain motifs: 24-27, 44-47,
70-73, 90-93, 142-145 [0153] PDZ domain motifs: 50-53, 59-62,
[0154] myristoylation sites: 16-21, 91-96 [0155] phosphorylation
sites: 78-80, 78-81, 86-88, 137-139, 137-140, 157-159, 158-161,
[0156] The following motifs are present in the beta chain precursor
of sheep lutropin: [0157] SH3 domain motifs: 24-27, 70-73, 90-93,
133-136 [0158] PDZ domain motifs: 50-53 [0159] myristoylation
sites: 17-22, 91-96, 121-126 [0160] phosphorylation sites: 60-62,
78-81, 86-88, 116-119
[0161] The following motifs are present in beta chain of rat
lutropin: [0162] SH3 domain motifs: 24-27, 70-73, 91-94, 133-136
[0163] PDZ domain motifs: 15-18, 50-53, 59-62 [0164] myristoylation
sites: 91-96, 121-126 [0165] phosphorylation sites: 78-80, 78-81,
86-88, 107-109, 116-119
[0166] The following motifs are present in the beta chain precursor
of pig lutropin: [0167] SH3 domain motifs: 24-27, 70-73, 90-93,
133-136 [0168] PDZ domain motifs: 50-53, 59-62 [0169]
myristoylation sites: 17-22, 91-96, 121-126 [0170] phosphorylation
sites: 78-80, 78-81, 86-88, 116-119
[0171] The following motifs are present in the beta chain of bovine
leutropin precursor: [0172] SH3 domain motifs: 24-27, 70-73, 90-93,
133-136 [0173] PDZ domain motifs: 50-53 [0174] myristoylation
sites: 17-22, 91-96, 121-126 [0175] phosphorylation sites: 60-62,
78-81, 86-88, 116-119
[0176] The following motifs are present in the beta chain of dog
leutropin precursor: [0177] SH3 domain motifs: 21-24, 67-70, 86-90,
130-133 [0178] PDZ domain motifs: 47-50, 56-59 [0179]
myristoylation sites: 13-18, 88-93, 118-123 [0180] phosphorylation
sites: 75-78, 83-85, 104-106, 113-116
[0181] The following motifs are present in the beta chain of cat
leutropin precursor: [0182] SH3 domain motifs: 26-29, 72-75, 92-95,
135-138 [0183] PDZ domain motifs: 52-55 [0184] myristoylation
sites: 18-23, 93-98, 123-128 [0185] phosphorylation sites: 22-24,
22-25, 80-82, 80-83, 109-111, 118-121
[0186] The following motifs are present in the beta chain of human
leutropin precursor: [0187] SH3 domain motifs: 24-27, 71-74, 91-94
[0188] PDZ domain motifs: 51-54 [0189] myristoylation sites: 16-21,
42-47, 91-96 [0190] phosphorylation sites: 78-80, 78-81, 86-88,
107-109, 114-117, 116-119
[0191] Based on the observations presented herein, the inventors
have established the following guidelines to for peptide selection:
[0192] peptides are preferably at least 5 amino acid residues in
length; and [0193] peptides preferably include one motif from each
of the following groups: [0194] Group A: SH3 and PDZ domain motifs
[0195] Group B: phosphorylation and myristoylation domain
motifs
[0196] Synthesis and screening of the peptides may be accomplished
using assays described herein or otherwise known in the art.
[0197] Preferred sites are found in mammalian LH or CG, preferably
from .beta.-hCG (SEQ ID NO: 1), horse .beta. CG (SEQ ID NO: 24),
sheep .beta. LH (SEQ ID NO: 25), pig .beta. LH (SEQ ID NO: 26), dog
.beta. LH (SEQ ID NO: 27), bovine .beta. LH (SEQ ID NO: 28), rat
.beta. LH (SEQ ID NO: 29), cat .beta. LH (SEQ ID NO: 30), and human
.beta. H (SEQ ID NO: 31).
[0198] Accordingly, the present invention provides a rationally
designed polypeptide comprising at least one amino acid segment
selected from the group consisting of SH3 and PDZ domain motifs;
and at least one amino acid segment selected from the group
consisting of phosphorylation and myristoylation domain motifs
("rationally designed polypeptides").
[0199] In conjunction with a Group A motif the Group B motif is
preferably positioned no more than 20 AA residues from the Group A
motif in either direction (i.e., towards the N terminus or towards
the C terminus) from the Group A motif. The sequence of the Group B
motif preferably begins no more than 20 amino acid residues from
the N or C terminus of the Group A motif.
[0200] Where the Group A motif is an SH3 and the Group B motif is a
phosphorylation site the amino acid sequence of the phosphorylation
site preferably begins no more than 10 amino acid residues from the
N or C terminus of the SH3 region.
[0201] In a preferred embodiment, the group A motif is an SH3 motif
and sequences contiguous with the SH3 motif include other proline
residues adjacent to the SH3 motif.
[0202] In a preferred embodiment, the phosphorylation site is
separated from the SH3 motif by no more than 5 amino acid residues,
more preferably no more than 3 amino acid residues, most preferably
no more than 1 amino acid. In other words, there are preferably no
more than 5 amino acid residues separating the phosphorylation site
from the SH3 motif, more preferably no more than 3 amino acid
residues, most preferably no more than 1 amino acid residue.
[0203] Where the Group A motif is an SH3 motif and the Group B
motif is a myristoylation motif the myristoylation motif is
preferably separated from either the N or C terminus of the SH3
motif by no more than 15 amino acid residues, more preferably no
more than 10 amino acid residues, most preferably no more than 5
amino acid residues.
[0204] In a preferred embodiment, the Group A motif is an SH3
motif, and the Group B motif is a myristoylation motif, the
myristoylation motif is immediately adjacent to with the SH3 motif,
i.e., no amino acid residues separate the two motifs.
[0205] In one embodiment of the invention, the rationally designed
polypeptide contains more than one Group A motif with one Group B
motif.
[0206] In another embodiment of the invention the rationally
designed polypeptide contains more than one Group A motif with more
than one Group B motif.
[0207] In an embodiment of the invention the rationally designed
polypeptide contains one Group A motif with more than one Group B
motif.
[0208] In a preferred embodiment, the rationally designed
polypeptide comprises at least one amino acid segment selected from
the group consisting of .beta.-hCG 4-7, 50-53, 70-73, 124-129,
141-144, 30-33, 41-44, and 139-142; and at least one amino acid
segment selected from the group consisting of .beta.-hCG 22-27,
47-52, 71-76, 66-68, 94-99, 109-112, and 120-122. Both components
of the rationally designed polypeptides are suitably provided in a
continuous segment of the foregoing CG and LH chains. For example,
one group of rationally designed polypeptides includes .beta.-hCG
6-37, 6-38, 6-39 and 6-40 (SEQ ID NOS: 13, 14, 15 and 16), each of
which includes a myristoylation site (22-27) and a PDZ domain
(30-33).
[0209] In an embodiment the rationally designed polypeptide
comprises a sequence selected from hCG 1-39, 1-29, 1-35, 41-54,
66-76, 93-130, 93-131, 93-132, 93-135, 93-144, 93-145, 41-54 linked
to 55-92, 41-54 linked to 59-89, or 41-54 linked to 55-76.
[0210] In another preferred embodiment, the rationally designed
polypeptide comprise a myristoylation and/or an SH3 domain selected
from the group consisting of the HUSI-II polypeptide,.sup.62
.beta.-hCG (SEQ ID NO: 1), horse .beta. CG (SEQ ID NO: 24), sheep
.beta. LH (SEQ ID NO: 25), pig .beta. LH (SEQ ID NO: 26), dog
.beta. LH (SEQ ID NO: 27), bovine .beta. LH (SEQ ID NO: 28), rat
.beta. LH (SEQ ID NO: 29), cat .beta. LH (SEQ ID NO: 30), and human
.beta. LH (SEQ ID NO: 31).
[0211] The rationally designed peptides are preferably at least 5
and less than 100 amino acid residues in length, more preferably at
least 5 and less than 50, and more preferably at least 5 and less
than 25, still more preferably at least 5 and less then 20, and
most preferably at least 5 and less than 15 amino acid residues in
length.
[0212] One or more rationally designed polypeptides may be provided
as component(s) of a fusion polypeptide, as discussed in section
8.2.3. Derivatives and analogues are also provided, as discussed in
section 8.2.4. The segments of the fusion need not be contiguous on
the native polypeptide, i.e., the segments may come from disparate
positions of the native polypeptide.
[0213] In a related aspect, the invention also provides methods
using the HUSI-II polypeptide,.sup.63 .beta.-hCG (SEQ ID NO: 1),
horse .beta. CG (SEQ ID NO: 24), sheep .beta. LH (SEQ ID NO: 25),
pig .beta. LH (SEQ ID NO: 26), dog .beta. LH (SEQ ID NO: 27),
bovine .beta. LH (SEQ ID NO: 28), rat .beta. LH (SEQ ID NO: 29),
cat .beta. LH (SEQ ID NO: 30), and human .beta. LH (SEQ ID NO: 31)
and fragments of these polypeptides. The data presented herein are
consistent with the proposition that the HUSI-II polypeptide and
fragments thereof will exhibit the same therapeutic efficacies as
the therapeutic polypeptides. Thus, while the ensuing discussion
speaks in terms of the therapeutic polypeptides, the discussion is
also applicable to the HUSI-II polypeptide,.sup.64 .beta.-hCG (SEQ
ID NO: 1), horse .beta. CG (SEQ ID NO: 24), sheep .beta. LH (SEQ ID
NO: 25), pig .beta. LH (SEQ ID NO: 26), dog .beta. LH (SEQ ID NO:
27), bovine .beta. LH (SEQ ID NO: 28), rat .beta. LH (SEQ ID NO:
29), cat .beta. LH (SEQ ID NO: 30), and human .beta. LH (SEQ ID NO:
31) and fragments of these polypeptides. Preferred fragments are
those comprising or consisting of the SH3 motif and its flanking
residues. Highly preferred fragments are AA-40-46, 40-60, 40-59,
40-58, 40-66, 40-67, 40-68. Moreover, in another highly preferred
embodiment, the fragment includes the SH3 motif and the
myristoylation site. Moreover, the invention includes fusion
peptides which may comprise one or more fragments of HUSI-II, and
may optionally comprise heterologous amino acid sequences.
[0214] 8.2.2 Polypeptides Containing MA Peptides
[0215] Where the therapeutic polypeptides are larger peptides that
comprise MA peptides corresponding to amino acid sequences found in
.beta.-hCG (SEQ ID NO: 1), such therapeutic polypeptides preferably
lack amino acid residues from the .beta.-hCG sequence (SEQ ID NO:
1) that are contiguous to the MA peptide sequence, i.e., the
extensions of the MA peptides are preferably not coextensive with
the corresponding contiguous amino acids from .beta.-hCG (SEQ ID
NO: 1). Moreover, the therapeutic polypeptides of the invention do
not include a polypeptide consisting of full-length human
.beta.-hCG (SEQ ID NO: 1).
[0216] In one aspect of the invention, the inventors' previously
discovered polypeptides and polypeptides comprising such peptides
are excluded from the therapeutic polypeptides of the invention,
e.g., Satellin A1 (SEQ ID NO: 19); the Satellin A1 branched
peptide: .beta.-hCG 45-57
[Leu-Gln-Dab(Pro)-Val-Leu-Pro-Dab(Pro)-Leu-Pro-Gln-Val-Val-Cys
((SEQ ID NO: 52) see SEQ ID NO: 19, for primary sequence), where
"Dab" represents diaminobutyric acid, and Dab(Pro) indicates a
proline peptide-bonded to the amino side chain of Dab], the
Satellin A2 circularized peptide [.beta.-hCG 45-57 (SEQ ID NO: 20)
with a cysteine residue added to the N-terminus, circularized via a
disulfide bond between the cysteine residues], and the Satellin B
peptide (SEQ ID NO: 23), as well as .beta.-hCG (SEQ ID NO: 1)
109-145, 47-55, 48-56, 45-57 fused to 109-119, and 45-57 in
combination with 109-119. The following peptides, previously
identified by the inventors, are also preferably excluded from the
therapeutic polypeptide of the invention: .beta.-hCG (SEQ ID NO: 1)
41-53, 41-54, 42-53, 42-53, 43-53, 44-53, 44-57, 45-53, 45-54,
45-55, 45-56, 45-57, 45-58, 46-53, 47-53, 47-54, 47-56, 47-58,
48-145, 58-145, and 109-119. However, these previously discovered
peptides may be included in various method and formulation aspects
of the invention, as discussed in more detail below.
[0217] 8.2.3 Fusion Polypeptides, Branched and Circularized
Polypeptides
[0218] The functional equivalents of the invention include fusion
polypeptides comprising two or more polypeptide segments covalently
linked together. The polypeptide segments of the fusion
polypeptides may include any combination of the following: MA
peptides, functional equivalents, and heterologous polypeptides.
For example, a fusion polypeptide of the invention may consist of
two or more MA peptides joined end-to-end by a peptide bond. As
another example, one or more MA peptides may be joined at the amino
and/or carboxy terminus of a heterologous polypeptide, and/or
joined to one or more side chains of one or more amino acid
residues within the heterologous polypeptide to form branches
(discussed more fully below). Fusion polypeptides of the invention
may also include additional MA peptides and/or heterologous
polypeptides as branches. The polypeptide segments of the fusion
proteins may or may not be contiguous to one another (i.e., an
intervening sequence may be present). The polypeptide segments of
the fusion polypeptides may also be linked by hydrocarbon
linkages.
[0219] A therapeutic polypeptide may be joined at its amino- or
carboxy-terminus via a peptide bond to another therapeutic
polypeptide. The invention also provides fusion polypeptides
comprising one or more therapeutic polypeptides joined to one or
more heterologous polypeptides. The heterologous polypeptides may
be selected from the group which comprises heterologous protein or
peptide therapeutics, and other pharmacologically active
polypeptides.
[0220] In a specific embodiment, the derivative is a fusion protein
comprising a therapeutic polypeptide joined at its amino or
carboxy-terminus to a chemokine (or active fragment, analogue, or
derivative thereof). Preferred chemokines are those which are
therapeutically useful in the treatment of HIV-infected subjects.
For example, the chemokine may be selected from the group
consisting of MIP-1.alpha., MIP-1.beta. and RANTES..sup.65 The
chimeric polypeptide may be produced by recombinant expression of a
nucleic acid encoding the fusion protein (comprising a MA peptide
coding sequence joined in-frame to a coding sequence for a
different protein).
[0221] A therapeutic polypeptide of the invention may also be
provided as a component of a fusion protein comprising a targeting
agent. For example, the targeting agent may be an antibody,
preferably a monoclonal antibody. In a specific embodiment, the
therapeutic polypeptide is linked to an antibody which targets a
specific cancer, such as prostate cancer, to facilitate contact
between the therapeutic polypeptide and the cancer cells. In
another embodiment, the antibody has specificity for specific blood
cells, such as cells expressing CD34. In this manner, specific
cells can be targeted for the proliferation-inducing effects of the
therapeutic polypeptides of the invention.
[0222] Where the therapeutic polypeptide is linked to a targeting
agent, the fusion polypeptide may also comprise other therapeutics,
such as a nucleic acid, gene, peptide, toxin, or a radioactive
molecule for tagging or to induce death in the target cell.
[0223] One or more polypeptide segments may be covalently bound to
one or more side chains of another of the polypeptide segments,
thereby forming a branched polypeptide. For example, one or more MA
peptides may be covalently bound to one or more side chains of a
heterologous polypeptide, thereby forming a branched polypeptide in
which each MA peptide forms a branch, which is linked to the
heterologous polypeptide at a side chain. Conversely, one or more
heterologous polypeptides may be joined as branch(es) to one or
more side chains of an MA peptide. One or more intervening amino
acid residues (e.g., an amino acid chain forming a flexible
polymer) may also be present between the branches and the base
sequence.
[0224] The functional equivalents of the invention also include
branched analogs. The branched analogs are analogous to the
branched polypeptides described in the preceding paragraph with the
exception that branching is facilitated by one or more amino acid
substitutions or insertions with amino acid(s) or amino acid
analog(s) having a free amino- or carboxy-side chain. The side
chain(s) may each form a peptide bond with a sequence of one or
more amino acids, e.g., a sequence of one or more prolines.
[0225] The functional equivalents also include circularized
polypeptides. Circularized polypeptides may be formed, e.g., by
disulfide bond formation. Where necessary, a cysteine residue may
be added to the therapeutic polypeptide to facilitate disulfide
bond formation. For example: [0226] one or more native amino acid
residues from the therapeutic polypeptide, e.g., an MA peptide, is
replaced with cysteine; [0227] one or more cysteines is inserted
between native residues of the therapeutic polypeptide, e.g.,
inserted between native residues of an MA peptide; and/or [0228]
one or more cysteines is added at one or both ends of the
therapeutic polypeptide, e.g., added at one or both ends of an MA
peptide sequence.
[0229] The therapeutic polypeptides can also be linked to a
suitable targeting agent or delivery vector to direct the
therapeutic polypeptides to a desired site of action.
[0230] In one aspect a therapeutic polypeptide is linked to a
monoclonal antibody, such as an antibody specific for a tumor
marker such as a monoclonal antibody against prostate specific
antigen (PSA), or a marker over-expressed in a tumor. The
monoclonal antibody delivers the therapeutic polypeptide directly
to the site of a tumor expressing PSA, e.g., to the site of a
primary prostate lesion or a metastasis. This allows the
polypeptide to be concentrated at the tumor site to increase its
activity, i.e., inducing apoptosis and/or exerting anti-angiogenic
activity.
[0231] In a specific embodiment the therapeutic peptides are linked
to an antibody that recognizes a cell surface marker or receptor.
In a preferred embodiment, the antibody is CD34 for targeting MA to
stem cells.
[0232] In another specific aspect the therapeutic polypeptide is
linked to a monoclonal antibody specific to a viral protein. In a
preferred aspect the therapeutic polypeptides is linked to a
monoclonal antibody against an HIV protein. This allows the
therapeutic peptide to be delivered directly to HIV expressing
cells targeting its action.
[0233] 8.2.4 Derivatives of the MA Peptides
[0234] The derivatives of the MA peptides can comprise
substitutions, additions or deletions that provide for
therapeutically effective molecules. In one aspect of the
invention, the derivatives have a primary amino acid sequence of a
therapeutic polypeptide, except that functionally equivalent amino
acid residues are substituted for residues within the sequence.
Such derivatives retain some or all of the bioactivity of the
corresponding therapeutic polypeptide, preferably a therapeutically
significant degree of activity.
[0235] One or more amino acid residues of a therapeutic polypeptide
can be substituted by another amino acid of a similar polarity that
acts as a functional equivalent, resulting in a silent alteration.
Conservative substitutions for an amino acid within the therapeutic
polypeptide may be selected from other members of the class to
which the amino acid belongs. For example, the nonpolar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan and methionine. The
polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine, and glutamine. The positively
charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid
and glutamic acid.
[0236] In a preferred embodiment the therapeutic polypeptides of
the invention are modified to increase their hydrophobicity in
order to enhance their penetration into the cell through the cell
membrane. This can be accomplished by the addition of one or more
hydrophobic amino acid residues at either the amino terminus,
carboxyl terminus or within the amino acid sequence of the
therapeutic polypeptide. The therapeutic polypeptide can also have
one or more hydrophobic residues within its sequence replacing
non-hydrophobic residues. The typical hydrophobic residues include
but are not limited to alanine, leucine, isoleucine, valine,
proline, phenylalanine, tryptophan and methionine.
[0237] Functional equivalents can be chemically synthesized..sup.66
For example, peptides can be synthesized by solid phase techniques,
cleaved from the resin, and purified by preparative high
performance liquid chromatography (HPLC)..sup.67 Functional
equivalents of MA peptides can also be synthesized using a peptide
synthesizer. The composition of the synthetic peptides may be
confirmed by amino acid analysis or sequencing..sup.68 Synthetic
production of the therapeutic polypeptides is discussed more fully
in 8.4.1.
[0238] Functional equivalents of the MA peptides can be prepared by
chemical peptide synthesis or by recombinant production from a
nucleic acid encoding the derivative. A nucleic acid encoding the
derivative can be prepared by various techniques known in the art
in light of the instant disclosure. For example, such a nucleic
acid may be prepared by mutation of a nucleic acid encoding the
corresponding therapeutic polypeptide. Various techniques for
mutagenesis are commonly practiced in the art, e.g., chemical
mutagenesis, in vitro site-directed mutagenesis, etc..sup.69
Recombinant production of the therapeutic polypeptides is discussed
more fully in 8.4.2.
[0239] The functional equivalents of the invention may also
comprise various nonclassical amino acids. For example, the
functional equivalents include therapeutic polypeptides with any
one or more of the following alterations: [0240] one or more native
amino acid residues from the therapeutic polypeptide, e.g., an MA
peptide is replaced with a non-classical amino acid residue; [0241]
one or more non-classical residues is inserted between native
residues of the therapeutic polypeptide, e.g., inserted between
native residues of an MA peptide; and [0242] one or more
non-classical residues is added at one or both ends of the
therapeutic polypeptide, e.g., added at one or both ends of an MA
peptide sequence.
[0243] Examples of non-classical amino acids include: D-isomers of
the common amino acids, 2,4-diaminobutyric acid, .alpha.-amino
isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,
.gamma.-Abu, .epsilon.-Ahx, 6-amino hexanoic acid, Aib, 2-amino
isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,
cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, .beta.-alanine, fluoro-amino acids, designer
amino acids such as .beta.-methyl amino acids, C.alpha.-methyl
amino acids, N.alpha.-methyl amino acids, and amino acid analogs in
general.
[0244] Also included within the scope of the invention are
derivatives comprising therapeutic polypeptides, which have been
differentially modified during or after synthesis, e.g., by
benzylation, glycosylation, acetylation, phosphorylation,
amidation, PEGylation, derivatization by known protecting/blocking
groups, proteolytic cleavage, linkage to an antibody molecule or
other cellular ligand, etc. In one embodiment, the therapeutic
polypeptides are acetylated at the N-terminus and/or amidated at
the C-terminus. In another embodiment, the derivatives are
conjugated to polymers, e.g., polymers known in the art to
facilitate oral delivery, decrease enzymatic degradation, increase
solubility of the polypeptides, or otherwise improve the chemical
properties the therapeutic polypeptides for administration to
humans or other animals. The polymers may be joined to the
therapeutic polypeptides by hydrolyzable bonds, so that the
polymers are cleaved in vivo to yield the active therapeutic
polypeptides.
[0245] Any of numerous chemical modifications may be carried out by
known techniques, including but not limited to acetylation,
formylation, oxidation, reduction; metabolic synthesis in the
presence of tunicamycin, etc.
[0246] In a preferred embodiment the therapeutic polypeptides of
the invention are produced in reverse order, substituting D-amino
acids for the naturally occurring L-amino acids in order to
increase stability and in vivo half-life on the polypeptide. In
this embodiment, the amino-terminus amino acid of the therapeutic
polypeptide becomes the carboxy-terminus amino acid and the
carboxy-terminus amino acid becomes the amino-terminus amino
acid.
[0247] 8.3 Antibodies
[0248] The invention also includes monoclonal and polyclonal
antibodies having binding affinity for one or more of the
therapeutic polypeptides of the invention. The term "antibodies" as
used herein is broadly construed to include (1) monoclonal and
polyclonal antibodies which bind to one or more therapeutic
polypeptides of the invention, as well as humanized analogues of
such antibodies and active fragments of such antibodies which bind
to one or more of the therapeutic polypeptides, and (2) antibodies
which bind to the variable regions of the foregoing antibodies,
humanized analogs and active fragments.
[0249] The antibodies can be manufactured by a wide variety of
known methods. As an example, antibodies may be produced by
immunizing a host animal by injection with MA peptides. Examples of
suitable animals include goats, sheep, donkeys, horses, hamsters,
chickens, rabbits, mice, rats, etc. Antibody production may be
performed according to a variety of methods known in the art. In a
preferred embodiment, antibodies are raised against therapeutic
polypeptides fused to a carrier protein, such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or ovalbumin (OVA).
The antibodies may, for example, be obtained from tissue culture
supernatants and/or cell lysates, ascite fluid, serum, plasma
and/or whole blood.
[0250] Once obtained, the antibodies may be cleaved to provide
F(Ab)2 and/or F(AB) fragments while still maintaining the activity
of the uncleaved antibodies. Antibodies can be immobilized on
resins, added in solution or coated on other solid support
surfaces.
[0251] Antibodies of the invention can be used in a variety of
assays, for example, enzyme linked immunosorbent assay (ELISA),
Western blot, and immuno-PCR assays, and are also useful in
solution or solid phase affinity quantification/qualification. In a
preferred embodiment the assay is an ELISA.
[0252] In one aspect of the invention, two or more of the
antibodies are used together in an in vitro assay for the
quantification or qualitative analysis of a therapeutic polypeptide
species. In a specific embodiment, one antibody is a monoclonal
antibody to the therapeutic polypeptide and one antibody is a
polyclonal antibody to the therapeutic polypeptide. In a related
embodiment, the antibodies are both monoclonal antibodies
recognizing different epitopes of a therapeutic polypeptide.
[0253] Various adjuvants may be employed in the production of
antibodies of the invention, to increase the immunological
response, depending on the host species, and including but not
limited to Freund's (complete and incomplete), mineral gels such as
aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanins, dinitrophenol, and potentially useful human
adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium
parvum. For preparation of monoclonal antibodies, any technique
that provides for the production of antibody molecules by
continuous cell lines in culture may be used. For example, the
hybridoma technique originally developed by Kohler and
Milstein,.sup.70 as well as the trioma technique, the human B-cell
hybridoma technique,.sup.71 and the EBV-hybridoma technique to
produce human monoclonal antibodies..sup.72 Monoclonal cells lines
can then be screened for binding to the particular MA peptides
peptides using the purified species in any type of immunoassay
available in the art..sup.73 The antibodies are preferably
monoclonal antibodies.
[0254] It will be recognized by one of skill in the art that the
antibodies of the invention have a wide range of uses, e.g., in the
isolation, qualitative characterization, and quantification of MA
peptide; isolation, qualitative characterization, and
quantification of natural and synthetic functional equivalents of
the MA peptide; as well as the isolation, qualitative
characterization, and quantification of immunologically
cross-reactive materials derived from other biological sources
e.g., primate, rodent, ovine, porcine and ovoid species.
[0255] The antibodies of the invention are useful for monitoring
serum levels of the therapeutic polypeptides by methods known in
the art. Antibodies to the therapeutic polypeptides are also useful
for tracking delivery of the therapeutic polypeptides. The
antibodies of the invention can be linked to a suitable tag to
allow visualization of sites where the antibodies have accumulated,
e.g., where they have bound to the therapeutic peptides or other
polypeptides. In one aspect of the invention, tagged antibodies are
administered to a subject to identify the site of accumulation or
expression of the therapeutic polypeptides.
[0256] Analysis of compounds using the antibodies of the invention
may be accomplished in biological fluids and physiological buffers.
Moreover, the antibodies may be immobilized by attachment to a
suitable support structure according to known methods for isolation
and purification of the MA peptide.
[0257] The antibodies of the invention are also useful for in vivo
therapeutic administration and manipulation of naturally occurring
levels of MA or pMA peptide, as well as in vitro analysis of
production levels, location and species, study of
pharmacokineticaly relevant levels of therapeutic polypeptides of
the invention, half-life and in vivo distribution, degradation,
manipulation and sites of production, depot and action. In this
regard, the antibodies of the invention may be bound to agents
having affinity for target tissues. Conversely, the antibodies
themselves are usefully employed in the targeting of therapeutics
to MA peptide-reactive sites, blocking of activity in vitro or in
vivo, modification of solution kinetics, and a wide variety of
other uses that would be apparent to one of skill in the art.
[0258] In one aspect of the invention, one or more antibodies of
the invention is administered to a subject to block the activity of
a therapeutic polypeptide or to block the activity of
cancer-produced hCG or a cancer-produced fragment of hCG (e.g.,
.beta.-core).
[0259] Moreover, the antibodies of the invention are useful for
identifying functional domains, folding patterns, sequence and in
investigating the effects of in vivo/in vitro post-translational
modifications on solubility of MA and pMA. The antibodies of the
invention also find a variety of uses in the investigation of
solubility, physio-chemical, biological, stability, degradation and
structural characteristics of the therapeutic polypeptides.
[0260] The antibodies of the invention are useful in the detection
and purification of MA peptides. The antibodies of the invention
may be linked to a variety of accessory molecules to aid in
purification, analysis, assay characteristics, as well as to
improve targeting, tracking and biological half-life, depoting,
location, and cofactor use. For example, antibodies can be tagged
by known methods with markers, such as with radioactive markers,
fluorescent markers, chemiluminescent markers or affinity tags,
such as biotin.
[0261] The antibodies of the invention provide a significant
advantage in the purification of MA peptides. Techniques used by
the inventors in the initial stages of this work included size
separation methodologies, i.e., SDS-PAGE, gel permeation
chromatography, size-exclusion centrifugation and membrane
separations, and non-specific charge effects, i.e., cation and
anion exchange chromatography, and RP-HPLC. The ability to isolate
the MA peptides using antibody-driven methods is a significant
advance over these original methods.
[0262] The antibodies of the invention are useful in qualitative
and quantitative assays for the presence of natural, synthetic and
modified MA peptide-derived sequences and also for immunologically
cross-reactive materials derived from other biological sources
i.e., primates, rodents, ovine, porcine and ovoid species. Examples
of suitable sources include urine, serum, plasma and whole-blood
collected from suitably qualified donors, tissue sections, biopsy
samples bacterial/tissue culture supernantants/lysates prepared
from transfected/transformed cell cultures, and partially purified
materials derived from these sources.
[0263] The described antibodies can be utilized in the purification
of synthetic and natural MA peptides and/or functional derivatives
from synthetic and natural sources. Examples of suitable starting
materials include, but are not limited to urine, serum, plasma and
whole-blood collected from suitably qualified donors, tissue
sections, biopsy samples bacterial/tissue culture
supernantants/lysates prepared from transfected/transformed cell
cultures, and partially purified to materials derived from these
sources.
[0264] In a specific embodiment antibodies to a therapeutic
polypeptide are immobilized to a support such as a silica or
sepharose bead in order to isolate the therapeutic peptide.
Antibodies may be positioned in a therapeutic polypeptide
manufacturing system (e.g., downstream from an incubator where a
therapeutic polypeptide is being produced by recombinant organisms)
for isolating the therapeutic polypeptide. Examples of suitable
production methods include chemical synthesis, expression in a
suitable recombinant expression vector/host cell culture systems,
and isolation from urine, serum or plasma (e.g., urine, serum or
plasma produced by a recombinant organism).
[0265] The antibodies may be manipulated according to various
techniques known in the art to achieve alteration of soluble
material concentrations, complexing material so as to reduce or
enhance half-life in solute, complexing to neutralize or otherwise
modify activity against target entities, modification of biological
target.
[0266] The antibodies of the invention are also useful for the
production of anti-idiotype antibodies, which will mimic the
therapeutic activities of the therapeutic polypeptides described
herein.
[0267] The antibodies of the invention also find use as immunogens
in suitable animal species (e.g., rat, mouse or rabbit) to produce
an anti-idiotype antibody that mimics the activity of a therapeutic
polypeptide. In a preferred aspect, the immunogen is a monoclonal
antibody that, upon incubation with a therapeutic polypeptide,
inactivates one or more of the therapeutic activities of the
therapeutic polypeptide.
[0268] 8.4 Preparation of Therapeutic Polypeptides
[0269] The therapeutic polypeptides can be prepared by any means
known in the art, for example, by known synthetic or recombinant
techniques. MA and pMA may be isolated from native sources of hCG,
such as early pregnancy urine.
[0270] 8.4.1 Preparation of Therapeutic Polypeptides by Synthetic
Methods
[0271] As an example, the therapeutic polypeptides can be
synthesized by employing the N-.alpha.-9-fluorenylmethyloxycarbonyl
or Fmoc solid phase peptide synthesis chemistry using a Rainin
Symphony Multiplex Peptide Synthesizer. The standard cycle used for
coupling of an amino acid to the peptide-resin growing chain
generally includes: (1) washing the peptide-resin three times for
30 seconds with N,N-dimethylformamide (DMF); (2) removing the Fmoc
protective group on the amino terminus by deprotection with 20%
piperidine in DMF by two washes for 15 minutes each, during which
process mixing is effected by bubbling nitrogen through the
reaction vessel for one second every 10 seconds to prevent
peptide-resin settling; (3) washing the peptide-resin three times
for 30 seconds with DMF; (4) coupling the amino acid to the peptide
resin by addition of equal volumes of a 250 mM solution of the Fmoc
derivative of the appropriate amino acid and an activator mix
consisting or 400 mM N-methylmorpholine and 250 mM
(2-(1H-benzotriazol-1-4))-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) in DMF; (5) allowing the solution to mix
for 45 minutes; and (6) washing the peptide-resin three times for
30 seconds of DMF. This cycle can be repeated as necessary with the
appropriate amino acids in sequence to produce the desired peptide.
Exceptions to this cycle are amino acid couplings predicted to be
difficult by nature of their hydrophobicity or predicted inclusion
within a helical formation during synthesis. For these situations,
the above cycle can be modified by repeating step 4 a second time
immediately upon completion of the first 45 minute coupling step to
"double couple" the amino acid of interest. In the first coupling
step in peptide synthesis, the resin can be allowed to swell to
effect more efficient coupling by increasing the time of mixing in
the initial DMF washes to three 15-minute washes rather than three
30 second washes.
[0272] After synthesis, the peptide can be cleaved from the resin
as follows: (1) washing the peptide-resin three times for 30
seconds with DMF; (2) removing the Fmoc protective group on the
amino terminus by washing two times for 15 minutes in 20%
piperidine in DMF; (3) washing the peptide-resin three times for 30
seconds with DMF; and (4) mixing a cleavage cocktail consisting of
95% trifluoroacetic acid (TFA), 2.4% water, 2.4% phenol, and 0.2%
trisopropysilane with the peptide-resin for two hours, then
filtering the peptide in the cleavage cocktail away from the resin,
and precipitating the peptide out of solution by addition of two
volumes of ethyl ether.
[0273] To isolate the peptide, the ether-peptide solution can be
allowed to sit at -20.degree. C. for 20 minutes, then centrifuged
at 6,000.times.G for 5 minutes to pellet the peptide. The peptide
can be washed with ethyl ether to remove residual cleavage cocktail
ingredients. The final peptide product can be purified by reversed
phase high pressure liquid chromatography (RP-HPLC) with the
primary solvent consisting of 0.1% TFA and the eluting buffer
consisting of 80% acetonitrile and 0.1% TFA. The purified peptide
can then be lyophilized to a powder.
[0274] Branched derivatives of the therapeutic polypeptides may be
prepared by any method known in the art for covalently linking any
naturally occurring or synthetic amino acid to any naturally
occurring or synthetic amino acid in a peptide chain. The amino
acid residue to which the branch is attached preferably has a side
chain group able to react with the amino or carboxyl group at a
terminus of the branch, so that the branch may be covalently bound
to the peptide chain.
[0275] Examples of amino acids with a free amino side chain group
include diaminobutyric acid, lysine, arginine, ornithine,
diaminopropionic acid and citrulline. Such amino acids can be
incorporated into a peptide so that an amino acid can form a branch
therewith, for example, by forming a peptide bond to the free amino
side group, from that residue.
[0276] Examples of amino acids with a free carboxyl side chain
group include glutamic acid, aspartic acid and homocitrulline. Such
amino acids can be incorporated into polypeptide (e.g., the
therapeutic polypeptide) so that an amino acid can form a branch
therewith, for example, by forming a peptide bond to the free
carboxyl side group, from that residue.
[0277] The amino acid from which the branch is formed can be linked
to a side chain group of an amino acid in the peptide chain by any
type of covalent bond. Preferred bonds include peptide bonds, ester
bonds and disulfide bonds Amino acids capable of covalently bonding
to a branch may also be substituted for residues within the MA
peptide sequence.
[0278] As an example, branched peptides can be prepared as follows:
(1) the amino acid to be branched from the main peptide chain can
be prepared as an N-.alpha.-tert-butyloxycarbonyl-protected
(Boc-protected) amino acid pentafluorophenyl (Opfp) ester and the
residue within the main chain to which this branched amino acid
will be attached can be an N-Fmoc-.alpha.-.gamma.-diaminobutyric
acid; (2) the coupling of the Boc protected amino acid to
diaminobutyric acid can be achieved by adding 5 grams of each
precursor to a flask containing 150 ml DMF, along with 2.25 ml
pyridine and 50 mg dimethylaminopyridine and allowing the solution
to mix for 24 hours; (3) the peptide can then be extracted from the
150 ml coupling reaction by mixing the reaction with 400 ml
dichlormethane (DCM) and 200 ml 0.12 N HCl in a 1 liter separatory
funnel, and allowing the phases to separate, saving the bottom
aqueous layer and re-extracting the top layer two more times with
200 ml 0.12 N HCl; (4) the solution containing the peptide can be
dehydrated by adding 2-5 grams magnesium sulfate, filtering out the
magnesium sulfate, and evaporating the remaining solution to a
volume of about 2-5 ml; (5) the dipeptide can then be precipitated
by addition of ethyl acetate and then 2 volumes of hexanes and then
collected by filtration and washed two times with cold hexanes; and
(6) the resulting filtrate can be lyophilized to achieve a light
powder form of the desired dipeptide. Branched peptides prepared by
this method will have a substitution of diaminobutyric acid at the
amino acid position which is branched. Branched peptides containing
an amino acid or amino acid analog substitution other than
diaminobutyric acid can be prepared in an analogous manner, using
is the N-F-moc coupled form of the amino acid or amino acid
analog.
[0279] The therapeutic polypeptides or functional equivalents of
the invention can also be prepared as cyclic polypeptides. For
example, in a segment containing a first cysteine residue, a second
cysteine residue may be inserted or substituted for a different
amino acid residue in the segment, and a disulfide bond may be
formed between the first and second cysteine residues. In a related
method, the therapeutic polypeptide comprises at least two cysteine
residues, disulfide bond is formed between two of the cysteine
residues. The therapeutic polypeptides may also be modified as
necessary by inserting one or more cysteine residues between two
other (preferably non-cysteine) amino acid residues; coupling at
least one cysteine residue at an end of the therapeutic
polypeptide; or replacing at least one non-cysteine amino acid
residue with a cysteine residue. Suitable modifications are those
which do not eliminate the therapeutic efficacy of the therapeutic
polypeptide.
[0280] Cyclic polypeptides may also be formed by establishing a
disulfide bond between two cysteine residues or by an amide
linkage. Disulfide bridge formation can be achieved by (1)
dissolving the purified polypeptide at a concentration of between
0.1.-0.5 mg/ml in 0.01 M ammonium acetate, pH 7.5; (2) adding 0.01
M potassium ferricyanide to the dissolved polypeptide dropwise
until the solution appears pale yellow in color and allowing this
solution to mix for 24 hours; (3) concentrating the cyclized
polypeptide to 5-10 ml of solution, repurifying the peptide by
reverse phase-high pressure liquid chromatography (RP-HPLC) and
finally lyophilizing the peptide. If the polypeptide to be cyclized
does not contain two appropriately situated cysteine residues,
cysteine residues can be introduced at the amino-terminus and/or
carboxy-terminus and/or internally such that the peptide to be
cyclized contains two cysteine residues spaced such that the
residues can form a disulfide bridge.
[0281] In another approach, cyclic peptides are obtained by
establishing an amide linkage. The amide linkage can be established
by various methods known in the art. In one method, an
allyl-protected amino acid, such as aspartate, glutamate,
asparagine or glutamine, can be incorporated into the peptide as
the first amino acid, and then the remaining amino acids coupled
onto the allyl-protected amino acid. The allyl protective group can
be removed by mixing the peptide-resin with a solution to of
tetrakistriphenylphosphine palladium (0) in a solution of
chloroform containing 5% acetic acid and 2.5% N-methylmorpholine.
The peptide resin can be washed with 0.5% N,N-diisopropylethylamine
(DIEA) and 0.5% sodium diethyldithiocabamate in DMF. The amino
terminal Fmoc group on the peptide chain can be removed by two
incubations for 15 minutes each in 20% piperidine in DMF, and
washed with DMF. The activator mix, N-methylmorpholine and HBTU in
DMF, can be brought onto the column and allowed to couple the free
amino terminal end to the carboxyl group generated by removal of
the allyl group to cyclize the peptide. The peptide can be cleaved
from the resin as described in the general description of chemical
peptide synthesis above. The peptide can be purified by reverse
phase-high pressure liquid chromatography (RP-HPLC). When the
peptide to be cyclized does not contain an allyl-protected amino
acid, an allyl-protected amino acid can be introduced into the
sequence of the peptide at the amino-terminus, carboxy-terminus or
internally, to permit the peptide to be cyclized.
[0282] The nucleic acid can be constructed by ligating the
appropriate nucleic acid sequences encoding the desired amino acid
sequences in the proper coding frame. The nucleic acid can be
induced to express the chimeric product by standard methods.
Alternatively, the chimeric product may be made by protein
synthetic techniques, e.g., by use of a peptide synthesizer.
[0283] 8.4.2 Preparation of Therapeutic Polypeptides by Recombinant
Expression Techniques
[0284] The therapeutic polypeptides of the invention can also be
obtained by recombinant expression techniques..sup.74 Examples of
recombinant expression systems that may be suitably employed in the
production of the therapeutic polypeptides of the invention include
prokaryotic cell systems, eukaryotic cell systems and artificial
expression systems.
[0285] An expression vector for expressing a nucleic acid sequence
encoding a therapeutic polypeptide can be introduced into a cell
for expression of the therapeutic polypeptide. In a preferred
embodiment, the nucleic acid is DNA. The vector can remain episomal
or become chromosomally integrated, as long as it can be
transcribed in the host cell to produce the desired RNA. Vectors
can be constructed by standard recombinant DNA technology methods.
Vectors can be plasmid, viral, or others known in the art, used for
replication and expression in, eukaryotic or prokaryotic cells.
[0286] For prokaryotic production, any expression vector that is
functional in the selected prokaryotic host cell may be used,
provided that the vector contains all of the necessary nucleic acid
components or elements to ensure expression of the therapeutic
polypeptide. Typically, the vector will contain a promoter, an
origin of replication element, a transcriptional termination
element, a ribosome binding site element, a polylinker region for
inserting the nucleic acid encoding the polypeptide to be
expressed, and a selectable marker element.
[0287] The promoter may be homologous (i.e., from the same
prokaryotic species and/or strain as the host cell), heterologous
(i.e., from a source other than the prokaryotic host cell species
or strain), or synthetic. As such, the source of the promoter may
be any unicellular prokaryotic or eukaryotic organism, any
vertebrate or invertebrate organism, or any plant, provided that
the promoter is functional in, and can be regulated by, the host
cell. The more preferred promoters of this invention are inducible
promoters, such as those of bacteriophage lambda origin, i.e.,
lambda promoters, the T5 promoter or the T7 promoter, bacterial
promoters such as lac, tac (a composite of the trp and lac
promoters), trp, and tna.
[0288] The promoter nucleic acid sequences useful in this invention
may be obtained by any of several methods well known in the art.
Typically, promoters useful herein will have been previously
identified by mapping and/or by restriction endonuclease digestion
and can thus be isolated from the proper tissue source using the
appropriate restriction endonucleases. In some cases, the promoter
may have been sequenced. For those promoters whose DNA sequence is
known, the promoter may be synthesized using the methods described
above for nucleic acid synthesis or cloning. Where all or only
portions of the promoter sequence are known, the promoter may be
obtained using PCR and/or by screening a genomic library with
suitable oligonucleotide and/or promoter sequence fragments from
the same or another species. Once isolated, the promoter may
optionally be sequenced and prepared synthetically.
[0289] Where the promoter sequence is not known, a fragment of DNA
containing the promoter may be isolated from a larger piece of DNA
that may contain, for example, a coding sequence or even another
gene or genes. Isolation may be accomplished by restriction
endonuclease digestion using one or more carefully selected enzymes
to isolate the proper DNA fragment. After digestion, the desired
fragment may be isolated by agarose gel purification, QIAGEN.TM.
column or other methods known to the skilled artisan. Selection of
suitable enzymes to accomplish this purpose will be readily
apparent to one of ordinary skill in the art.
[0290] An origin of replication is typically a component of
commercially available prokaryotic expression vectors. The origin
of replication aids in the amplification of the vector in a host
cell. Amplification of the vector to a desirable copy number (e.g.,
a number which results in maximum production of the therapeutic
polypeptide and effective maintenance of the plasmid in the cell
culture) can, in some cases, be important for optimal expression of
the therapeutic polypeptide. If the vector of choice does not
contain an origin of replication site, one may be chemically
synthesized based on a known sequence, and ligated into the
vector.
[0291] A transcription termination element is typically located 3'
to the end of the Polypeptide coding sequence and serves to
terminate transcription of the polypeptide. Usually, the
transcription termination element in prokaryotic cells is a G-C
rich fragment followed by a poly T sequence. While the element is
easily cloned from a library or even purchased commercially as part
of a vector, it can also be readily synthesized using methods for
nucleic acid synthesis such as those described above.
[0292] Selectable marker genes encode proteins necessary for the
survival and growth of a host cell grown in a selective culture
medium. Typical selection marker genes encode proteins that (a)
confer resistance to antibiotics or other toxins, e.g., ampicillin,
tetracycline, or kanamycin for prokaryotic host cells, (b)
complement auxotrophic deficiencies of the cell; (c) supply
critical nutrients not available from complex media; or (b) result
in fluorescence or other observable qualities. Preferred selectable
markers are the kanamycin resistance gene, the ampicillin
resistance gene, and the tetracycline resistance gene.
[0293] A ribosime binding site element may also be present. This
element, commonly called the Shine-Dalgarno sequence, facilitates
translation initiation of a mRNA. The element is typically located
3' to the promoter and 5' to the coding sequence of the polypeptide
to be synthesized. The Shine-Dalgarno sequence is varied but is
typically a polypurine (i.e., having a high A-G content). Many
Shine-Delgarno sequences have been identified, each of which can be
readily synthesized using methods set forth above.
[0294] All of the elements set forth above, as well as others
useful in this invention, are well known to the skilled artisan and
are described, for example, in Sambrook et al.,.sup.75 Berger et
al..sup.76 and other references..sup.77
[0295] For eukaryotic expression, any promoter known to be
effective in the cells in which the vector will be expressed can be
used to initiate expression of the therapeutic polypeptide.
Suitable promoters may be inducible or constitutive. Examples of
suitable eukaryotic promoters include the SV40 early promoter
region,.sup.78 the promoter contained in the 3N long terminal
repeat of Rous sarcoma virus,.sup.79 the HSV-1 (herpes simplex
virus-1) thymidine kinase promoter,.sup.80 the regulatory sequences
of the metallothionein gene,.sup.81 etc., as well as the following
animal transcriptional control regions, which exhibit tissue
specificity and have been utilized in transgenic animals: elastase
I gene control region (active in pancreatic acinar cells);.sup.82
insulin gene control region (active in pancreatic beta
cells),.sup.83 immunoglobulin gene control region (active in
lymphoid cells),.sup.84 mouse mammary tumor virus control region
(active in testicular, breast, lymphoid and mast cells),.sup.85
albumin gene control region (active in liver cells),.sup.86
alpha-fetoprotein gene control region (active in liver
cells),.sup.87 alpha 1-antitrypsin gene control region (active in
liver cells),.sup.88 beta-globin gene control region (active in
erythroid cells),.sup.89 myelin basic protein gene control region
(active in oligodendrocyte cells in the brain),.sup.90 myosin light
chain-2 gene control region (active in skeletal muscle
cells),.sup.91 and gonadotropin releasing hormone gene control
region (active in cells of the hypothalamus)..sup.92
[0296] The therapeutic polypeptides of the invention can be
prepared for secretion using recombinant DNA technology. This may
be accomplished by creating a nucleic acid construct wherein the
DNA encoding the therapeutic polypeptide is attached at its 5' end
to a naturally occurring or synthetic DNA sequence encoding a
signal peptide. For secretion, the signal peptide sequence selected
must be one that is recognized by, and therefore capable of being
processed by, the host cell into which this construct is to be
inserted and expressed. Thus, for example, a signal peptide
obtained from a naturally secreted bacterial polypeptide can be
attached to a polypeptide from a source such as human tissue
thereby creating a hybrid precursor polypeptide that can be
synthesized in, and secreted from, those bacterial (and other
prokaryotic) cell species that recognize and are able to process
the signal peptide. The hybrid construct can be introduced into the
host cell to provide the host cell with the capability of
manufacturing and secreting the therapeutic polypeptide.
[0297] In one aspect of the invention, a mammal is genetically
modified to produce the therapeutic polypeptide in its milk
Techniques for performing such genetic modifications are described
in U.S. Pat. No. 6,013,857, issued Jan. 11, 2000, for "Transgenic
Bovines and Milk from Transgenic Bovines." The genome of the
transgenic animal is modified to comprise a transgene comprising a
DNA sequence encoding a therapeutic polypeptide operably linked to
a mammary gland promoter. Expression of the DNA sequence results in
the production of the therapeutic polypeptide in the milk. The
therapeutic polypeptide may then be isolated from milk obtained
from the transgenic mammal (e.g., using a column comprising an
antibody which binds to the therapeutic polypeptide). The
transgenic mammal is preferably a bovine species.
[0298] 8.4.3 Preparation of MA (SEQ ID NO: 2) and pMA (SEQ ID NO:
3) and Other Native MA Peptides by Isolation from Native hCG
Sources
[0299] The MA peptides can be prepared from certain native sources
of hCG. Native preparations (i.e. derived from naturally occurring
sources and not recombinantly produced) of hCG and .beta.-hCG
preparations can be obtained from a variety of sources. Both hCG
and .beta.-hCG are commercially available (e.g., Sigma Chemical
Company) and hCG is commercially available in a form suitable for
therapeutic use in humans (e.g., from Fujisawa, Wyeth-Ayerst
Laboratories (APL.TM.), Organon, Inc. (PREGNYL.TM.) and Serono
Laboratories, Inc. (PROFASIT.TM.)). MA peptides are also present in
the urine of women most abundantly in their first trimester of
pregnancy ("human early pregnancy urine"). Other sources include,
but are not limited to, urine from women in the second and third
trimesters of pregnancy, urine from patients with proteinuria,
urine from patients having hCG secreting tumors or other cancer
patients, from pituitary glands, and from urine of post-menopausal
women
[0300] MA peptide can be isolated and/or partially purified from
native sources of hCG. For example, the inventors have isolated MA
peptide from human early (i.e. first trimester) pregnancy urine.
Other native sources of hCG include, but are not limited to, urine
from women in the second and third trimester of pregnancy, urine
from proteinuria patients (both pregnant women with preeclampsia
and patients with nephrotic syndromes), urine from patients with
hCG-secreting tumors, cultures of hCG-producing cells (e.g.,
trophoblasts.sup.93) and pituitary glands.
[0301] MA peptide can be isolated and/or partially purified using
conventional techniques, such as affinity chromatography. For
example, antibodies prepared against MA peptide can be used to
prepare an affinity chromatography column that can be used to
purify the polypeptides by well-known techniques..sup.94
Antibodies, either polyclonal or, preferably, monoclonal, with
specificity for MA peptide can be generated by the methods
described herein or by other methods known in the art..sup.95
[0302] Fractions of human early pregnancy urine or other source of
MA can be assayed for the presence of the MA peptide species using
a monoclonal antibody specific for the MA peptides. The assay can
be performed by known methods. For example, an immunoradiometric
assay (IRMA) can be used..sup.96 Briefly, the IRMA assay is
performed by adsorbing an antibody against the MA peptides onto the
surface of wells of a microtiter plate by incubation in a coating
buffer (0.2 M sodium bicarbonate, pH 9.5) overnight at 4.degree. C.
The residual non-specific binding sites are blocked by the addition
of a 1% bovine serum albumin solution (with 0.1% sodium azide) to
the wells for 3 hours at room temperature, and the wells of the
microtiter plate are then washed with deionized water. An aliquot
of the fraction in assay buffer (0.01 M sodium phosphate, 0.15 M
NaCl, 0.01 M EDTA, 0.1% sodium azide, 0.1% bovine .gamma.-globulin,
pH 7.4) is incubated in the wells for 24 hours at room temperature.
The sample is then removed and the wells washed with deionized
water. A solution of a second antibody specific for the MA peptide,
which antibody has been iodinated with 1.sup.125, (approximately
40,000 cpm/well) is incubated in the wells for 24 hours at room
temperature. The iodinated antibody solution is removed and the
wells washed five times with deionized water. The level of
radioactivity in each well is then determined in a scintillation
counter which can measure .gamma.-irradiation.
[0303] In an alternative embodiment, MA peptides can be obtained by
proteolysis of .beta.-hCG (SEQ ID NO: 1) followed by purification
using standard techniques such as chromatography (e.g., HPLC),
electrophoresis, etc. Moreover, MA peptides and fragments thereof
can be obtained by treating the .beta.-core of .beta.-hCG to remove
the residual sugar residues and treating .beta.-hCG to break 6-40
away from 55-89 and treating the deglycosylated beta core to break
the 6-40 and 55-92 apart.
[0304] 8.5 Therapeutic Methods
[0305] The therapeutic polypeptides of the invention have multiple
therapeutic and diagnostic uses, as more fully described in the
ensuing sections. It will be appreciated that the therapeutic
polypeptides of the invention may be administered to a specific
subject for any one or more of the therapeutic uses discussed
herein. The therapeutic methods of the invention include
administration of the therapeutic polypeptides of the invention, as
well as administration of a nucleic acid encoding a therapeutic
polypeptide and/or a functional equivalent thereof. The therapeutic
polypeptides of the invention may also be administered as
components of cells transformed to express the therapeutic
polypeptides of the invention. The therapeutic polypeptides may be
expressed with signal peptides, directing excretion of the
polypeptides and/or functional equivalents from the cells. The
signal peptides may be enzymatically cleaved upon excretion to
provide free therapeutic polypeptides. The therapeutic polypeptides
may also be administered by means which cause a subject's own cells
to express the polypeptide from the subject's native genome. In all
of the therapeutic methods of the invention, the therapeutic
polypeptide is administered in a therapeutically effective
amount.
[0306] The therapeutic methods of the invention may all include
administration of multiple therapeutic polypeptides [e.g.,
combinations of MA (SEQ ID NO: 2); pMA (SEQ ID NO: 3); MA.sub.S1
(SEQ ID NO: 4); MA.sub.S2 (SEQ ID NO: 5); MA.sub.S3 (SEQ ID NO: 6);
MA.sub.S5 (SEQ ID NO: 7); MA.sub.S9 (SEQ ID NO: 8); MA.sub.S10 (SEQ
ID NO: 9); and MA.sub.S11 (SEQ ID NO: 10)] in a regimen that may or
may not include other therapeutic agents.
[0307] Moreover, the therapeutic methods of the invention may also
include administration of other .beta.-hCG (SEQ ID NO: 1) fragments
either alone, or in a therapeutic regimen that also includes
administration of one or more therapeutic polypeptides of the
invention--though in a preferred embodiment, use of such fragments
without also using a therapeutic polypeptide of the invention is
excluded from the therapeutic methods of the invention. Preferred
fragments of .beta.-hCG, which are not therapeutic polypeptides of
the invention, are those with demonstrated anti-HIV, anti-cancer
and/or pro-hematopoietic efficacy. Such polypeptides and peptide
fragments are specifically described in U.S. Pat. Nos. 5,968,513
and 5,997,871; U.S. patent application Ser. Nos. 09/220,415 and
08/709,948; and International Patent Publications PCT/US97/11209
and PCT/US97/11202; the entire disclosure of each of these
patents/applications is incorporated herein by reference. Examples
include .beta.-hCG fragments consisting of amino acid sequences
41-54, 45-54, 47-53, 45-57, and 109-119 of .beta.-hCG (SEQ ID NO:
1), as well as 45-57::109-119, 110-119::45-57, and 47-57::108-119.
Preferred .beta.-hCG therapeutics include the Satellins, e.g.:
[0308] Satellin A1: .beta.-hCG 45-57
[Leu-Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys (SEQ ID NO:
19); [0309] branched Satellin A1: .beta.-hCG 45-57
[Leu-Gln-Dab(Pro)-Val-Leu-Pro-Dab(Pro)-Leu-Pro-Gln-Val-Val-Cys
((SEQ ID NO: 52) see SEQ ID NO: 19 for primary sequence)], where
"Dab" represents diaminobutyric acid, and Dab(Pro) indicates a
proline peptide-bonded to the amino side chain of Dab); [0310]
circularized Satellin A2: .beta.-hCG 45-57 with a cysteine residue
added to the N-terminus, circularized via a disulfide bond between
the systeine residues
[Cys-Leu-Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys (SEQ ID
NO: 20)]; and [0311] Satellin B: .beta.-hCG 109-119
[Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser (SEQ ID NO: 23).
[0312] 8.5.1 Treatment of Retrovirus Infections
[0313] The invention provides for the treatment of diseases and
disorders associated with retrovirus infections in a subject. The
method generally comprises administering to a subject infected with
a retrovirus a therapeutically effective amount of one or more
therapeutic polypeptides of the invention to treat or prevent the
infection.
[0314] In a central embodiment of the invention, the therapeutic
polypeptides are used to treat or prevent HIV infection. The
anti-HIV activity of MA peptides has been demonstrated in both in
vivo and in vitro studies. In vitro cell culture studies conducted
by the inventors show that at least one anti-HIV property exhibited
by the MA peptides is the inhibition of HIV replication.
[0315] In HIV transgenic mice and rats, MA peptides suppress HIV
expression and prevent death. When MA peptides are administered to
nursing mice, the homozygous transgenic pups develop normally as
compared to placebo-treated pups, which fail to develop and then
die within two weeks of birth.
[0316] In monkeys, crude fractions of MA peptides reduce SIV viral
load and prevent AIDS-like disease, increas CD4.sup.+ T cell
counts, promote weight gain and improve bone marrow function.
[0317] The invention provides for treatment or prevention of
diseases and disorders associated with retroviral infection by a
method that comprises administering to an infected subject one or
more therapeutic polypeptides of the invention. For example, MA
peptides exhibit properties that are useful in reversing the bone
marrow dysplasia seen in AIDS patients.
[0318] The therapeutic polypeptides can also be used to prevent the
progression of HIV infection to AIDS or to treat a subject with
AIDS. In one aspect of the invention, the therapeutic polypeptides
of the invention are administered to an HIV-infected subject having
a T cell count that is below 500.
[0319] The therapeutic polypeptides of the invention also exhibit
properties that are useful in the treatment of individuals acutely
exposed to HIV (e.g., rape or inadvertent needle sticks). In such
cases, one or more therapeutic polypeptides of the invention can be
administered to prevent the establishment of an HIV infection
following an initial inoculation event. Administration of a
multi-drug regimen of anti-HIV therapeutics following such an
inoculation event is known to be useful in reducing the probability
of establishing an active HIV infection. The therapeutic
polypeptides can be used as a component of such a multi-drug "day
after" treatment, to reduce the probability that an HIV infection
will become established.
[0320] The therapeutic polypeptides can be administered in a
monotherapeutic treatment regimen, e.g., a therapeutic polypeptide
can be administered alone in the treatment of HIV without also
administering other anti-HIV therapeutics. Alternatively, one or
more therapeutic polypeptides can be administered as component(s)
of a multi-drug regimen, which includes one or more other anti-HIV
compounds. Examples of other anti-HIV compounds that can be used in
such multi-drug regimens include: protease inhibitors (e.g.,
CRIXIVAN.TM. (indinavir); FORTOVASE.TM. and INVIRASE.TM.
(saquinavir); NORVIR.TM. (ritonavir); and VIRACEPT.TM.
(nelfinavir)); non-nucleoside reverse transcriptase inhibitors
(e.g., RESCRIPTOR.TM. (delavirdine); SUSTIVA.TM. (efavirenz); and
VIRAMUNE.TM. (nevirapine)); and nucleoside reverse transcriptase
inhibitors (e.g., VIDEX.TM. (didanosine, also known as DDI);
EPIVIR.TM. (lamivudine, also known as 3TC); ZERIT.TM. (stavudine,
also known as d4T); HIVID.TM. (Zalcitabine, also known as DDC);
RETROVIR.TM. (zidovudine, also known as AZT or ZDV); and
COMBIVIR.TM. (lamivudine and zidovudine)).
[0321] A multi-drug regimen may also include multiple therapeutic
polypeptides of the present invention [e.g., combinations of MA
(SEQ ID NO: 2); pMA (SEQ ID NO: 3); MA.sub.S1 (SEQ ID NO: 4);
MA.sub.S2 (SEQ ID NO: 5); MA.sub.S3 (SEQ ID NO: 6); MA.sub.S5 (SEQ
ID NO: 7); MA.sub.S9 (SEQ ID NO: 8); MA.sub.S10 (SEQ ID NO: 9);
MA.sub.S11 (SEQ ID NO: 10); .beta.-hCG 55-88 (SEQ ID NO: 11);
.beta.-hCG 55-90 (SEQ ID NO: 12); .beta.-hCG 55-91 (SEQ ID NO: 13);
.beta.-hCG 55-74 (SEQ ID NO: 14); .beta.-hCG 6-37 (SEQ ID NO: 15);
.beta.-hCG 6-38 (SEQ ID NO: 16); .beta.-hCG 6-39 (SEQ ID NO: 17);
and .beta.-hCG 6-40 (SEQ ID NO: 18) and/or their functional
equivalents] in a regimen that may or may not include other
anti-HIV compounds.
[0322] A multi-drug regimen may also include therapeutic agents for
treating various side effects of HIV-infection or AIDS, such as
therapy for treating opportunistic infections and/or malignancies.
For example, a multi-drug therapeutic regimen may include
antibiotics for bacterial infections, chemotherapy and/or radiation
therapy for malignancies, and/or ativiral treatments for
opportunistic viral infections (e.g., treatments for
Cytomegalovirus, Herpes zoster, and/or Herpes simplex infection).
[0323] Moreover, the therapeutic polypeptides of the invention may
be administered with other .beta.-hCG (SEQ ID NO: 1) fragments,
and/or polypeptides comprising such fragments, with demonstrated
anti-HIV, anti-cancer and/or pro-hematopoietic efficacy.
[0324] The therapeutic polypeptides are also useful in combination
with one or more chemokines, functional equivalents of chemokines,
and/or genes encoding chemokines or their functional equivalents.
Examples of suitable cytokines include macrophage-derived
chemokine, monocyte chemotactic protein 1, monocyte chemotactic
protein 2, monocyte chemotactic protein 3, monocyte chemotactic
protein 4, activated macrophage specific chemokine 1, macrophage
inflammatory protein 1 alpha, macrophage inflammatory protein 1
beta, macrophage inflammatory protein 1 gamma, macrophage
inflammatory protein 1 delta, macrophage inflammatory protein
2.alpha., macrophage inflammatory protein 3.alpha., macrophage
inflammatory protein 3.beta., regulated upon activation, normal T
cell expressed and secreted (and its variants), I-309, EBI1-ligand
chemokine, pulmonary and activation regulated chemokine, liver and
activation-regulated chemokine, thymus and activation regulated
chemokine, eotaxin (and variants), human CC chemokine 1, human CC
chemokine 2, human CC chemokine 3, IL-10-inducible chemokine,
Liver-expressed chemokine, 6Ckine, exodus 1, exodus 2, exodus 3,
thymus-expressed chemokine, secondary lymphoid tissue chemokine,
lymphocyte and monocyte chemoattractant, monotactin,
chemokine-related molecule, myeloid progenitor inhibitory factor-1,
myeloid progenitor inhibitory factor-2, stromal cell-derived factor
1.alpha., stromal cell-derived factor 1.beta., B-cell-attracting
chemokine 1, HuMIG, H174, Interferon-stimulated T-cell
.alpha.-chemoattractant, interleukins, IP-10, platelet factor 4,
growth-regulated gene-.alpha., growth-regulated gene-.beta.,
growth-regulated gene-.gamma., neutrophil-activating protein 2,
ENA-78, granulocyte chemotactic protein 2, lymphotactin,
fractalkine/neurotactin, viral chemokines and functional
equivalents of the foregoing chemokines. Other specific examples of
suitable components Steel factor (SLF) and adult PB plasma.
Preferred factors are those which cause proliferation or, less
preferably, differentiation of cells that are CFU-GEMM or earlier
cells, e.g., IL-3 and GM-CSF. Preferred chemokines include C--C
type chemokines, such as RANTES, MIP-1.alpha., MIP-1.beta. and
MDC.
[0325] A preferred embodiment of the invention relates to methods
of using one or more therapeutic polypeptides of the invention for
treatment or prevention of HIV infection, preferably HIV-1
infection, in a human subject. In a specific embodiment, one or
more therapeutic polypeptides of the invention is administered to a
subject with a set of one or more conditions which does not include
a cancer which secretes hCG or hCG fragments to treat or prevent
HIV infection in the subject. In another specific embodiment, one
or more therapeutic polypeptides of the invention is used for the
treatment or prevention of HIV infection in a human subject with a
set of one or more conditions that does not include Kaposi's
sarcoma (KS). In the treatment of HIV infection, the therapeutic
polypeptides of the invention can be used to prevent or slow
progression of HIV infection to AIDS in a human subject, or to
treat a human subject with AIDS.
[0326] In another aspect the therapeutic polypeptide is
administered to an infant born to an HIV infected mother in order
to reduce the risk of HIV infection in the newborn and to increase
weight gain and blood cell development. Moreover, the therapeutic
polypeptides may also (or alternatively) be administered to the
HIV-infected mother, before or during birth of the infant, to
reduce the risk of HIV infection in the newborn. In a preferred
embodiment, one or more therapeutic polypeptides are administered
to an HIV infected mother to prevent wasting, promote hematopoiesis
and to reduce the risk of infecting the unborn. In a specific
embodiment only one or more therapeutic polypeptides is
administered, i.e., therapeutic polypeptide(s) in the absence of
other anti-HIV, anti-wasting and/or pro-hematopoietic therapeutic
agents. In an additional embodiment the therapeutic polypeptides
are administered as part of a multi-drug therapy regimen.
[0327] The therapeutic polypeptides of the invention are also
useful in the treatment of retroviral infection in non-human
animals. For example, one or more therapeutic polypeptides of the
invention is suitably administered, alone or in combination with
one or more other pharmaceutical agents in a therapeutically
effective amount, to (1) a horse species (e.g., Equus caballus) to
treat or prevent an infection and/or symptoms associated with
equine infectious anemia virus; (2) a bovine species (e.g., Bos
Laurus or Bos indicus) to treat or prevent the infection and/or
symptoms associated with bovine immunodeficiency virus; (3) an Ovis
species to treat or prevent the infection and/or symptoms
associated with visna virus; or (4) a feline species to prevent the
infection and/or symptoms associated with feline immunodeficiency
virus and/or feline leukemia virus (FELV).
[0328] 8.5.2 Treatment of Non-HIV Viral Infection
[0329] The therapeutic polypeptides are also useful in the
treatment or prevention of viral diseases other than HIV. In this
embodiment, the therapeutic polypeptides are suitably administered
to a subject to slow or stop the progression of viral diseases,
such as hepatitis and herpes infection. In one aspect of the
invention, the therapeutic polypeptides of the invention are
administered to a subject infected with both HIV and hepatitis C to
directly combat the virus and/or to combat symptoms of the
infection. The therapeutic polypeptide can be used in a
monotherapeutic treatment regimen or in combination with other
anti-viral compounds in a multi-drug regimen.
[0330] To assess the utility of treating a subject infected with a
virus, one skilled in the art can evaluate the anti-viral activity
of any of the therapeutic polypeptides in any in vitro system as
described herein or as otherwise known in the art. After
demonstration of in vitro activity, or if in vitro testing systems
are not available, the therapeutic polypeptides may also evaluated
in animal models, e.g., as described herein, or in any of a variety
of known in vitro assays.
[0331] To effect treatment or prevention, one or more therapeutic
polypeptides of the invention is administered to a subject, either
alone or in combination with another drug, or drugs for the
prevention and/or treatment of an infection, symptoms of an
infection, and/or conditions resulting from an infection by a
virus, such as hepatitis C virus, herpesvirus 1, herpesvirus 4, is
encephalomyocarditis virus (EMCV), rubella virus and influenza
virus. In a related embodiment, one or more therapeutic
polypeptides of the invention is administered to a subject, either
alone or in combination with one or more other therapeutic agents
for the prevention and/or treatment of viral meningitis.
[0332] 8.5.3 Treatment of Bacterial Infections
[0333] The therapeutic polypeptides can be administered alone or in
combination with another drug or drugs. The effectiveness of
treating a specific bacterial infection in a subject can be
assessed by in vitro and/or in vivo methods available in the art.
The bacterial infection may be an intracellular bacterial infection
and/or an extracellular bacterial infection.
[0334] In a preferred embodiment, the therapeutic polypeptides are
administered either alone or in combination with another drug or
drugs to a subject to treat or prevent infection with Treponema
pallidum (i.e., a subject with syphilis).
[0335] Helicobacter pylori, the causative agent of gastric ulcers,
may also be treated or prevented by administration of a therapeutic
polypeptide of the invention, either alone or in combination with
one or more other therapeutic agents. In a preferred embodiment,
the therapeutic polypeptide is delivered in combination with
Flagil, Amoxycylin and/or a drug to reduce production of acid such
as ranitidine (e.g., ZANTAC.TM.), famotidine (e.g., PEPCID.TM. or
omeprazole (e.g., PRILOSEC.TM.)
[0336] The therapeutic polypeptides of the invention are also
suitably employed in the treatment or prevention infection with
Escherichia coli and/or infection with Mycobacterium
tuberculosis.
[0337] 8.5.4 Treatment of Wasting Syndromes
[0338] The inventors have discovered that MA peptides mediate a
significant reversal in wasting syndromes. For example, the
empirical work undertaken by the inventors has demonstrated
reversal in wasting associated with HIV infection, as well as
wasting associated with various malignancies.
[0339] The invention provides a method for treating wasting
syndromes comprising administering a therapeutically effective
amount of one or more therapeutic polypeptides to a subject with a
wasting syndrome.
[0340] The methods are applicable to the treatment of any disease
or disorder characterized by an undesired loss of body cell mass.
For example, the therapeutic polypeptides are useful in the
treatment of: wasting associated with viral infection, such as
HIV-associated wasting; wasting associated with bacterial infection
or other types of infection or sepsis; cachexia associated with
cancer, chemotherapy, and/or radiation therapy; wasting associated
with chronic cardiovascular disease; wasting caused by exposure to
toxic substances; and wasting associated with diarrhea and other
gastrointestinal disorders.
[0341] In a preferred embodiment, one or more therapeutic
polypeptides of the invention is administered to treat or prevent a
wasting syndrome associated with HIV infection. In a related
embodiment, one or more therapeutic polypeptides of the invention
is administered to treat or prevent a wasting syndrome associated
with cancer.
[0342] The therapeutic polypeptides of the invention may also be
administered to treat and/or prevent chronic diarrhea and/or its
associated effects.
[0343] 8.5.5 Treatment of Hematopoietic Deficiencies
[0344] The invention also relates to therapeutic methods for
treating or preventing hematopoietic deficiencies. MA promotes both
in vivo and in vitro hematopoiesis of multiple lineages, e.g.,
T-cells, erythrocytes, granulocytes, platelets, and macrophages, of
mice, rats, monkeys, and human bone marrow. MA can rescue lethally
irradiated animals and restore their blood counts to normal, even
when administered before or after exposure to radiation (e.g., 24
hours after the radiation exposure). Equally remarkable, MA
protects against side effects of cancer chemotherapy (TAXOL.TM.
(paclitaxel)) at lethal doses, and facilitates recovery of acutely
bled rats and monkeys. None of these hematopoietic effects have
been observed with pure native glycosylated .beta.-core,
.beta.-hCG, .alpha.-hCG, hCG scrambled MA (see SCRAM I and SCRAM II
in Table 1), nor with a variety of synthetic peptides corresponding
to many other regions of .beta.-hCG.
[0345] The methods of the invention for treating hematopoietic
deficiency can be employed in the treatment of any disease or
disorder in which increased amounts of hematopoietic cells are
desirable. Examples include disorders associated with reduced
numbers of one or more hematopoietic cell types. The treatment
methods of the invention comprise administering to a subject with a
hematopoietic deficiency a therapeutically effective amount of one
or more therapeutic polypeptides of the invention.
[0346] The invention also provides methods for expansion of
hematopoietic cells in vitro by contact with a therapeutic
polypeptide of the invention. In a related aspect, the invention
provides for treatment or prevention of hematopoietic cell
deficiencies by administering hematopoietic cells, the numbers of
which have been increased in vitro by contact with a therapeutic
polypeptide of the invention.
[0347] The therapeutic polypeptides are useful in treating
conditions characterized by failure or dysfunction of normal blood
cell production and maturation. Examples of such conditions include
aplastic anemias, cytopenias and hypoproliferative stem cell
disorders. These disorders entail failure of stem cells in bone
marrow to provide normal numbers of functional blood cells.
[0348] Aplastic anemias result from the failure of stem cells to
give rise to the intermediate and mature forms of red cells, white
cells, and platelets. While red cell production is usually most
seriously affected, a production of other mature blood cell
elements, such as white cells and/or platelets is also relatively
common. The majority of these anemias are acquired during adult
life and are not related to any apparent genetic predisposition.
About half of these acquired anemias arise in the absence of any
obvious causative factor such as exposure to poisons, drugs or
disease processes that impair stem cell function; these are termed
idiopathic aplastic anemias. The remaining cases are associated
with exposure to an extremely diverse array of radiation,
chemotherapy, chemicals and drugs and also occur as a consequence
of viral infections, such as HIV infection, and after
pregnancy.
[0349] Other specific types of aplastic anemias, which may be
treated by administration of the therapeutic polypeptides, include
agranulocytosis and thrombocytopenia, in which the major deficiency
lies in particular white cells and in platelet production,
respectively. These non-red blood cell deficiencies are also often
associated with HIV infection. Idiopathic Thrombocytopenic Purpura
(ITP), a severe platelet deficiency is also associated with HIV
infection. Additionally, agranulocytosis may be associated with
autoimmune syndromes such as systemic lupus erythematosus (SLE) or
with other infections, such as neonatal rubella.
[0350] In addition, immune deficiencies that are the primary or
secondary result of infection by pathogenic microorganisms can be
treated by administration of one or more of the therapeutic
polypeptides. For example, immune deficiencies caused by microbes
that are intracellular pathogens of hematopoietic cells can be
treated by providing new hematopoietic cells. In one aspect of the
invention, these new hematopoietic cells are generated by
contacting hematopoietic stem and/or progenitor cells in vitro with
the therapeutic polypeptides to cause proliferation of the cells.
Examples of microbes which cause such immune deficiencies include
gram-negative bacilli such as Brucella or Listeria; Mycobacterium
tuberculosis, Mycobacterium leprae; parasites such as Plasmodium
and Leishmania; and fungi (such as those that cause pneumonia and
other lethal infections secondary to
immunodeficiencies)..sup.97
[0351] The therapeutic polypeptides can be administered to a
subject to treat a cytopenia associated with HIV infection. The
hematopoietic deficiencies associated with HIV infection include
reduction in CD4.sup.+ T cells and other lymphocytes, red blood
cells, platelets, specifically ITP, and neutrophils.
[0352] In one aspect of the invention, HIV-infected subjects are
treated by contacting hematopoietic stem and/or progenitor cells in
vitro with one or more therapeutic polypeptides; the resulting
hematopoietic cells can then be directly infused into the subject
in need of treatment. The cells can be obtained from the subject or
from a suitable donor. Hemeotopoetic deficiency can also be treated
by directly administering one or more therapeutic polypeptides or
functional equivalents to the subject in need of treatment.
[0353] In one aspect of the invention, hematopoietic deficiency is
treated by administering autologous hematopoietic cells (obtained
from the subject or its identical twin) to the subject after in
vitro expansion. This embodiment may also be employed as a step in
a gene therapy protocol. For example, nucleic acid(s) of interest
(e.g., comprising a gene(s) which provides a function desired in a
subject) can be introduced into hematopoietic cells, before or
after expansion of the cells by contact with one or more
therapeutic polypeptides. Cell subpopulations can be isolated for
use, before or after expansion in vitro. For example, blood cells
can be isolated and expanded, and optionally also differentiated,
in vitro, followed by introduction of all or a therapeutically
effective portion of the cells (e.g, purified platelets, red blood
cells, lymphocytes, etc.) into a subject.
[0354] Hematopoeitic deficiencies that can be treated by the
therapeutic polypeptides can be generally divided into five broad
categories. These categories should not be understood to be
all-inclusive, as conditions not within these categories may be
treatable using the therapeutic polypeptides of the invention.
First are diseases resulting from a failure or dysfunction of
normal blood cell production and maturation (i.e., aplastic anemia,
cytopenias and hypoproliferative stem cell disorders). The second
group includes neoplastic, malignant diseases in the hematopoietic
organs (e.g., leukemia and lymphomas). The third group of disorders
comprises disorders found in subjects with a broad spectrum of
malignant solid tumors of non-hematopoietic origin. Induction of
hematopoietic cell proliferation or administration of replacement
hematopoietic cells in these subjects serves as a bone marrow
rescue procedure, which is provided to a subject following
otherwise lethal chemotherapy or irradiation of the malignant
tumor. The fourth group of diseases consists of autoimmune
conditions; in subjects affected with these conditions the
hematopoietic cells serve as a source of replacement of an abnormal
immune system. The fifth group of diseases comprises a group of
genetic disorders which can be corrected by infusion of
hematopoietic stem cells, preferably syngeneic, which prior to
transplantation have undergone gene therapy.
[0355] In one aspect of the invention, the disease or disorder
results from a failure or dysfunction of normal blood cell
production and maturation. For example, the therapeutic
polypeptides of the invention are useful in the treatment of
hyperproliferative stem cell disorders, aplastic anemia,
pancytopenia, agranulocytosis, thrombocytopenia, red cell aplasia,
Blackfan-Diamond syndrome due to drugs, radiation, or infection,
and idiopathic anemia.
[0356] The therapeutic polypeptides of the invention are also
useful in the treatment of hematopoietic malignancies, such as
acute lymphoblastic (lymphocytic) leukemia, chronic lymphocytic
leukemia, acute myelogenous leukemia, chronic myelogenous leukemia,
acute malignant myelosclerosis, multiple myeloma, polycythemia
vera, agnogenic myelometaplasia, Waldenstrom's macroglobulinemia,
Hodgkin's lymphoma, and non-Hodgkin's lymphoma.
[0357] Furthermore, the therapeutic polypeptides of the invention
are useful in the treatment of immunosuppression in subjects with
malignant, solid tumors, such as malignant melanoma, carcinoma of
the stomach, ovarian carcinoma, breast carcinoma, small cell lung
carcinoma, retinoblastoma, testicular carcinoma, glioblastoma,
rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma and lymphoma.
[0358] The therapeutic polypeptides of the invention are also
useful in the treatment of autoimmune diseases, such as rheumatoid
arthritis, diabetes type I, chronic hepatitis, multiple sclerosis
and systemic lupus erythematosus.
[0359] Moreover, the therapeutic polypeptides of the invention are
usefully employed in the treatment of anemias resulting from
various genetic (congenital) disorders, such as congenital anemias,
familial aplastic anemia, Fanconi's syndrome, Bloom's syndrome,
pure red cell aplasia (PRCA), dyskeratosis congenita,
Blackfan-Diamond syndrome, congenital dyserythropoietic syndromes
I-IV, Chwachmann-Diamond syndrome, dihydrofolate reductase
deficiencies, formamino transferase deficiency, Lesch-Nyhan
syndrome, congenital spherocytosis, congenital elliptocytosis,
congenital stomatocytosis, congenital Rh null disease, paroxysmal
nocturnal hemoglobinuria, G6PD (glucose-6-phosphate dehydrogenase)
deficiency, pyruvate kinase deficiency, congenital erythropoietin
sensitivity deficiency, sickle cell disease and trait,
hemoglobinemias, congenital disorders of immunity, severe combined
immunodeficiency disease (SCID), bare lymphocyte syndrome,
ionophore-responsive combined immunodeficiency, combined
immunodeficiency with a capping abnormality, nucleoside
phosphorylase deficiency, granulocyte actin deficiency, infantile
agranulocytosis, Gaucher's disease, adenosine deaminase deficiency,
Kostmann's syndrome, reticular dysgenesis and congenital leukocyte
dysfunction syndromes.
[0360] In a preferred aspect, one or more therapeutic polypeptides
of the invention is used to treat a disease resulting from a
failure or dysfunction of normal blood cell production and
maturation, such as an aplastic anemia, a cytopenia or a
hypoproliferative stem cell disorder.
[0361] The therapeutic polypeptides also promote the in vitro
growth of all hematopoietic progenitors in human cord blood and
bone marrow, as well as bone marrow of other animals, such as
monkeys, rats, digs, cats, pigs, sheep, cattle and horses.
[0362] The therapeutic polypeptides are also useful as
radioprotective and/or chemoprotective agents. In animal studies
(mouse and rat), in which animals were treated with a lethal dose
of radiation or chemotherapy, administration of a single dose of a
therapeutic polypeptide either 24 hours before or 24 hours after
the lethal dose significantly increased survival time. Animals
treated with a single dose of therapeutic polypeptide survived for
15-18 days, compared to survival times of 3-4 days for untreated
animals. More dramatically, when radiation or chemotherapy-treated
animals received MA peptides every other day for two weeks, the
animals survived and their blood cell counts returned to normal
within 28-30 days. In contrast, lethally exposed animals who did
not receive MA peptides died in 3-4 days.
[0363] The present invention therefore provides methods of treating
subjects before, during or after exposure to radiation to protect
blood cells and other cells and/or increase blood cell counts, the
methods comprising administering one or more therapeutic
polypeptides of the invention to the subject before and/or during
and/or after exposure to the radiation. The therapeutic
polypeptides are preferably administered in an amount sufficient to
decrease the recovery time of subjects administered chemotherapy or
radiation. In cases of accidental exposure to chemical agents or
radiation, the therapeutic polypeptides are preferably administered
in an amount sufficient to decrease the recovery time or, in cases
of lethal exposure, to lengthen survival time and/or to improve the
likelihood of survival of the subject so exposed. Administration of
the therapeutic polypeptides of the invention permits increased
dosing of radiation and/or chemotherapy, thereby enabling more
aggressive therapy with less harm to the subject.
[0364] The invention also provides methods for treating subjects
who have sustained or may be expected to sustain blood loss. MA
(SEQ ID NO: 2) promotes blood cell formation in animal models
(monkeys and rats) where blood loss was induced. In an animal
(monkey and rat) bled by 1/3 to 1/2 its blood volume, treatment
with MA peptides increases survival rates, while also reducing the
time necessary to restore normal blood counts. This surprising
discovery is consistent with the conclusion that the therapeutic
polypeptides of the invention are useful in trauma settings, where
massive blood loss has occurred. The invention thus provides a
method for increasing survival rates, reducing complications and/or
reducing recovery times comprising administering one or more
therapeutic polypeptides of the invention to a subject who has
sustained blood loss, such as a trauma victim.
[0365] The therapeutic polypeptides of the invention have utility
in any medical condition or circumstance in which a subject has
sustained a medically important amount (i.e., an amount which is
detrimental to the health of the subject) of blood loss. In such
cases, the therapeutic polypeptides of the invention are
administered to assist in the restoration of normal blood counts.
For example, the therapeutic polypeptides of the invention may be
administered in surgical settings to reduce dependency on
heterologous blood transfusions. The therapeutic polypeptides of
the invention may also be administered prior to surgery to induce
hematopoiesis and thereby increase blood cell counts, thus reducing
or eliminating the need for blood transfusions during or after
surgery.
[0366] The therapeutic polypeptides of the invention may also be
administered to a subject before and/or after blood loss (e.g.,
anticipated blood loss, such as donation of blood or planned
surgery) to reduce the severity of or eliminate the adverse effects
of blood loss. The therapeutic polypeptides may be administered to
induce production of excess blood cells, so that sufficient amounts
of blood may be removed and stored for transfusions that may become
necessary during or after anticipated blood loss.
[0367] In a related aspect of the invention one or more of the
therapeutic polypeptides is delivered to a donor of a blood cell
component or bone marrow to decrease the time necessary for cell
counts to return to normal level following such donation to
decrease the time between successive donations of cells.
[0368] The therapeutic polypeptides can also be administered to
eliminate and/or reduce the need for or increase the efficacy of a
blood or bone marrow transfusion in trauma or non-trauma settings.
The therapeutic polypeptides can be administered in trauma settings
where blood loss threatens a subject's health. Administration of
the therapeutic polypeptides can be used to reduce or eliminate the
need for blood transfusions. For example, administering one or more
therapeutic polypeptides during or after a blood transfusion can
reduce the time required to restore normal blood cell counts and
function. Such administration can limit the need for additional
transfusions, and/or reduce complications associated with blood
loss as well as those associated with blood transfusion.
Administration one or more of the therapeutic polypeptides of the
invention can therefore increase survival rates and reduce recovery
time.
[0369] In a specific embodiment the therapeutic peptides are made
available to military forces in combat settings. The therapeutic
peptides are then administered at the first threat of biological
and/or chemical exposure to reduce or prevent damage to the bone
marrow and circulating blood cells. The therapeutic peptides are
preferably administered as soon as practicable after an injury to
promote hematopoiesis and to reduce the risk of infections.
[0370] The therapeutic polypeptides are suitably delivered with one
or more other pro-hematopoietic agents to promote hematopoiesis, in
vitro or in vivo. As an example MA can be administered with
erythropoietin to a subject or applied ex-vivo with erythropoietin
to cells to increase the numbers of red cells. In a specific
embodiment MA is provided as a component of a pharmaceutical
formulation which also comprises the other agent.
[0371] Examples of pro-hematopoietic agents that can be formulated
with or administered with MA to promote hematopoiesis can be
identified by one skilled in the art using assays as described
herein and/or by any other assay known in the art to quantify or
otherwise characterize hematopoietic production.
[0372] In one aspect one or more therapeutic polypeptides is
administered with one or more cytokines, preferably a growth or
differentiation factor.
[0373] In a preferred embodiment MA is formulated with
erythropoietin and administered to a subject to increase numbers of
red cells in a subject presenting with renal failure.
[0374] In another preferred embodiment MA is formulated with colony
stimulating factor and administered to a subject to increase the
numbers of white cells.
[0375] One or more of the therapeutic polypeptides may also be
added to stored blood to extend the viability of the collected
blood product and to increase the time the product can be used for
transfusion into a subject. In one aspect, the therapeutic
polypeptide is added to a blood collection vessel prior to
collection of the blood.
[0376] One or more of the therapeutic polypeptides of the invention
may also be administered to a subject who has been, or may have
been, exposed to an infectious agent. Such administration will
promote hematopoiesis and improve the subject's immune response to
the infectious agent. In a preferred embodiment, the therapeutic
polypeptides are administered to a subject prior to, during and/or
after possible exposure to an infectious agent as a result of
traveling to an area known to be endemic for infectious agents.
[0377] In a related aspect of the invention, the therapeutic
polypeptides may be administered to a subject to enhance normal
physiological functioning. As an example, the therapeutic
polypeptides may be administered to a high altitude climber to
increase red blood cells. The therapeutic polypeptides may be
administered to a subject to enhance athletic performance.
[0378] 8.5.6 Expansion of Stem and Progenitor Cells
[0379] The invention provides a method for the expansion of stem
and/or progenitor cells. Stem cells and progenitor cells can be
expanded ex-vivo and/or in vivo by contacting the cells with a
therapeutic polypeptide of the invention. One or more therapeutic
polypeptides of the invention may be administered as a component of
a therapeutic regimen with other pharmaceutical agents to induce
proliferation and/or differentiation of stem and/or progenitor
cells into desired cell types and in sufficient numbers prior to
implantation in a subject.
[0380] Stem and/or progenitor cells may, for example, be isolated
from a subject's own organ system or from a donor and expanded
ex-vivo prior to implantation in a subject. In a preferred
embodiment, stem or progenitor cells are isolated from the bone
marrow of a subject or a donor. As an alternative, the therapeutic
polypeptide can be directly administered to a subject to promote
expansion of stem and progenitor cells in vivo.
[0381] Stem cells are also suitably isolated from cord blood at
birth and then expanded by contacting with a therapeutic
polypeptide and stored for future use by the donor or by another
subject. Isolated stem cells may also be frozen prior to expansion
and thawed for expansion as needed.
[0382] In one embodiment, one or more therapeutic polypeptides is
applied to stem cells and/or progenitor cells with one or more
cytokine(s) to increase cell numbers and to stimulate
differentiation and/or migration. Examples of cytokines that can be
usefully combined with a therapeutic polypeptide of the invention
include EGF, FGF-2, IL-2, GM-CSF, G-CSF, EPO, IGF and TGF.
Preferred cytokines are those which induce the proliferation and/or
differentiation of stem and/or progenitor cells. Cytokines and
other pharmaceutical agents useful in the expansion,
differentiation and/or migration of stem and progenitor cells can
be identified by combining them with the therapeutic polypeptide
and screening them using any in vitro and/or in vivo assay known in
the art.
[0383] The stem cells may also be embryonic stem cells. In a
specific embodiment, the therapeutic polypeptide is utilized to
increase numbers of stem and progenitor cells giving rise to
cartilage cells, bone cells, fat cells, muscle cells, neural cells
(e.g., astrocytes), and/or cells of the hematopoietic system.
[0384] The therapeutic polypeptides may also be applied to stem
and/or progenitor cells giving rise to neural cells to provide
neural cells for treating a subject presenting with Parkinson's,
Alzheimer's disease, multiple sclerosis, stroke and/or injuries to
the neural system.
[0385] The therapeutic polypeptides are usefully administered to a
subject to increase numbers of stem and/or progenitor cells giving
rise to pancreatic islet cells (in vivo or in vitro) for the
treatment of diabetes.
[0386] The therapeutic polypeptides are also suitably administered
to a subject who has sustained an injury to a bone, such as a
broken bone, to promote healing.
[0387] One or more therapeutic polypeptides of the invention may be
brought into contact with stem and/or progenitor cells, either in
vivo or in vitro, to increase number of stem and/or progenitor
cells giving rise to heart, smooth and/or skeletal muscle.
[0388] In a preferred embodiment, the therapeutic polypeptide is
administered to a subject to maintain or increase the numbers of
stem and/or progenitor cells to slow the aging process and its
associated complications.
[0389] 8.5.7 Treatment of Cancer
[0390] The invention further relates to methods for treating and/or
preventing cancer. The methods comprise administration of one or
more therapeutic polypeptides of the invention in an amount
sufficient to treat and/or prevent cancer. Like the crude hCG
preparations discussed in Section 4, MA and pMA induce apoptotic
death of human cancer cells in vitro and in vivo, e.g., Kaposi's
sarcoma, other sarcomas, carcinomas of the breast, prostate, lung,
pancreas, brain, and kidney, and some hematopoietic tumors as well
as carcinomas, melanomas, and sarcomas of rodents.
[0391] The pharmacological properties of the MA peptides are
consistent with their efficacious use in cancer therapy. Examples
of activities of MA peptides useful in cancer therapy include:
[0392] induction of apoptisis of cancer cells; [0393] promotion of
hematopoiesis; [0394] protection of bone marrow, blood and/or other
cells from side effects of chemotherapy and/or radiation therapy;
[0395] direct boosting of the immune system; [0396] induction of
anti-angiogenesis; and [0397] reversal of wasting associated with
cancer.
[0398] The therapeutic polypeptides are useful in the treatment of
Kaposi's sarcoma (KS). MA peptides kill KS cells in vitro and block
tumor formation when such tumor cells are transplanted into immune
deficient mice. As described more fully in Section 9, systemic or
intralesional inoculation with MA peptides induces apoptosis of KS
cells.
[0399] The therapeutic polypeptides are also useful in the
treatment of brain, breast, lung, pancreas, prostate, renal and
hematopoietic cancers. The MA peptides exhibit anti-cancer activity
against brain, breast, lung, pancreas, prostate, and renal cancers
as well as in some hematopoietic cancers grown in vivo as
xenotransplants in immune deficient mice. The same range of
activity is seen with cultured human tumor cells.
[0400] Examples of cancers treatable by the methods of the
invention include: KS, brain, breast, lung, pancreas, prostate, and
renal cancer, and hematopoietic malignancies.
[0401] In a specific embodiment the therapeutic polypeptides are
administered to a subject suffering from a cancer that secretes hCG
or one of its subunits.
[0402] The therapeutic polypeptides are also useful in the ongoing
treatment of diagnosed and treated cancer patients to prevent
reoccurrence of disease, and in preventing the spread of a
malignancy in a dormant cancer, such as the spread of prostate
cancer.
[0403] The therapeutic polypeptides of the invention are useful as
a monotherpay or in combination with other therapies including
radiation or chemotherapy. Moreover, one or more therapeutic
polypeptides of the invention is usefully administered as a
component of a cancer vaccine composition to boost the immune
response to a cancer vaccine and optionally to induce apoptosis
and/or angiogenesis.
[0404] The therapeutic polypeptides are also usefully employed in
the treatment of leukemias. Specific leukemias which may be treated
using the therapeutic polypeptides of the invention include, for
example, acute leukemia, such as acute lymphocytic leukemia and
acute myelocytic leukemias (e.g., myeloblastic, promyelocytic,
myelomonocytic, monocytic and erythroleukemia), and chronic
leukemia (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia).
[0405] The therapeutic polypeptides of the invention are usefully
employed in the treatment of polycythemia vera, lymphoma (e.g.,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's macroglobulinemia, and heavy chain disease.
[0406] Additionally, the therapeutic polypeptides of the invention
are useful in the treatment of solid tumors, including sarcomas and
carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, Kaposi's sarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, uterine cancer,
testicular tumor, lung carcinoma, small cell lung carcinoma,
bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
melanoma, neuroblastoma, retinoblastoma, and virally-induced
cancers.
[0407] In specific embodiments, a therapeutic polypeptide of the
invention is used in the treatment of neoplasms, such as
gestational trophoblastic tumor, testicular germ cell tumor,
adenocarcinoma, virally induced cancers, as well as cancer of the
bladder, pancreas, cervix, lung, liver, ovary, colon, prostate
and/or stomach.
[0408] One or more therapeutic polypeptides of the invention may be
administered to a subject as a treatment for neuroblastoma or
carcinoma of the ovary or stomach.
[0409] One or more therapeutic polypeptides of the invention may be
administered to a subject as a treatment for Kaposi's sarcoma or
carcinoma of the breast, lung, prostate, or kidney (renal).
[0410] In one aspect of the invention, one or more therapeutic
polypeptides of the invention is administered in conjunction with
other cancer therapy, such as chemotherapy (e.g., treatment with
tamoxifen, adriamycin, etoposide, bleomycin, vincristine,
vinblastine, doxorubicin, paclitaxel and/or docetaxal). Examples of
other suitable antineoplastics for use in combination therapies
with the therapeutic polypeptides of the invention include:
[0411] adrenocorticotrophic hormones (e.g., corticotropin);
antibiotic antineoplastics (e.g., plicamycin); miscellaneous
antineoplastics (e.g., gallium nitrate); bone resorption
suppression agents (e.g., etidronate disodium and pamidronate
disodium); and glucocorticoids (e.g., adrenal cortex;
betamethasone; budesonide; cloprednol; cortisone acetate;
cortivazol; deflazacort; dexamethasone; fluprednisolone;
hydrocortisone; hydrocortisone acetate; hydrocortisone cypionate;
hydrocortisone hemisuccinate; hydrocortisone sod phosphate;
hydrocortisone sod succinate; meprednisone; methylprednisolone;
methylprednisolone acetate; methylprednisolone hemisucc;
methylprednisolone sod succ; paramethasone acetate; prednisolone;
prednisolone; prednisolone acetate; prednisolone hemisuccinate;
prednisolone Na Met-Sul-Benz; prednisolone sod phosphate;
prednisolone sod succinate; prednisone; prednylidene;
prednylidene).
[0412] The therapeutic polypeptides of the invention are also
useful for treating premalignant conditions to prevent progression
to a neoplastic or malignant state (e.g., the malignant disorders
listed above). Such prophylactic or therapeutic use is indicated in
conditions known or suspected of preceding progression to neoplasia
or cancer. Preferred conditions for treatment according to this
aspect of the invention include those characterized by
non-neoplastic cell growth, such as hyperplasia, metaplasia, or
most particularly, dysplasia..sup.98 Prophylactic treatment is also
warranted where the subject is determined to have a genetic
predisposition to a cancer. As an example, such a determination may
be made by identification of a mutation that predisposes the
subject to a cancer, e.g., using the BRCA1 test (Myriad Genetics,
Salt Lake City).
[0413] Hyperplasia is a form of controlled cell proliferation
involving an increase in cell number in a tissue or organ without
significant alteration in structure or function. As an example,
endometrial hyperplasia often precedes endometrial cancer.
[0414] Metaplasia is a form of controlled cell growth in which one
type of adult or fully differentiated cell substitutes for another
type of adult cell. Metaplasia can occur in epithelial or
connective tissue cells. Atypical metaplasia involves a somewhat
disorderly metaplastic epithelium.
[0415] Dysplasia is frequently a forerunner of cancer and is found
mainly in the epithelia; it is the most disorderly form of
non-neoplastic cell growth, involving a loss in individual cell
uniformity and abnormal changes in the architectural orientation of
cells. Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation, and
is often found in the cervix, respiratory passages, oral cavity,
and gall bladder.
[0416] Alternatively, or in addition to, the presence of abnormal
cell growth characterized as hyperplasia, metaplasia, or dysplasia,
the presence of one or more characteristics of a transformed
phenotype, or of a malignant phenotype, displayed in vivo or
displayed in vitro by a cell sample from a subject, can indicate
the desirability of prophylactic/therapeutic administration of one
or more therapeutic polypeptides of the invention. Examples of
characteristics of a transformed phenotype include morphology
changes, looser substratum attachment, loss of contact inhibition,
loss of anchorage dependence, protease release, increased sugar
transport, decreased serum requirement, expression of fetal
antigens, etc.
[0417] In other embodiments, a subject who exhibits one or more of
the following predisposing factors for malignancy is treated by
administration of an effective amount of one or more therapeutic
polypeptides of the invention: a chromosomal translocation
associated with a malignancy (e.g., the Philadelphia chromosome for
chronic myelogenous leukemia, t(14;18) for follicular lymphoma,
etc.); familial polyposis or Gardner's syndrome (possible
forerunners of colon cancer); benign monoclonal gammopathy (a
possible forerunner of multiple myeloma); and a first degree
kinship with persons having a cancer or precancerous disease
showing a Mendelian (genetic) inheritance pattern (e.g., familial
polyposis of the colon, Gardner's syndrome, hereditary exostosis,
polyendocrine adenomatosis, medullary thyroid carcinoma with
amyloid production and pheochromocytoma, Peutz-Jeghers syndrome,
neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid
body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma,
xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi
syndrome, albinism, Fanconi's aplastic anemia, and Bloom's
syndrome)..sup.99
[0418] The therapeutic polypeptides of the invention are also
usefully administered to a human subject to prevent progression of
prostate, breast, colon, lung, pancreatic, uterine cancer, melanoma
or sarcoma.
[0419] Hyperproliferative malignant stem cell disorders, as well as
non-hematopoietic malignancies, can be treated with chemotherapy or
radiation therapy along with rescue of hematopoietic cells by
direct in vivo administration of one or more therapeutic
polypeptides of the invention. Such conditions may also br treated
by administration of hematopoietic cells induced to proliferate by
contacting the cells in vitro with one or more therapeutic
polypeptides of the invention.
[0420] Hyperproliferative stem cell disorders are currently treated
by, inter alia, chemotherapy and, when feasible, allogeneic bone
marrow transplantation. However, allogeneic HLA identical sibling
bone marrow rarely available. Moreover, transplantation is
associated with various complications, such as immunodeficiency and
graft versus host disease. Induction of hematopoietic cell
proliferation in vivo or provision of autologous hematopoietic stem
and progenitor cells expanded by administration of one or more
therapeutic polypeptides of the invention in vitro permits
hematopoietic reconstitution in subjects lacking suitable
allogeneic donors and eliminates the risks of graft versus host
disease arising from allogeneic marrow transplantation.
[0421] The therapeutic polypeptides of the invention are usefully
employed in vitro to induce proliferation in hematopoietic cells
which are then administered to a subject who has undergone, and/or
will undergo, chemotherapy or radiation therapy for treatment of
cancer or an immunological disorder. In another embodiment, one or
more therapeutic polypeptides of the invention is directly
administered to a subject who has undergone, and/or will undergo,
chemotherapy or radiation therapy for treatment of cancer or an
immunological disorder.
[0422] Radiation therapy and chemotherapy (e.g., radiation therapy
and chemotherapy associated with cancer therapy) commonly result in
damage to bone marrow and/or blood cells. One or more therapeutic
polypeptides of the invention can be administered to a subject
before, during and/or after chemotherapy or radiation therapy to
prevent, limit and/or control hematopoietic damage or dysfunction.
Administration of one or more therapeutic polypeptides of the
invention before, during and/or after chemotherapy and/or radiation
therapy will reduce damage to the bone marrow and circulating blood
cells. Such administration may thereby reduce the need for blood
and bone marrow transfusions, and eliminate or reduce the risks
associated with such transfusions (e.g., the risk of contracting an
infectious disease such as HIV or hepatitis from a donor). Such
administration can enable the use of more aggressive radiation
therapy than would be tolerated by a subject in the absence of the
pro-hematopoietic effects of the therapeutic polypeptides of the
invention.
[0423] 8.5.8 Treatment of Impaired Immune Function and Autoimune
Conditions
[0424] The therapeutic polypeptides can also be administered to a
subject before, during and/or after chemotherapy or radiation
therapy to prevent, limit or control hematopoietic or other cell
damage or dysfunction and thereby reduce or eliminate the risks of
infection and bleeding disorders, increase survival rates and
reduce recovery time.
[0425] The inventors have surprisingly discovered that the MA
peptides exhibit anti-angiogenic effects in animal systems.
Accordingly, the therapeutic polypeptides are useful to prevent or
reduce pathological angiogenesis, e.g., in subjects with cancer,
macular degeneration, Crohn's disease or glaucoma. Direct treatment
of a subject with one or more therapeutic polypeptides of the
invention can prevent, limit or reduce medically detrimental
angiogenesis.
[0426] Many chronic inflammatory and degenerative diseases are
characterized by autoimmunity. Examples of such autoimmune
disorders include rheumatoid arthritis and other inflammatory
osteopathies, diabetes type I, chronic hepatitis, multiple
sclerosis, and systemic lupus erythematosus.
[0427] Autoimmune disorders are often treated by lymphoid
irradiation. Administration of one or more therapeutic polypeptides
of the invention, and/or cells produced by in vitro exposure to one
or more therapeutic polypeptides of the invention can be used to
repopulate the hematopoietic system after radiotherapy.
[0428] Anti-inflammatory drugs, such as steroids, retard
inflammatory cells, which are activated by autoreactive T cells.
However, such drugs do not prevent self-recognizing T cells from
activating new inflammatory cells. A more direct approach to
treating autoimmune diseases requires eradication of T cells by
irradiation of the lymphoid tissues, and repopulation of the
subject's hematopoietic system by administering stem cells from the
unirradiated bone marrow. The rationale for this approach is that
the formation of new populations of mature T cells from bone marrow
stem cells may result in absence of T cells that have reactivity to
self-specific antigens. This procedure, called total lymphoid
irradiation (TLI), has been used to treat intractable rheumatoid
arthritis..sup.100 Clinical trials have shown that in the majority
of otherwise intractable cases, joint disease is significantly
alleviated for at least 2-3 years. The major drawback to such
treatment is failure of stem cells in the bone marrow to
efficiently repopulate the hematopoietic system, resulting in
infections and bleeding disorders.
[0429] Other researchers have studied the use of TLI as an
alternative to cytotoxic drugs for treatment of SLE..sup.101
Studies of the use of TLI to treat intractable SLE have shown that
this treatment alleviates disease activity but is severely limited
by failure of bone marrow stem cells to rapidly and efficiently
repopulate the hematopoietic system after irradiation.
[0430] The therapeutic polypeptides of the invention can be
administered to promote proliferation of pluripotential stem cells
and other hematopoietic cells to increase the success of TLI
therapy. Additionally, hematopoietic stem and progenitor cells can
be isolated from the subject before treatment or obtained from a
suitable donor, induced to proliferate in vitro, and then
introduced into the subject after TLI treatment to repopulate the
hematopoietic system. Alternatively, hematopoietic stem and
progenitor cells can be isolated from the subject prior to therapy
or obtained from a suitable donor and infused into the patent and
expanded in vivo by direct administration of one or more
therapeutic polypeptides of the invention. Thus, the therapeutic
polypeptides of the invention can be administered to promote
proliferation of the remaining hematopoietic cells to increase the
success of TLI therapy. Additionally, hematopoietic stem and
progenitor cells can be isolated from the subject before treatment,
induced to proliferate ex vivo and then introduced into the subject
after TLI treatment to repopulate the hematopoietic system.
[0431] The therapeutic polypeptides may also be delivered to a
subject undergoing radiation and/or chemotherapy for the treatment
of a cancer to prevent damage to the oral and/or gastrointestinal
tract. Damage to the cells lining the oral cavity and the
gastrointestinal tract add greatly to the discomfort of patients
undergoing anti-cancer therapy and greatly increases the risk of
loss of weight and fungal infections.
[0432] Subjects with impaired immune function are prone to
infections, such as fungal infections. Impairment of immune
function can be associated with a variety of causes, such as cancer
therapy (e.g., radiation and/or chemotherapy), stress to or
reduction in the capacity of the hematopoietic system, and
infections (e.g., viral and/or bacterial infections).
Administration of one or more therapeutic polypeptides of the
invention to such persons can reduce the risk of developing fungal
infections. The therapeutic polypeptides are also usefully
administered to a subject presenting with a fungal infection to
eliminate the infection.
[0433] The therapeutic polypeptides of the invention are suitably
administered to a subject presenting with a fungal infection to
eliminate or reduce the severity of such infection. The therapeutic
peptide(s) may, for example, be administered locally by direct
inoculation into the site of infection or topically to the site of
infection. The therapeutic peptide(s) may also be administered
systemically to eliminate or reduce the severity of the infection
and/or to prevent the spreading of the infection to uneffected
tissue.
[0434] The therapeutic polypeptides are also usefully administered
to a subject with a wound or burn. One or more therapeutic
polypeptides may be administered to a subject who has sustained a
burn or laceration to reduce inflammation, promote hematopoiesis,
and/or to reduce the risk of infection. For example, one or more of
the therapeutic polypeptides may be topically or systemically
applied to the site of a wound or burn in order to reduce
inflammation, reduce the risk of infection and to promote
healing.
[0435] In another embodiment, one or more of the therapeutic
polypeptides is administered to a subject to reduce inflammation of
the cornea and/or to promote development of new corneal tissue.
[0436] In one aspect of the invention, the therapeutic polypeptides
are administered to a subject who has an infection to boost the
subject's capacity to combat the infection. The therapeutic
polypeptides may be administered in conjunction with antibiotics to
combat infection.
[0437] 8.5.9 Treatment Methods Employing Gene Therapy
Techniques
[0438] The invention provides gene therapy methods that employ the
therapeutic properties of the therapeutic polypeptides. In the gene
therapy aspects of the invention, therapy is effected by
administering to a subject genetic material comprising a nucleic
acid sequence encoding a therapeutic polypeptide. The therapeutic
polypeptide is then expressed in the subject, thereby mediating a
therapeutic effect. For a general treatment of gene therapy, see
Lemoine, ed., Understanding Gene Therapy..sup.102
[0439] The invention provides treatment methods comprising
administering to a subject a nucleic acid comprising a nucleotide
sequence encoding one or more therapeutic polypeptides of the
invention. The gene therapy methods of the invention are usefully
employed in the treatment of any of the diseases and conditions
described herein (e.g., see Sections 4.1, 4.2, 4.3, 4.4, 8.5.1,
8.5.4, 8.5.5 and 8.5.7). For example, such methods are usefully
employed in the treatment and/or prevention HIV infection, wasting,
cancer, and/or hematopoietic deficiency.
[0440] The invention provides a nucleic acid for use in gene
therapy methods. The nucleic acid encodes one or more therapeutic
polypeptides of the invention. The nucleic acid is preferably
provided as a component of an expression vector that produces the
therapeutic polypeptide in a suitable host cell.
[0441] The nucleic acid may also be provided as a component of an
expression cassette, for insertion into an expression vector. The
nucleic acid comprises a promoter operably linked to a nucleic acid
sequence coding for a therapeutic polypeptide. The promoter may be
inducible or constitutive and is optionally tissue-specific.
[0442] The nucleic acid sequence encoding the therapeutic
polypeptide and encoding any other desired sequences may be flanked
by regions that promote homologous recombination at a desired site
in the genome, thus providing for intrachromosomal expression of
the therapeutic polypeptide..sup.103
[0443] Delivery of the nucleic acid into a subject may be either
direct, in which case the subject is directly exposed to the
nucleic acid or nucleic acid-carrying vector, or indirect, in which
case, cells are first transformed with the nucleic acid in vitro,
then administered to the subject. These two approaches are known,
respectively, as in vivo and ex vivo gene therapy.
[0444] In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. Delivery can be accomplished by any of numerous methods
known in the art. For example, the nucleic acid can be included as
a component of an appropriate nucleic acid expression vector and
administered by an intracellular delivery method. Examples of
suitable intracellular delivery methods include infection using a
defective or attenuated retroviral or other viral vector,.sup.104
direct injection of naked DNA, microparticle bombardment (e.g., a
gene gun; Biolistic, Dupont), coating with lipids or cell-surface
receptors or transfecting agents, encapsulation in liposomes,
microparticles, or microcapsules, or linkage to a peptide which is
known to enter the cell or nucleus (e.g., by administering it in
linkage to a ligand subject to receptor-mediated
endocytosis.sup.105), which can be used to target cell types
specifically expressing the receptors, etc. In a specific
embodiment, the nucleic acid can be targeted in vivo for
cell-specific uptake and expression, by targeting a specific
receptor..sup.106 In another embodiment, a nucleic acid-ligand
complex can be formed in which the ligand comprises a fusogenic
viral peptide to disrupt endosomes, allowing the nucleic acid to
avoid lysosomal degradation. Alternatively, the nucleic acid can be
introduced intracellularly and incorporated by homologous
recombination within host cell DNA for expression..sup.107 For a
recent review of non-viral gene therapy, see Ledly, "Nonviral Gene
Therapy The Promise of Genes as Pharmaceutical
Products.".sup.108
[0445] In a specific embodiment, the gene therapy methods employ a
viral vector that comprises a nucleic acid sequence encoding one or
more therapeutic polypeptides of the invention. For example, the
viral vector may comprise a retroviral vector..sup.109 Suitable
retroviral vectors are those which have been modified to delete
retroviral sequences that are not necessary for packaging of the
viral genome. Retroviral vectors are maintained in infected cells
by integration into genomic sites upon cell division. The nucleic
acid to be used in gene therapy is cloned into the vector, which
facilitates delivery of the gene into a subject. More detail
concerning retroviral vectors can be found in Boesen et al..sup.110
which describes the use of a retroviral vector to deliver the mdrl
gene to hematopoietic stem cells in order to make the stem cells
more resistant to chemotherapy..sup.111
[0446] Adenoviruses may also be suitably employed in gene therapy
methods of the invention. Adenoviruses naturally infect respiratory
epithelia and are therefore especially attractive vehicles for
delivering genes to respiratory epithelia. Other targets for
adenovirus-based delivery systems are liver, the central nervous
system, endothelial cells, and muscle. Adenoviruses have the
advantage of being capable of infecting non-dividing cells.
Kozarsky and Wilson,.sup.112 present a review of adenovirus-based
gene therapy. Bout et al..sup.113 demonstrated the use of
adenovirus vectors to transfer genes to the respiratory epithelia
of rhesus monkeys. Other instances of the use of adenoviruses in
gene therapy can be found in Rosenfeld et al.;.sup.114 and
Mastrangeli et al..sup.115
[0447] Adeno-associated virus (AAV) and Herpes virus vectors are
also suitably employed in the gene therapy methods of the
invention..sup.116 For recent reviews of viral vecotors in gene
therapy, see Smith, "Viral Vectors in Gene Therapy";.sup.117
Amalfitano, et al., "Isolation and characterization of packaging
cell lines that coexpress the adenovirus E1, DNA polymerase, and
preterminal proteins: implications for gene therapy";.sup.118 Hitt
et al., "Human Adenovirus Vectors for Gene Transfer into Mammalian
Cells";.sup.119 Ali et al. The use of DNA viruses as vectors for
gene therapy";.sup.120 Yeh et al. "Advances in adenoviral vectors:
from genetic engineering to their biology";.sup.121 Robbins et al.,
"Viral Vectors for Gene Therapy.".sup.122
[0448] DNA and/or RNA encoding the therapeutic polypeptides can be
administered to a subject by introduction of the specific DNA or
RNA sequence in a live bacterial delivery system as is described
for DNA in U.S. Pat. No. 5,877,159, March, 1999. titled "Method for
Introducing and Expressing Genes in Animal Cells and Live Invasive
Bacterial Vectors for use in the Same."
[0449] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. Cells that have taken up and are
expressing the transferred gene are then isolated. The isolated
cells may then be delivered to the subject.
[0450] In an ex vivo gene therapy method, the vector comprising the
nucleic acid encoding one or more therapeutic polypeptides of the
invention may be introduced into a cell in vitro, prior to
administration in vivo of the resulting recombinant cell. Such
introduction can be carried out by any method known in the art.
Examples of suitable methods include transfection, electroporation,
microinjection, infection with a viral vector containing the
nucleic acid sequences, cell fusion, chromosome-mediated gene
transfer, microcell-mediated gene transfer, spheroplast fusion,
etc. Numerous techniques are known in the art for the introduction
of foreign genes into cells.sup.123 and may be used in accordance
with the invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0451] The resulting recombinant cells can be delivered to a
subject by various methods known in the art. In a preferred
embodiment, recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are administered intravenously. Epithelial cells
can be injected, e.g., subcutaneously, or recombinant skin cells
(e.g., keratinocytes) may be applied as a skin graft onto the
subject. The amount of cells envisioned for use depends on the
desired effect, subject state, etc., and can be determined by one
skilled in the art.
[0452] In an embodiment in which recombinant cells are used in gene
therapy, a nucleic acid sequence coding for therapeutic
polypeptides of the invention or functional equivalents thereof is
introduced into the cells such that it is expressible by the cells
or their progeny. The recombinant cells are then administered in
vivo where they will produce the therapeutic polypeptides to
provide the desired therapeutic effect. In a specific embodiment,
stem or progenitor cells, preferably hematopoietic stem or
progenitor cells, are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro are suitable for use in
accordance with this embodiment of the invention.
[0453] In a preferred aspect of the invention, hematopoietic cells
(preferably hematopoietic stem and progenitor cells) are
administered to a subject. Prior to such administration, the cells
are induced to proliferate with one or more therapeutic
polypeptides of the invention and genetically manipulated to stably
incorporate a heterologous gene capable of expression by their
progeny cells. This particular aspect of the invention is highly
useful in the treatment of diseases and disorders affecting cells
of hematopoietic lineage. In one embodiment, hematopoietic
reconstitution with such recombinant hematopoietic cells can be
used in the treatment of genetic disorders of the hematopoietic
system. Such genetic disorders include but are not limited to those
described herein.
[0454] Genetic deficiencies or dysfunctions of hematopoietic cells
can be treated by supplying recombinant stem and progenitor cells
to a subject. In a specific embodiment, subjects having
hematopoietic cells which lack a gene or have a mutant gene, can be
provided stem and progenitor cells that comprise a functional
counterpart of the deficient gene. Examples of genes that can be
usefully employed in this embodiment of the invention include genes
for hemoglobin or for enzymes which mediate the synthetic pathway
for hemoglobin. Such genes are useful in the treatment of anemias,
such as .beta.-thalassemia and sickle-cell syndrome.
[0455] In another specific embodiment, subjects with infections by
pathogenic microorganisms which occur in or affect a hematopoietic
cell lineage can be treated with recombinant hematopoietic cells.
Such recombinant hematopoietic cells can comprise a heterologous
gene which is expressed as a product which ameliorates disease
symptoms, is toxic to the pathogen without undue detriment to the
host, or which interferes with the pathogen's life cycle, etc.
Examples of infectious agents that cause infections treatable with
recombinant stem cells according to this embodiment of the
invention include lymphotropic viruses such as HIV; gram-negative
bacilli such as Brucella or Listeria; Mycobacterium tuberculosis;
Mycobacterium leprae; parasites, such as Plasmodium and Leishmania;
and fungi (such as those that cause pneumonia and other lethal
infections secondary to immunodeficiencies)..sup.124
[0456] The invention also provides stem or progenitor cells that
express a sequence that is "anti-sense" to the nucleic acid of a
hematopoietic cell pathogen. An antisense polynucleotide, which is
complementary to the pathogen's RNA or DNA, can hybridize to and
inactivate all or a critical component of such RNA or DNA. For
example, the antisense preferably inhibits the expression of a
protein or peptide that is critical to the pathogen's life
cycle.
[0457] Recombinant hematopoietic cells can be used in the treatment
of HIV infection. Recombinant stem and progenitor cells which
express an antisense nucleic acid that is complementary to a
critical region (e.g., the long-terminal repeat or polymerase
sequence) of the HIV genome.sup.125 can be used for in the
treatment of HIV infection.
[0458] A nucleotide encoding a sequence which is complimentary to a
sequence encoding MA (SEQ ID NO: 2) (MA antisense) may be
administered as a treatment for cancers.
[0459] 8.5.10 Use of the Therapeutic Polypeptides in Vaccines
[0460] The prohematopoietic effects of the MA peptides are
consistent with the conclusion that the therapeutic polypeptides of
the invention are usefully administered to a subject to enhance the
subject's immune response to an antigen, particularly a vaccine.
Accordingly, the invention provides a method for enhancing the
efficacy of a vaccine. This method comprises administering one or
more therapeutic polypeptides of the invention to a subject with an
antigen against which an immune response is desired. The
therapeutic polypeptides of the invention are also useful in
modulating Th1 vs Th2 immune response.
[0461] The therapeutic polypeptides of the invention may be
administered with the antigen and/or within a time period before
and/or after administration of the antigen. The window during which
the therapeutic polypeptides of the invention may be administered
(in relation to the administration of the antigen) is any time
during which the administration of the therapeutic polypeptides of
the invention enhances the subject's immune response to the
antigen. Enhancement of the immune response is determined by
comparison with a subject receiving an identical dose of the
antigen in the absence of the therapeutic polypeptide.
[0462] The efficacy of a vaccine can also be enhanced by
administering to a subject a nucleic acid comprising a nucleotide
sequence encoding the therapeutic polypeptide. The nucleic acid can
be administered with an antigen against which an immune response is
desired. Alternatively, the nucleic acid can be administered with a
second nucleic acid comprising a nucleotide sequence encoding an
antigen against which an immune response is desired, such that the
therapeutic polypeptide and the antigen are expressed in a
coordinated manner upon introduction into a suitable cell.
Additionally, one or more nucleotide sequences encoding one or more
therapeutic polypeptides of the invention and one or more
nucleotide sequences encoding one or more antigens, against which
an immune response is desired, may be provided on the same nucleic
acid.
[0463] 8.5.11 Other Treatment Methods
[0464] The therapeutic polypeptides are usefully added as component
of part of the fluid to allow a sperm and egg to fuse for the
purpose of implantation to promote fertilization and growth of the
embryo. In a specific embodiment, one or more of the therapeutic
polypeptides is administered to a subject prior to and/or during
and/or after implantation of an embryo to promote retention and
development of the placenta and embryo.
[0465] The therapeutic polypeptides of the invention are usefully
administered to a subject who has sustained an injury to the spinal
cord, nerves or the brain to reduce inflammation and to promote
growth of stem cells and nerve tissue and ultimately to reduce
neural damage and the resulting effects of such damage.
[0466] The inventors have also observed that humans exhibit a
feeling of wellbeing and increased libido when administered the
therapeutic polypeptides of the invention. Accordingly,
administration of one or more of the therapeutic polypeptides of
the invention to a subject to induce such a feeling of wellbeing
and/or to increase libido forms another aspect of the invention. In
a related embodiment the therapeutic polypeptide is administered to
a subject in order to treat impotence.
[0467] Further examples of diseases which may be treated using the
therapeutic polypeptides of the invention include osteopetrosis,
myelosclerosis, acquired hemolytic anemias, acquired
immunodeficiencies, infectious disorders causing primary or
secondary immunodeficiencies, bacterial infections (e.g.,
Brucellosis, Listeriosis, tuberculosis, leprosy), parasitic
infections (e.g., malaria, Leishmaniasis), fungal infections,
disorders involving disproportions in lymphoid cell sets and
impaired immune functions due to aging, phagocyte disorders,
Kostmann's agranulocytosis, chronic granulomatous disease,
Chediak-Higachi syndrome, neutrophil actin deficiency, neutrophil
membrane GP-180 deficiency, metabolic storage diseases,
mucopolysaccharidoses, mucolipidoses, miscellaneous disorders
involving, immune mechanisms, Wiskott-Aldrich Syndrome and alpha
1-antitrypsin deficiency.
[0468] In another aspect of the invention, one or more therapeutic
polypeptides is administered to a woman in perimenopause or
menopause to prevent or treat osteoporosis.
[0469] 8.5.12 In Vitro Preparation of Hematopoietic Cells
[0470] The invention provides methods for inducing hematopoietic
stem and progenitor cells to proliferate. Sources for such cells
are known in the art and include, for example, bone marrow, fetal
and neonatal blood (preferably from the umbilical cord and/or
placenta), peripheral blood, neonatal thymus, and neonatal spleen.
Suitable cells also include cryopreserved cells, cell lines, and
long-term cell cultures derived from the foregoing sources.
Preferred cells are mammalian cells, e.g., dog, cat, sheep, rat,
cow, horse, primate, monkey, and most preferably, human.
[0471] Techniques for obtaining such stem and progenitor cells are
well known in the art. For example, in one particular embodiment,
human bone marrow cells can be obtained from the posterior iliac
crest by needle aspiration..sup.126 Neonatal blood can be obtained
at birth by direct drainage from the umbilical cord and/or by
needle aspiration from the delivered placenta at the root and at
distended veins..sup.127 Fetal blood can be obtained, e.g., by
taking it from the fetal circulation at the placental root with the
use of a needle guided by ultrasound,.sup.128 by
placentocentesis,.sup.129 by fetoscopy,.sup.130 etc.
[0472] Methods of the invention which comprise contacting
hematopoietic stem and/or progenitor cells (or other hematopoietic
cells) with one or more therapeutic polypeptides of the invention
have already been described. Such methods can be carried out on
unseparated, partially separated, or purified cell populations. The
methods are usefully employed before and/or after cryopreservation
(and thawing) or in vitro culturing of such cell populations.
Moreover, the methods may also be employed before and/or after
introduction of a recombinant gene, and any other desired
manipulations of the cells. In a preferred aspect, samples (e.g.
bone marrow or adult blood or neonatal blood) can be subjected to
physical and/or immunological cell separation procedures to enrich
for hematopoietic stem and progenitor cells. For example, such
separation procedures may be carried out prior to culturing in the
presence of a therapeutic polypeptide of the invention to induce
proliferation of the cells.
[0473] Various procedures for enriching for stem and progenitor
cells are known in the art. For example, suitable procedures
include equilibrium density centrifugation, velocity sedimentation
at unit gravity, immune rosetting and immune adherence, counterflow
centrifugal elutriation, T lymphocyte depletion, and
fluorescence-activated cell sorting, alone or in combination.
Procedures have been reported for the isolation of very highly
enriched populations of stem/progenitor cells. U.S. Pat. No.
5,061,620 dated October 1991 discloses a method for isolation of
human hematopoietic stem cells. Several groups have purified murine
CFU-S using slightly different procedures..sup.131 Studies using
human.sup.132 or murine.sup.133 fetal liver cells have yielded
highly enriched progenitor cells with up to 90% of them being
colony forming cells for multi-, erythroid-, and
granulocyte-macrophage lineages. CFU-E have also been very highly
enriched..sup.134 Purification of adult mouse marrow CFU-GM with
cloning efficiencies of up to 99% in semi-solid medium has been
accomplished by pretreatment of mice three days prior to sacrifice
with cyclophosphamide, density separation of cells on
Ficoll-Hypaque, and counterflow centrifugal elutriation..sup.135
The resulting fraction of cells contained no detectable CFU-GEMM,
BFU-E or CFU-MK, but up to 10% of the cells formed CFU-S measured
at day 12. These procedures, or modifications thereof, can be
used.
[0474] Human stem and progenitor cells are present in the
non-adherent, low density, T-lymphocyte-depleted fraction of bone
marrow, spleen, and adult and cord blood cells. Low density
(density less than 1.077 gm/cm.sup.3) cells can be separated by use
of Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, N.J.) or
Percol..sup.136 In this procedure, the mature cells of the
granulocytic series, which are not needed for transplantation, are
removed in the dense fraction that goes to the bottom of the tube.
An adherence/nonadherence separation protocol can also be used for
enrichment of hematopoietic stem and progenitor cells.
[0475] It is also possible to use cell separation procedures that
entail immunological recognition of cells. Stem and progenitor
cells can be isolated by positive or negative selection using
antibodies that recognize antigenic determinants on the surface of
cells. One means is to separate the cells by using monoclonal
antibodies that recognize cell surface determinants on these cells,
in conjunction with separation procedures such as
fluorescence-activated cell sorting or panning..sup.137 Human
hematopoietic stem and progenitor cells contain antigenic
determinants that are not present on all other cells. These
antigenic determinants can be used in antibody selection protocols
for enrichment purposes. Such antigens include, for example, those
known to be present on human stem/progenitor cells,.sup.138 as well
as those used to distinguish progenitors of the
granulocyte-macrophage lineage,.sup.139 and others known in the
art..sup.140
[0476] The numbers of the hematopoietic stem and/or progenitor
cells (or precursor cells thereof) may be expanded by exposing the
cells or contacting them with a composition comprising a
therapeutic polypeptide of the invention time sufficient to obtain
the desired number of cells. Preferably the cells are contacted
while under appropriate culture conditions for a period of time
which ranges from 1 to 21 or, more preferably, from 7 to 21
days.
[0477] The composition comprising the therapeutic polypeptides of
the invention, to which the stem and progenitor cells are exposed,
may comprise other growth factors (i.e., polypeptides that
stimulate cell division) and/or cytokines or cell culture
materials. Examples of suitable growth factors include but are not
limited to heregulin (ERG), insulin, insulin-like growth factors I
and II (IGF-I and IGF-II), epidermal growth factor (EGF),
insulin-like growth factors I and II (IGF-I and IGF-II), epidermal
growth factor (EGF), interleukins (e.g., IL-8), macrophage
colony-stimulating factor (M-CSF), erythropoietin (EPO),
platelet-derived growth factor (PDGF), fibroblast growth factor
(FGF), transforming growth factors alpha and beta (TGF-alpha and
TGF-beta), hepatocyte growth factor (HGF), and nerve growth factor
(NGF). Examples of suitable cytokines include macrophage-derived
chemokine, monocyte chemotactic protein 1, monocyte chemotactic
protein 2, monocyte chemotactic protein 3, monocyte chemotactic
protein 4, activated macrophage specific chemokine 1, macrophage
inflammatory protein 1 alpha, macrophage inflammatory protein 1
beta, macrophage inflammatory protein 1 gamma, macrophage
inflammatory protein 1 delta, macrophage inflammatory protein
2.alpha., macrophage inflammatory protein 3.alpha.., macrophage
inflammatory protein 3.beta., regulated upon activation, normal T
cell expressed and secreted (and its variants), I-309, EBI1-ligand
chemokine, pulmonary and activation regulated chemokine, liver and
activation-regulated chemokine, thymus and activation regulated
chemokine, eotaxin (and variants), human CC chemokine 1, human CC
chemokine 2, human CC chemokine 3, IL-10-inducible chemokine,
Liver-expressed chemokine, 6Ckine, exodus 1, exodus 2, exodus 3,
thymus-expressed chemokine, secondary lymphoid tissue chemokine,
lymphocyte and monocyte chemoattractant, monotactin,
chemokine-related molecule, myeloid progenitor inhibitory factor-1,
myeloid progenitor inhibitory factor-2, stromal cell-derived factor
1.alpha., stromal cell-derived factor 1.beta., B-cell-attracting
chemokine 1, HuMIG, H174, Interferon-stimulated T-cell
.alpha.-chemoattractant, interleukins, IP-10, platelet factor 4,
growth-regulated gene-.alpha., growth-regulated gene-.beta.,
growth-regulated gene-.gamma., neutrophil-activating protein 2,
ENA-78, granulocyte chemotactic protein 2, lymphotactin,
fractalkine/neurotactin, viral chemokines and functional
equivalents of the foregoing chemokines. Other specific examples of
suitable components include Steel factor (SLF) and adult PB plasma.
Preferred factors are those which cause proliferation or, less
preferably, differentiation of cells that are CFU-GEMM or earlier
cells, e.g., IL-3, GM-CSF.
[0478] The stem and/or progenitor cells are preferably contacted
with the therapeutic polypeptide(s) and/or functional equivalent(s)
of the invention during cell culture. Thus, the therapeutic
polypeptide is preferably added to the cell culture medium being
used to culture the hematopoietic stem and/or progenitor cells.
[0479] The cells may be cultured by any method known in the art.
Examples of suitable methods for culturing the cells include
growing cells in culture dishes, test tubes, roller bottles,
bioreactors (perfusion system machines wherein cells are grown on a
surface with continual perfusion by medium; e.g., as sold by
Aastrom Biosciences, Inc., Ann Arbor, Mich.), etc. Various
protocols have been described for the in vitro growth of cord blood
or bone marrow cells..sup.141 Such procedures, or modifications
thereof, may be employed in the methods of the invention. The cell
culture medium is preferably supplemented to contain an effective
concentration of one or more therapeutic polypeptides of the
invention. Culturing may also be achieved by using any of a variety
of cross-flow filter culturing systems known in the art.
[0480] Progeny cells of hematopoietic stem and progenitor cells can
be generated in vitro. Such differentiated progeny cells can be
therapeutically useful. For example, in one embodiment of this
aspect of the invention, hematopoietic stem cells and/or CFU-GEMM
progenitor cells, can be induced to differentiate into platelets.
Such platelets can be used, for example, for infusion into a
subject with thrombocytopenia (e.g., a subject with HIV-associated
ITP or a subject undergoing radiation and/or chemotherapy for
cancer).
[0481] In another embodiment, granulocytes can be generated in
vitro prior to infusion into a subject. One or more of the
hematopoietic progeny cells can be generated in vitro, allowing for
the in vitro production of blood components. The generation of
differentiated blood components may be accompanied by expansion of
the hematopoietic stem and progenitor cell pool to permit
production of a greater quantity of differentiated cells.
[0482] Various growth factors can be used to promote expansion
and/or differentiation of hematopoietic stem and progenitor cells,
such as cytokines (growth factors) including, but not limited to,
G-CSF, CSF-1, IL-3, IL-5, tumor necrosis factor-.beta., and
.alpha.-interferon.
[0483] The blood components produced by the methods of the
invention have a variety of in vitro uses, e.g., for the production
and isolation of hematopoietic cell products such as growth
factors, antibodies, etc.
[0484] A specific embodiment of the invention relates to a method
of increasing the amount of hematopoietic cells, which method
comprises contacting in vitro a non-terminally differentiated
hematopoietic cell with a composition comprising an amount of a
therapeutic polypeptide of the invention effective to increase
proliferation of the cell, under conditions suitable and for a time
sufficient to increase the numbers of the hematopoietic cell. For
example, hematopoietic cell numbers can be increased by contacting
a non-terminally differentiated hematopoietic cell (e.g., a cell
isolated from bone marrow or blood, adult or fetal or umbilical
cord blood) with a composition comprising a therapeutic polypeptide
of the invention and culturing the cell for at least ten days.
[0485] 8.6 Assays for Therapeutic Activity
[0486] The ensuing description is described with regard to the
assaying of an individual therapeutic polypeptide for therapeutic
activity; however, it will be appreciated that the same methods can
be used to assay multi-drug combinations that include combinations
of the therapeutic polypeptides, and which optionally include other
therapeutic compounds. A kit may be provided for any of the
following assay methodologies. Such a kit suitably comprises one or
more therapeutic polypeptides of the invention along with
instructions and/or other reagents and/or supplies for performing
the assay.
[0487] 8.6.1 Assays for Anti-HIV Activity
[0488] The invention provides a method for screening therapeutic
polypeptides of the invention for anti-HIV activity. The assay
comprises assaying the therapeutic polypeptides of the invention
for the ability to inhibit HIV replication or expression of HIV RNA
or protein. In one specific embodiment, the therapeutic polypeptide
of the invention is assayed by a method comprising: (1) contacting
HIV-1-infected cultured hematopoietic cells with one or more
therapeutic polypeptides to of the invention, and (2) measuring
HIV-1 p24 antigen levels in the cells. The measured HIV-1 p24
antigen levels in the cells is preferably compared to a
corresponding group of cells for which the contacting step has not
been performed. A lower level of HIV-1 p24 antigen levels in the
contacted cells indicates that the therapeutic polypeptide has
anti-HIV activity.
[0489] The therapeutic polypeptide of the invention may also be
assayed by a method comprising: (1) contacting HIV-1-infected
cultured hematopoietic cells with one or more therapeutic
polypeptides of the invention; (2) measuring the activity of a
reporter gene product expressed from a construct in which the HIV-1
LTR is operably linked to the reporter gene; and (3) comparing the
measured expression of the reporter gene in the contacted cells
with the levels in cells not contacted with the therapeutic
polypeptide of the invention. A lower level in the contacted cells
indicates that the therapeutic polypeptide has anti-HIV
activity.
[0490] A therapeutic polypeptide of the invention may also be
assayed by a method comprising measuring HIV-1 derived RNA
transcripts or HIV-1 antigen levels in HIV-1 transgenic mice
administered the therapeutic polypeptide. The measured transcript
or antigen levels in the mice which have been administered the
therapeutic polypeptide of the invention may be compared with the
levels in mice not administered the therapeutic polypeptide of the
invention. A lower level of transcript or antigen in mice to which
the therapeutic polypeptide of the invention was administered
indicates that the therapeutic polypeptide has anti-HIV
activity.
[0491] The therapeutic polypeptides of the invention may also be
assayed by a method comprising measuring SIV p27 antigen levels in
the peripheral blood mononuclear cells of SIV infected monkeys. A
lower level of SIV p27 antigen in monkeys administered the
therapeutic polypeptide of the invention indicates that the
therapeutic polypeptide has anti-HIV activity.
[0492] A therapeutic polypeptide of the invention in vitro can be
assayed by examining the effect of the therapeutic polypeptide on
HIV replication in cultured cells. Briefly, cultured hematopoietic
cells (e.g., primary PBMCs, isolated macrophages, isolated
CD4.sup.+ T cells or cultured 119 human T cells) are acutely
infected with HIV-1 using titers known in the art to acutely infect
cells in vitro, such as 10.sup.5 TCID.sub.50/ml. Then, appropriate
amounts of the therapeutic polypeptide are added to the cell
culture media. Cultures are assayed 3 and 10 days after infection
for HIV-1 production by measuring levels of p24 antigen using a
commercially available ELISA assay. Reduction in p24 antigen levels
over levels observed in untreated controls indicates the
therapeutic polypeptide is effective for treatment of HIV
infection.
[0493] Additionally, assays for HIV-1 LTR driven transcription are
useful for testing the efficacy of therapeutic polypeptides of the
invention. Such assays employ a reporter gene, i.e., a gene with a
detectable protein or RNA product. A preferred reporter gene is the
gene for chloramphenicol acetyltransferase (CAT). The reporter gene
is cloned into a DNA plasmid construct, so that the transcription
of the reporter gene is driven by the HIV-1 LTR promoter. The
resulting construct is then introduced by transfection, or any
other method known in the art, into a cultured cell line. A
preferred cell line is the human CD4.sup.+ T cell line HUT 78.
After exposure of the transformed cells to the therapeutic
polypeptide, transcription from the HIV-1 LTR is determined by
measurement of CAT activity using techniques which are routine in
the art. Reduction in HIV-1 LTR driven transcription demonstrates
utility of the therapeutic polypeptide for treatment and/or
prevention of HIV infection.
[0494] Exemplary tests in animal models are described briefly as
follows: First, a therapeutic polypeptide of the invention is
administered to mice transgenic for HIV-1 (e.g., mice which have
integrated molecular clone pNL4-3 containing 7.4 kb of the HIV-1
proviral genome deleted in the gag and pot genes)..sup.142 Skin
biopsies taken from the mice are tested for HIV-1 gene expression
by RT-PCR (reverse transcription-polymerase chain reaction) or for
HIV-1 antigen expression, such as expression of gp120 or NEF, by
immunostaining. Additionally, the mice are examined for reduction
in the cachexia and growth retardation usually observed in HIV-1
transgenic mice..sup.143
[0495] The efficacy of therapeutic polypeptides of the invention
can also be determined in SIV infected rhesus monkeys..sup.144 The
rhesus monkeys are preferably infected with SIV.sub.mac251, which
induces a syndrome in the experimentally infected monkeys that is
very similar to human AIDS..sup.145 Monkeys can be infected with
cell free SIV.sub.mac251, for example, with virus at a titer of
10.sup.4.5 TCID.sub.50/ml. Infection is monitored by the appearance
of SIV p27 antigen in PBMCs. Utility of the therapeutic polypeptide
is characterized by normal weight gain, decrease in SIV titer in
PBMCs and/or an increase in CD4.sup.+ T cells.
[0496] Once the therapeutic polypeptide has been tested in vitro,
and also preferably in a non-human animal model, the utility of the
therapeutic polypeptide can be determined in human subjects. The
efficacy of treatment with a therapeutic polypeptide can be
assessed by measurement of various parameters of HIV infection and
HIV associated disease. Specifically, the change in viral load can
be determined by quantitative assays for plasma HIV-1 RNA using
quantitative RT-PCR.sup.146 or by assays for viral production from
isolated PBMCs. Viral production from PBMCs is determined by
co-culturing PBMCs from the subject with H9 cells and subsequent
measurement of HIV-1 titers using an ELISA assay for p24 antigen
levels.sup.147 Another indicator of plasma HIV levels and AIDS
progression is the production of inflammatory cytokines such as
IL-6, IL-8 and TNF-.alpha.; thus, efficacy of the therapeutic
polypeptide can be assessed by ELISA tests for reduction of serum
levels of any or all of these cytokines.
[0497] Administration of the therapeutic polypeptide can also be
evaluated by assessing changes in CD4.sup.+ T cell levels, body
weight, or any other physical condition associated with HIV
infection or AIDS or AIDS Related Complex (ARC). Reduction in HIV
viral load or production, increase in CD4.sup.+ T cell or
amelioration of HIV-associated symptoms demonstrates utility of a
therapeutic polypeptide for administration in treatment/prevention
of HIV infection.
[0498] 8.6.2 Assays for Anti-Wasting Activity
[0499] The invention provides a method for screening a therapeutic
polypeptide of the invention for anti-wasting activity. Such method
generally comprises assaying the preparation for the ability to
promote weight gain in an animal model that exhibits a wasting
syndrome.
[0500] In one specific embodiment, the therapeutic polypeptide of
the invention is screened by a method comprising: (1) administering
the therapeutic polypeptide of the invention to an offspring of an
HIV-1 transgenic mouse; (2) measuring the weight of the offspring;
and (3) comparing the weight of the offspring therapeutic
polypeptide of the invention with the weight of an offspring not so
exposed. A greater weight in the exposed offspring indicates that
the preparation has anti-wasting activity. The same method can be
employed using an SIV infected monkey.
[0501] Any animal model in which wasting occurs can be used.
Exemplary tests in animal models are described briefly as follows:
First, a therapeutic polypeptide of the invention is assayed in
mice transgenic for HIV-1 (e.g., mice which have integrated
molecular clone pNL4-3 containing 7.4 kb of the HIV-1 proviral
genome deleted in the gag and pol genes)..sup.148 These mice
exhibit cachexia and growth retardation..sup.149 Reversal of the
cachexia and growth retardation in the HIV transgenic mice is
consistent with a conclusion that the therapeutic polypeptide is
useful for treating or preventing wasting syndromes.
[0502] Similarly, the efficacy of therapeutic polypeptide of the
invention can be assayed in SIV infected rhesus monkeys..sup.150
The rhesus monkeys are preferably infected with SIV.sub.mac251,
which induces a syndrome in experimentally infected monkeys that is
very similar to human AIDS and results in weight loss in the
infected monkeys..sup.151 Specifically, monkeys are infected with
cell free SIV.sub.mac251, for example, with virus at a titer of
10.sup.4.5 TCID.sub.50/ml and SIV infection is monitored by the
appearance of SIV p27 antigen in PBMCs. An increase in the weight
of infected monkeys administered the therapeutic polypeptide of the
invention indicates that the therapeutic polypeptide of the
invention has utility in the treatment of wasting syndrome.
[0503] Compounds for use in therapy are preferably tested in
suitable animal model systems prior to testing in humans, including
but not limited to rats, mice, chicken, cows, monkeys, rabbits,
etc. For in vivo testing, prior to administration to humans, any
animal model system known in the art may be used.
[0504] Once the therapeutic polypeptide of the invention has been
tested in a non-human animal model, the utility of the therapeutic
polypeptide of the invention can be determined in human subjects.
Improvement in wasting syndrome, i.e. and increase in body cell
mass, can be assessed by any well known clinical techniques
available in the art. Examples of suitable techniques include
measuring body weight, determining total body potassium content,
determining intracellular water volume, bioelectrical impedance
analysis, anthropometrics and determining total body nitrogen
content..sup.152 Therapeutic polypeptides of the invention which
increase body weight or cell mass are concluded to have utility in
treating wasting syndrome.
[0505] 8.6.3 Assays for Anti-Cancer Activity
[0506] The invention provides a method for screening a therapeutic
polypeptide of the invention for anti-cancer activity. The method
comprises assaying the therapeutic polypeptide of the invention for
the ability to inhibit the survival or proliferation of malignant
cells.
[0507] In one embodiment, the preparation is screened by a method
comprising: (1) contacting malignant cells with the therapeutic
polypeptide of the invention; (2) measuring the survival or
proliferation of malignant cells; and (3) comparing the survival or
proliferation of the cells contacted with the therapeutic
polypeptide of the invention with the survival or proliferation of
cells not so contacted (e.g., cells contacted with a control). A
lower level of survival or proliferation in the contacted cells
indicates that the preparation has anti-cancer activity. Examples
of suitable cells are those which are derived from or display
characteristics associated with a malignant disorder.
[0508] Cells may also be screened for the ability of a therapeutic
polypeptide of the invention to convert cells having an abnormal
phenotype to a more normal cell phenotype. For example, suitable
cells may include pre-neoplastic or pre-malignant cells. A more
normal phenotype in the contacted cells indicates that the
preparation has anti-cancer activity.
[0509] Many assays standard in the art can be used to assess
whether a pre-neoplastic state, neoplastic state, or a transformed
or malignant phenotype, is present. For example, characteristics
associated with a transformed phenotype (a set of in vitro
characteristics associated with a tumorigenic ability in vivo)
include a more rounded cell morphology, looser substratum
attachment, loss of contact inhibition, loss of anchorage
dependence, release of proteases such as plasminogen activator,
increased sugar transport, decreased serum requirement, expression
of fetal antigens, disappearance of the 250,000 dalton surface
protein, etc..sup.153
[0510] A therapeutic polypeptide of the invention may also be
assayed the ability to inhibit Kaposi's Sarcoma cell proliferation
or promote Kaposi's Sarcoma cell apoptosis. The therapeutic
polypeptide of the invention may be screened by a method comprising
measuring proliferation or colony formation in cultured KS Y-1 or
KS-SLK cells contacted with the therapeutic polypeptide of the
invention. Contacted cells are compared with cells which have not
been contacted with the therapeutic polypeptide of the invention. A
lower level of proliferation or colony formation in the contacted
cells indicates that the preparation has anti-Kaposi's Sarcoma
activity.
[0511] A therapeutic polypeptide of the invention can be tested for
efficacy in the treatment or prevention of Kaposi's sarcoma (1) by
any the method described herein; (2) by the Lunardi-Iskandar et
al..sup.154 method; and/or (3) by any other method known in the
art. Briefly, KS cell lines, KS Y-1 (Ibid.) or KS-SLK,.sup.155
which will produce malignant tumors in immunodeficient mice, are
used to perform in vitro proliferation and clonogenic
assays..sup.156 Methods for performing such assays are well known
in the art. A therapeutic polypeptide of the invention that reduces
proliferation or colony formation in the cultured cells can be used
in the methods of the invention for treatment or prevention of
KS.
[0512] The method may also comprise measuring apoptosis in a
Kaposi's Sarcoma tumor in an immunodeficient mouse. Kaposi's
Sarcoma tumors may be induced by injection with KS Y-1 or KS-SLK
cells. Following exposure to the therapeutic polypeptide of the
invention, the degree of apoptosis in the tumor of a mouse which
has been exposed to the therapeutic polypeptide of the invention
may be compared with the degree of apoptosis in a mouse with a
tumor in a mouse not so exposed. A higher in level of apoptosis in
the tumor of the exposed mouse indicates that the therapeutic
polypeptide of the invention has anti-Kaposi's Sarcoma
activity.
[0513] In vitro assays can be used to determine whether
administration of a specific therapeutic polypeptide of the
invention is indicated in a specific subject. For example, the
invention provides in vitro cell culture assays. A tissue sample
obtained from a subject is grown in culture and exposed to or
otherwise administered a therapeutic polypeptide of the invention.
The effect of the therapeutic polypeptide of the invention upon the
tissue sample is observed. In one embodiment, where the subject has
a malignancy, a sample of cells from the malignancy is plated out
or grown in culture. The cells are then exposed to a therapeutic
polypeptide of the invention. A therapeutic polypeptide which
inhibits survival or growth of the malignant cells is selected for
therapeutic use in vivo.
[0514] Many assays standard in the art can be used to assess such
survival and/or growth. For example, cell proliferation can be
assayed by measuring .sup.3H-thymidine incorporation, by direct
cell count, by detecting changes in transcriptional activity of
known genes such as proto-oncogenes (e.g., fos, myc) or cell cycle
markers. Cell viability can be assessed by trypan blue staining.
Cell differentiation can be assessed visually based on changes in
morphology, etc.
[0515] In various specific embodiments, in vitro assays can be
carried out with representative cells of cell types involved in a
subject's specific disorder, to determine if a therapeutic
polypeptide has a desired effect upon such cell types.
[0516] In other specific embodiments, the in vitro assays described
supra can be carried out using a cell line, rather than a cell
sample derived from the specific subject to be treated. The cell
line is preferably derived from or displays characteristic(s)
associated with the malignant, neoplastic or pre-neoplastic
disorder desired to be treated or prevented, or is derived from the
cell type upon which an effect is desired, according to the
invention.
[0517] Efficacy of a therapeutic polypeptide can also be determined
by administration of the a therapeutic polypeptide or functional
equivalent to immunodeficient mice injected with either the KS-Y-1
or KS-SLK cells, which cause tumor formation in the mice. The
degree of apoptosis and angiogenesis of tumor cells after treatment
with the therapeutic polypeptide is measured. Apoptosis is detected
by staining fixed tissue samples from the tumor for the presence of
cells with DNA fragmentation. For example, this detection is
accomplished by treating tissue slides from formalin-fixed tumors
with terminal deoxynucleotide transferase for extension of DNA ends
(3' hydroxyl ends) and incorporation of digoxigenin-11-dUTP.
Anti-digoxigenin antibody conjugated with the enzyme peroxidase
allows detection of apoptotic cells that stain brown whereas viable
cells stain blue. An increase in KS tumor cell apoptosis and a
decrease in angiogenesis indicates that the therapeutic polypeptide
has utility in treatment of KS.
[0518] A therapeutic polypeptide can also be assessed in clinical
trials in human subjects presenting with KS or any other cancer. To
test the efficacy of the therapeutic polypeptide in KS patients,
either local, i.e. intralesional, or systemic administration of the
therapeutic polypeptide can be used. Tumors can be examined
physically for regression in response to administration of the
therapeutic polypeptide. Additionally, tissue biopsies can be taken
from the tumors and examined for apoptosis, as described above.
[0519] Compounds for use in therapy can be tested in suitable
animal model systems prior to testing in humans, including but not
limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For in
vivo testing, prior to administration to humans, any animal model
system known in the art may be used.
[0520] 8.6.4 Assays for Pro-Hematopoietic Activity
[0521] The invention provides a method for testing a therapeutic
polypeptide for pro-hematopoietic activity. The therapeutic
polypeptide is preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. Any in vitro or in vivo assay known in the art to measure a
pro-hematopoietic effect may be used. For example, suitable assays
include those which measure the ability to induce hematopoietic
cell proliferation in vitro or production of one or more
hematopoietic cell types in vivo.
[0522] A specific embodiment provides a method for screening a
therapeutic polypeptide for pro-hematopoietic activity, the method
comprising assaying the preparation for the ability to induce an
increase in hematopoietic cell numbers. The preparation may be
screened by a method comprising measuring the number of colonies
formed from hematopoietic stem or progenitor cells. The number of
colonies formed from cells contacted with the therapeutic
polypeptide is compared with the number of colonies formed from
cells not so contacted. A higher number of colonies formed from the
contacted cells indicates that the preparation has
pro-hematopoietic activity.
[0523] In one embodiment, the cells are CD4.sup.+ T cells in an SIV
infected monkey. The monkey is exposed to the therapeutic
polypeptide. The number of CD4.sup.+ T cells in the monkey, which
has been exposed to the therapeutic polypeptide, with the number of
CD4.sup.+ T cells in a monkey not so exposed. A higher number of
CD4.sup.+ T cells in the exposed monkey indicates that the
preparation has pro-hematopoietic activity.
[0524] The effect of the therapeutic polypeptide can also be
measured on proliferation of hematopoietic cells in vitro. For
example, hematopoietic cells may be cultured for an appropriate
amount of time, such as 5 to 20 days and preferably 10 days, in the
presence of (or otherwise exposed to) the therapeutic polypeptide
of the invention. Colony assays are then performed to determine the
number of colonies formed in comparison to the number of colonies
formed by cells cultured in the absence of the therapeutic
polypeptide of the invention. For example, hematopoietic progenitor
cells can be isolated from bone marrow or cord blood, seeded in
methylcellulose in the presence of absence of the therapeutic
polypeptide, and then colony numbers determined after 10 days of
culture. An increase in colony numbers in cells contacted with the
therapeutic polypeptide of the invention indicates that the
therapeutic polypeptide of the invention has activity in inducing
proliferation of hematopoietic cells. Thus, for example, depending
on the progenitor cell desired to be assayed, CFU-GM, CFU-GEMM,
etc., assays can be performed using this method.
[0525] A therapeutic polypeptide of the invention can also be
tested in vivo for the ability to increase numbers of hematopoietic
cells. Preferably, the therapeutic polypeptide of the invention is
tested in animal models of hematopoietic disorders before testing
them in human subjects. For example, a therapeutic polypeptide can
be tested in rhesus monkeys infected with SIV, particularly
SIV.sub.mac251 (as described above). Blood or bone marrow of the
infected monkeys can be examined for an increase in CD4.sup.+ T
cells or any other hematopoietic cell type for which the monkey is
deficient. An increase in numbers of the hematopoietic cell
demonstrates that the therapeutic polypeptide is useful for
treating diseases and disorders associated with hematopoietic
deficiencies. Any animal model of an anemia can be similarly used
for testing.
[0526] A therapeutic polypeptide of the invention can be tested in
human subjects, preferably after tests in vitro and/or in vivo in
an animal model. The subjects tested may be affected by
hematopoietic deficiencies. Such deficiencies may include
deficiencies associated with HIV infection such as anemia,
neutropenia, thrombocytopenia, or CD4.sup.+ T cell lymphocyte
deficiency. In such cases, the therapeutic polypeptide of the
invention may be screened for activity in increasing numbers of
hematopoietic cells for which the subject is deficient. Briefly,
the therapeutic polypeptide is administered, for example by
intramuscular injection two to three times per week, to the subject
presenting with the hematopoietic deficiency. The subject's blood
or bone marrow is assayed before and after treatment with the
therapeutic polypeptide for an increase in the hematopoietic cell
numbers. A therapeutic polypeptide of the invention that causes an
increase in hematopoietic cell numbers is useful for treatment of
diseases and disorders associated with hematopoietic
deficiencies.
[0527] Assays for hematopoietic cell proliferation in the blood or
bone marrow can be accomplished by any method well known in the
art. For example, blood can be drawn and blood cell numbers can be
determined by routine clinical laboratory tests for red blood
cells, platelets, neutrophils, lymphocytes, etc. Additionally,
colony assays on isolated bone marrow can be performed to assess
increases in stem or progenitor cells. For example, bone marrow can
be sampled and bone marrow cells evaluated for stem and progenitor
cell colony formation. Briefly, cells are seeded in
methylcellulose, cultured for 12 to 14 days, and then scored for
colony formation where aggregates containing more than 50 cells are
counted as a colony..sup.157 Bone marrow progenitors that can be
evaluated by this colony assay include, but are not limited to,
CFU-Mix, BFU-e and CFU-GM.
[0528] As an alternative to colony assays for detection and
quantitation of stem and/or progenitor cells, immunological
detection methods can be employed, based on the antigens expressed
by the particular cell type (see, e.g., the relevant discussion
hereinabove).
[0529] 8.7 Therapeutic Compositions and Methods of
Administration
[0530] The invention provides methods of treatment and prevention
by administration to a subject in need of such treatment of a
therapeutically or prophylactically effective amount of one or more
therapeutic polypeptides of the invention. The subject is
preferably an animal, including, but not limited to, animals such
as monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is
preferably a mammal, and most preferably human. In a specific
embodiment, the subject is a human not afflicted with a cancer
which secretes hCG or hCG fragments and, more particularly, not
afflicted with KS.
[0531] Various delivery systems are known and can be used to
administer therapeutic polypeptides of the invention. For example,
suitable systems include: encapsulation in liposomes,
microparticles and/or microcapsules; recombinant cells capable of
expressing the therapeutic polypeptide; receptor-mediated
endocytosis;.sup.158 plasmids encoding one or more therapeutic
polypeptides; viral vector delivery systems, etc. The therapeutic
polypeptides can be delivered in a vesicle, in particular a
liposome..sup.159
[0532] Methods of introduction include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and/oral routes. The compounds
may be administered by any convenient route, for example by
infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.) and may be administered together with other
biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compositions of the invention into the central
nervous system by any suitable route, including intraventricular
and intrathecal injection. An intraventricular catheter may be used
to facilitate intraventricular injection, for example, attached to
a reservoir, such as an Ommaya reservoir. Pulmonary administration
can also be employed, e.g., by use of an inhaler or nebulizer, and
formulation with an aerosolizing agent.
[0533] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment. For example, local administration may be
achieved by topical application, by injection, by means of a
catheter, by means of a suppository, or by means of an implant, the
implant being of a porous, non-porous, or gelatinous material,
including membranes, such as sialastic membranes, or fibers.
[0534] In yet another embodiment, the therapeutic polypeptide can
be delivered in a controlled release system. A pump may be used as
needed..sup.160 Polymeric materials may also be employed in a
controlled release system, according to methods known in the
art..sup.161 In yet another embodiment, a controlled release system
can be placed in proximity of the therapeutic target, thus
requiring only a fraction of the systemic dose..sup.162 Other
controlled release systems are discussed in the review by
Langer..sup.163
[0535] In a specific embodiment a nucleic acid encoding one or more
therapeutic polypeptides of the invention is administered by gene
therapy methods as described herein, or as otherwise known in the
art.
[0536] The pharmaceutical compositions comprise a therapeutically
effective amount of one or more therapeutic polypeptides of the
invention, and a pharmaceutically acceptable carrier.
[0537] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the therapeutic polypeptide is administered
to a subject. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of one or more therapeutic polypeptides of the invention,
preferably in purified form, together with a suitable amount of
carrier so as to provide the form for proper administration to the
subject. The formulation should suit the mode of administration. In
a preferred embodiment, the composition is formulated in accordance
with routine procedures as a pharmaceutical composition adapted for
intravenous administration to human beings. The compositions may
also be formulated for vetrinary use.
[0538] Examples of suitable pharmaceutical carriers include sterile
liquids, such as water and oils, including those of petroleum,
animal, vegetable or synthetic origin, such as peanut oil, soybean
oil, mineral oil, sesame oil and the like. Water is a preferred
carrier when the pharmaceutical composition is administered
intravenously. Saline solutions and aqueous dextrose and glycerol
solutions can also be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical excipients include
starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,
chalk, silica gel, sodium stearate, glycerol monostearate, talc,
sodium chloride, dried skim milk, glycerol, propylene glycol,
water, ethanol and the like.
[0539] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion,
tablets, pills, capsules, powders, sustained-release formulations
and the like.
[0540] The composition can be formulated as a suppository, with
traditional binders and carriers such as triglycerides.
[0541] Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate,
etc.
[0542] Typically, compositions for intravenous administration are
solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may also include a solubilizing agent and a local
anesthetic such as lignocaine to ease pain at the site of the
injection.
[0543] Generally, the ingredients are supplied either separately or
mixed together in unit dosage form, for example, as a dry
lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where the composition is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline.
Where the composition is administered by injection, an ampoule of
sterile water for injection or saline can be provided so that the
ingredients may be mixed prior to administration.
[0544] The therapeutic polypeptides of the invention can be
formulated as neutral or salt forms. Pharmaceutically acceptable
salts include those formed with free amino groups such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with free carboxyl groups such as
those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, etc.
[0545] The amount of the therapeutic polypeptide(s) and/or
functional quivalent(s) of the invention that will be effective in
the treatment of a particular disorder or condition depends on
various factors and can readily be determined by one of skill in
the art using standard clinical techniques with reference to the
instant disclosure. For example, dosage amounts will depend on the
nature of the disorder or condition. In vivo and/or in vitro assays
may optionally be employed to help predict optimal dosage ranges.
Effective doses may also be extrapolated from dose-response curves
derived from the in vitro and in vivo experiments described herein.
In general, the dose for administration to a human is 0.01 to 5.0
mg/24-48 hours, preferably 0.1-4 mg/24-48 hours, more preferably
0.25 to 2.5 mg/24-48 hours. In non-human animals preferred dosages
are 0.00014 to 0.071 mg/kg/24-48 hours, more preferably 0.0014 to
0.057 mg/kg/24-48 hours, most preferably 0.0036 to 0.036
mg/kg/24-48 hours. These ranges will vary depending on the route of
administration, the seriousness of the disease or disorder, the
size of the subject, and other factors known in the art.
[0546] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0547] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
9. EXAMPLES
[0548] Except as specifically described herein, methods used in the
ensuing examples have been previously published..sup.164
[0549] 9.1 Isolation and Characterization of the MA Peptides
[0550] The active factor was isolated and purified from three
sources with similar results: [0551] a 2 day freshly collected
sample of urine from women in early (first trimester) pregnancy
kept at 4.degree. without any additions, and used almost
immediately after collection; [0552] urine collections made over
several weeks from women in early pregnancy and stored at
-80.degree. with sodium azide; and [0553] commercial preparations
of hCG pre-tested for activity.
[0554] 9.1.1 Preparation of MA Peptides from Urine
[0555] Three different sources of the therapeutic polypeptides,
previously shown to contain all the biological activities
previously described in the filing, were used for the starting
materials: A. Commercial hCG preparations. B. 24 hr urine
collection from 2 different volunteers in the first trimester of
pregnancy [3.about.4 weeks] and processed after storage at
4.degree. C. without any preservative. C. Two-week urine collection
from two women in first their trimester of pregnancy stored at
-80.degree. C. with sodium azide[1 g/l] as
preservative..degree.
[0556] A). Commercials hCG preparations: APL (Wyeth Ayerst),
Pregnyl (Organon) and CG10 (Sigma) contain mixtures of peptides
(i.e. .alpha.+.beta. hCG, free a hCG, free .beta. hCG, .beta. core,
nicked .beta. hCG and unknown cleavage or degraded .beta. hCG
peptides). 10 vials of commercials grade hCG-CG10 [10.000
IU/vials.about.1 mg of total proteins], APL [20.000
IU/vials.about.2 mg of total proteins] or Pregnyl[10.000
IU/ml.about.1 mg of total proteins] were dissolved in PBS without
Ca2+ Mg2+ [10.000 IU/1 ml of PBS, pH 7.2] and were ultra-filtered
and concentrated using three steps of size exclusions: 30 kDa, 10
kDa and 3 kDa of Macrocep [Pall Gelman Laboratory, Ann Arbor,
Mich., USA]. Macrosep macro-concentrators provide rapid and
convenient concentration, purification and desalting of small
volume biological samples. A starting sample of 15 ml can be
concentrated to 0.5 ml within [centrifugation at 1000 to 5000 g,
typically for 30-90 minutes].
[0557] Retentate and filtrate at each step were tested for
biological activity (i.e. anti-HIV-1, anti-Tumor and
hematopoiesis). The material was then purified on a SUPERDEX.TM.
peptide column HR 10/30 column run on HPLC (size exclusion from
100-7000 Da, Pharmacia). Concentrate sample load volume was 0.5 ml.
Samples after HPLC were analyzed on SDS page gel [non-reducing
conditions] and also by an agarose gel technique..sup.165 The gels
showed major bands [silver staining] at approximately >3
[Pp1.about.MA] and >6 kDa [Pp2.about.pMA]. The gels were divided
into seven 0.5 cm sections, each section was then electro-eluted
(Hoefer GE 200 eluter, Pharmacia), using eluter buffer (BIO-RAD,
CA), and tested for biological activity after removal of SDS and
desalting. The eluted material was analyzed by MALDI-TOF mass
spectrometry or SELDI using the matrix: CHCA and SA. Sample was
also further purified by either DEAE or direct reverse phase [C18]
column chromatography. Purity was again confirmed by MALDI-TOF mass
spectrometry or SELDI. Purified samples were then sequenced
utilizing N-terminal sequencing by Edman degradation.
[0558] B) From 24 hr collections of urine from two different
volunteers in the early first trimester of pregnancy [.about.3-4
weeks] we proceeded within days after storage at 4.degree. C.
without any preservatives. Fifty ml/tube [1 L.about.20 tubes] of
urine were centrifuged at 1000 g for 30 min, the supernatant was
collected and the pellets were discarded. The pooled supernatants
were then filtered through a 0.45 micron membrane filter [D50200,
Nalgene, Rochester, N.Y.]. One L of urine was processed at a time
and 10 runs were pooled because of equipment limitations. 1 L of
fresh urine yielded about 10 ug of Maternin. The material was then
processed as described above starting with the 30 kDa, then the 10
kDa and finally the 3 kDa cutoff Pall Filtron Macrocep [PALL Gelman
Laboratory, Ann Arbor, Mich.]. C) Two-week collections of urine
from 2 different women in their first trimester of pregnancy were
stored at -80.degree. C. with sodium azide [1 g/l] as a
preservative. The frozen urine was thawed and the pH was adjusted
to 7.2-7.4 with sodium hydroxide and allowed to sediment for 1 hour
at room temperature. Approximately 75% of the supernatant was
decanted and the remaining supernatant was centrifuged to remove
sediment and added to the rest of the supernatant. The supernatant
was then filtered through a 0.45 filter [D50200, Nalgene,
Rochester, N.Y.]. From that point on the frozen urine was processed
as described above
[0559] FIG. 1, Panel A illustrates the peak fractions on
HPLC-superdex peptide column chromatography, which followed
stepwise size fractionation utilizing filters of varying size
porosity and was followed by either porous DEAE or reverse phase
(C18) column chromotography.
[0560] Active peaks were pooled for SDS PAGE analysis. The
activities on SDS PAGE (Panel B) migrated as two distinct bands,
the size of which are only crude estimates in view of their low
molecular weights, but in general they ran with markers of about
4-6 kDa and 3-4 kDa for the larger and smaller polypeptides
respectively. These samples were electroeluted and analyzed. In
other experiments, affinity columns of polyclonal antibodies to the
purified bioactive polypeptides were used.
[0561] After the urine samples were centrifuged at 1000 g for 30
min, the pellet was discarded, and the supernatant then filtered
through a 0.45 micron filter to further remove particulate. The hCG
preparations were simply dissolved in PBS. The purification scheme
employed a series of microcep size exclusion filters: 30 kDa, 10
kDa, and 3 kDa. The activities were recovered in the 30 and 10 kDa
filtrates but were retained with the 3 kDa. The active fractions
were lyophilized, dissolved in 2.5 ml of H.sub.2O, and 0.5 ml
aliquots were applied in series five times to an HPLC-superdex
(HR10/30) peptide column (range 100-7000 Da) with PBS. The peak
activities (FIG. 1, Panel A) were pooled, lyophilized, and the
samples dissolved in H.sub.2O. One microgram was then applied to
each lane of SDS polyacrylamide or agarose slab gels for
electrophoresis. The gels showed major bands (silver staining) at
approximately 4-6 kDa and 3-4 kDa (FIG. 1, Panel B). The gels were
divided into seven 0.5 cm sections, each section electroeluted with
a Hoefer GE 200 eluter (Pharmacia) using eluter buffer (Bio Rad,
Ca.), and assayed after removal of the SDS with SDS removal kit
(Pierce Co., Ill.) and removal of salt (Bio Rad, desalting kit).
The activities migrated precisely with these bands.
[0562] For generating larger amounts of MA, 20 or more lanes were
used and the area corresponding to known size of MA were
electroeluted and pooled. One microgram of material from each band
region was inoculated i.m. into male NZW rabbits every 2 days for
10 days with Freund's adjuvant. After 10 weeks sera was collected
and tested by immunoblotting.
[0563] The polyclonal antibodies so generated reacted with
homologous antigen and gave reactivity with crude hCG preparations
known to contain MA activity and with sera from early pregnancy but
had no cross reactivity with pure hCG, .beta.-hCG, .alpha.-hCG,
native glycosylated (S-core (FIG. 2). There was little cross
reactivity by Western blot between MA and pMA, but cross
reactivities were found on ELISA (not shown).
[0564] These polyclonal antibodies were then used in an affinity
column as part of a larger scale purification scheme. In addition,
neither the 6 kD nor the 3-4 kDa polypeptide had reactivity with
various monoclonal antibodies to .beta.-hCG, (B108) or with three
monoclonal antibodies (B-201, B-204, and B-210) to the native
glycosylated .beta.-core (FIG. 2) (the .beta.-core antibodies were
generously supplied by Dr. S. Birken, Columbia University).
Purification form commercial hCG preparations followed similar
procedures.
[0565] MoAbs were generated by injecting 6 week-old BALB/C with 300
ug of MA or pMA in Freund's adjuvant by the I.P. route. Repeated
immunizations were done every few weeks for 6 months. At peak Ab
titer (measured by ELISA), spleens were removed and splenocytes
fused with NS1 plasmacytoma cells (6). Hybrid clones were selected
for specificity of reactions in limiting dilution cultures,
injected I.P. into nude mice and the ascites fluid collected after
3 weeks. The MoAbs were then purified by column chromatography
(ref). Fractions containing the activity and single H and L chains,
as shown by Comassie blue staining of SAS-AGE, were pooled.
Isotyping was with isostrips (mouse isotyping kit, Roche).
[0566] Assays were performed with 1 ug of Ab and 10 ug of antigen
electrophorese on a 10-20% acrylamide denaturing gel by
electroblotting onto a nitrocellulose membrane (Bio-Rad). Blots
were blocked with nonfat dry milk on TBS-tween 20 for 1 hr at room
temperature, incubated with the indicated MoAb (5 to 10 ug) for 3
hrs, washed extensively with 1% TBS, and then incubated with a
1:1000 dilution of horseradish peroxidase-conjugated secondary
antibody (Sigma) for 1 hr. Blots were washed with TBS and developed
using the enhanced chemiluminesence method (Amersham) or by
immunoblot kits (Bio-Rad). In addition polyclonal antibodies
(Po-Ab) were also prepared. One ug of purified MA or pMA with 0.5
ml Freund's complete adjuvant were injected i.p. into 4 kg New
Zealand male rabbits, and the sera were collected 21/2 months
later.
[0567] MA peptides were tested for their sensitivity to proteolytic
enzymes as well as for thermosensitivity. Peptides were incubated
with pronase, trypsin or chymotrypsin in PBS for 1 h at 37.degree.
C. The active material was protease-sensitive and lipase-(Sigma
kit) and glycosidase-(Bio Rad kit) resistant. Proteolytic enzyme
treatment eliminated the inhibitory effect of MA peptides in both
the KS and human carcinoma (prostate and breast) clonogenic assays
as well as in the HIV-1 replication assays as measured by HIV p24
ELISA. The purified material resisted boiling for 3 min but was
destroyed after boiling 1 hr. Purity was estimated by the above gel
procedures (Panel B), mass spectroscopy using MALDI-TOF and a CHCA
matrix (Panel C), and N-terminal sequence analysis by Edman
degradation (Panel A insert), and the sequence identity was
confirmed by peptide synthesis (Panel D) with demonstrable
reproducibility of bioactivities.
[0568] These analyses revealed that the material which migrated at
3-4 kDa was a polypeptide of size varying from 30 to 35 AA. The
molecular masses correspond to 3.33 kDa to 3.8 kDa. The variation
in size appears to be the result of variable C-terminal cleavage.
However, the most frequently isolated polypeptide with these
activities was the 35-mer identical to .beta.-hCG AA 55-89, and
smaller forms were identical but with varying degrees of C-terminal
truncation. We named these polypeptides MA. The polypeptide of
about 4-6 kDa contained the MA sequences but linked to other yet to
be defined sequences. We tentatively refer to the larger
polypeptide as pro-MA (pMA). The conclusion that MA is the active
material was proven by demonstrating all of the biological
activities with synthetic versions of MA as well as with more
truncated forms. We note that MA activity is susceptible to
4.degree. C. storage in water and to freeze thawing. Reproducible
results were best obtained when the peptides were used soon after
dissolving in aqueous solutions.
[0569] After isolation and confirmation of activity the material
was sent to two independent laboratories for sequencing. N-terminal
protein sequence analysis was performed by automated Edman
degradation as well as on a combination of mass spectroscopy and
Edman degradation.
[0570] 9.1.2 Synthetic Production of MA peptides
[0571] MA (SEQ ID NO: 2) and derivatives of MA were produced
synthetically by solid-phase synthesis using an automated
synthesizer employing Fmoc and, t-Butyl, on a Wang resin.
[0572] N.alpha.-Fmoc-protected amino acids (10 equivalents) were
added sequentially using HTBU
(0-Benzotriazole-N,N,N',N'-tetramethyl-uronium hexafluorophosphate)
and N-methylmorpholine as coupling reagents. Side chain protected
N.alpha.-Fmoc amino acids were purchased from AnaSpec. The side
chain is Trityl for Cys and Gln, t-Butyl for Asp, Thr, Ser and Pbf
(pentamethyldihydrobenzofuran-5-sulfonyl). Peptides were
deprotected and cleaved from the resin using reagent B for two
hours. Peptides were purified to >95% by reverse-phase HPLC.
[0573] Isolated sequenced MA (SEQ ID NO: 2) is 35 AA residues in
length. The inventors have produced synthetic peptides that exhibit
the activities of MA (SEQ ID NO: 2). One active peptide, MA.sub.S1
(SEQ ID NO: 4) is only 15 AA acid residues in length. Another,
MA.sub.S2, (SEQ ID NO: 5) is 30 AA residues in length.
[0574] The following designations will be used for the polypeptides
(experimental and various controls) used in the work described
herein: [0575] pMA (SEQ ID NO: 3): natural peptide 38 amino acids
in length; [0576] MA (SEQ ID NO: 2): natural peptide 35 amino acids
in length; [0577] MA.sub.S1 (SEQ ID NO: 4): MA synthetic peptide 15
amino acids in length; [0578] MA.sub.S2 (SEQ ID NO: 5): MA
synthetic peptide 30 amino acids in length; [0579] MA.sub.S3 (SEQ
ID NO: 6): MA synthetic peptide 35 amino acids in length (same
sequence as MA, but MA.sub.S3 is the synthetic version as opposed
to MA, which is the isolated version); [0580] CP: control peptide
32 amino acids in length, consisting of AA 21-52 of .beta.-hCG (SEQ
ID NO: 1); [0581] CR 127: highly purified native hCG; and [0582]
beta core: purified hCG beta core (a well known and substantiated
degradation product of the beta chain of hCG, consisting of an
N-terminal polypeptide of the beta chain: AA 6-42 joined by a
disulfide bridge to a C-terminal polypeptide formed also from
.beta.-hCG (SEQ ID NO: 1): AA 55-92 with AA 43-54 having been
removed).
[0583] 9.2 Anti-Cancer Effects of MA
[0584] The anti-tumor activities of MA were assessed in several in
vitro and in vivo systems using published methods..sup.166 In vitro
studies were with a wide variety of human tumor cell lines as well
as short-term cultures of cells from biopsy samples obtained from
tumors. The assays included effects of MA on colony formation, cell
number and viability (trypan blue), and apoptosis. Four measures of
microscopy were used. In vivo studies included the effects of MA on
tumors induced in immunodeficient mice by xenotransplanted various
human neoplastic cells, spontaneous neoplasms of immuno-competent
mice and rats, and a few standard tumors in immuno-competent mice.
Representative examples are presented.
[0585] 9.2.1 MA Selectively Kills Human Tumor Cells In Vitro
[0586] Since the crude active hCG preparations.sup.167 and
partially purified fractions.sup.168 were previously shown to
inhibit growth of KS tumor cells, the effects of native, synthetic
full-length, and various truncated forms of MA were all first
tested on KS cells. The procedures used were as previously
described..sup.169 A dose-dependent inhibition of colony formation
was observed. At concentrations of approximately 30 nM there was
60-80% inhibition (FIG. 3, Panel A) and evidence (DNA laddering and
Annexin V staining) of marked apoptosis (FIG. 3, Panel B).
[0587] For colony formation assays (Panel A): Cells
(5.times.10.sup.5/ml) were seeded in modified Eagle's medium
supplemented with 10% FCS, 10% conditioned medium form PHA
stimulated human lymphocytes, 2 mM glutamine in 0.8% V/V
methylcellulose with or without various treatments in a final
volume of 1.0 ml. Then 0.1 ml aliquots were seeded in 96 well flat
bottom microtest plates and incubated at 37.degree. C. in 5%
CO.sub.2 in air for 7 days. Aggregates containing 50 cells or more
were scored as colonies. The results show a representative
experiment and are mean of triplicate values.
[0588] For Annexin V assays (Panel B): 5.times.10.sup.5 cells were
seeded in triplicate on 24 gelatin coated wells in conditions as
described for Panel A and treated with the indicated test
materials. After 3 days the cells were dislodged from their
monolayers by gentle pipetting with cell dissociation buffer (Life
Technologies), counted after trypan blue staining and aliquots
examined for Annexin V staining by indirect IFA (ref).
[0589] Studies of the anti-tumor cell effects of MA were extended
to other carcinomas, melanoma, some hematopoietic tumors, and
carcinomas of the colon, prostate, breast, lung, brain, pancreas,
and kidney-using cell lines from these tumors. In all cases a dose
dependent inhibition of growth (FIG. 3, Panels A-D) and induction
of apoptosis (FIG. 3, Panels E-H) was again obtained, illustrated
here only for studies with carcinomas of the prostate and breast.
Less than 30 nM native and synthetic MA, including the truncated
forms, produced near maximum effects. Many more types of tumor
cells were studied utilizing other assays (see below).
[0590] More extensive studies of the effects of MA and with
truncated synthetic forms of MA (MA.sub.S) on a wide range of other
human tumor cells were carried out using three-dimensional confocal
microscopy. Cells were cultured and then seeded onto gelatinized
glass chamber slides and incubated 24 hrs with the various test
materials. The slides were then fixed with formalin and treated
serially with Triton X-100 (0.01%) for 10 min at 25.degree., 0.4
ug/ml fluorescein isothiocyanate (FITC)-labeled Phalloidin (Sigma
Chem. Co.) at room temperature for 30 min to bind actin, washed
with PBS and the DNA stained with Propidium iodide (PI) (0.5 ugml)
(Sigma Chemical Co.), for 15 min. The stained slides were washed
and mounted on slow fade (Molecular Probes, Inc., Eugene, Oreg.)
and examined through a dual emission filter XF53 (Omega, Inc.,
Brattlesboro, Va.) by fluorescent microscopy. In some experiments
only DNA staining was performed. In these instances Triton X-100
was not used. The slides were stained with PI and examined for cell
morphology and chromatin degradation under 3D confocal
microscopy.
[0591] These results provided morphological conformation of the
induction of apoptosis of almost all tested human tumor cells by
MA. Prominent nuclear pyknotic changes were observed with all tumor
cells (FIG. 4, Panels B through K; Panel A shows normal cells).
Untreated cells (first line) show normal morphology. The confocal
microscopy results also verified the specificity of MA in killing
tumor cells. Concentrations of MA (2000 ng/ml which are 10.times.
higher than needed for inducing apoptosis of tumor cells) had no
effect on the morphology of primary human fibroblasts, cord blood
derived CD34+ cells, PBMC derived CD4+ T cells, or macrophages
(FIG. 4, Panel A).
[0592] To be certain that this specificity for tumor cells was not
simply the consequences of a difference between cycling and
non-cycling cells, the following cell-types were also used:
proliferating human normal fibroblasts, endothelial cells (HUVEC),
and PHA-stimulated lymphocytes. Again, no inhibition of growth or
evidence of apoptosis was observed. In fact, MA actually promoted
growth of some of these cells.
[0593] Moreover, 200 ng/ml of MA, MA.sub.S1 (a synthetic 15-mer
lacking the first 7 AA and last 13AA of MA), and MA.sub.S2 (a
synthetic 30-mer lacking the first 3AA and last 2AA of MA) induced
apoptosis of: [0594] KS Y-1 cells: provided by NIH (Panel B);
[0595] other sarcoma cell lines: fibrosarcoma 4TB 166, and Ewing's
sarcoma from ATCC (not shown); [0596] two prostate carcinoma cell
lines: PC3 and PC4 (Panels C and F); [0597] gliobastoma: HTB16/ATCC
(Panel D); [0598] carcinoma of the breast: HTB124/ATCC (Panel E);
[0599] carcinoma of the pancreas: CRL 1682/ATCC (Panel G); [0600]
carcinoma of the colon: CRL 1682 (not shown); [0601] carcinoma of
the lung: HTB184/ATCC (Panel H); and [0602] carcinoma of the
kidney: HTB46/ATCC (Panel I); [0603] melanoma: HTB67/ATCC (not
shown); and [0604] some leukemia and lymphoma cell lines:
HUT78/ATCC, DAVDI, HG,
[0605] SULTAN, T-cell lymphoma (not shown).
[0606] 9.2.2 MA Inhibits Growth and Induced Apoptosis in Primary
Human Cancer Cells
[0607] Because cell lines might be more susceptible to growth
inhibition and apoptosis, the effects of MA on short-term cultured
human tumor cells were investigated. The cells were obtained from
biopsied specimens and immediately cultured (primary cultures). The
results were similar to the results with tumor cell lines. MA
inhibited growth and induced apoptosis at similar concentrations to
those concentrations that inhibited growth and induced apoptosis of
neoplastic cell lines. FIG. 3, Panels D and H are representative
results with primary breast carcinoma cells showing inhibited
growth and induced apoptosis respectively. These pro-apoptotic
effects of MA on human tumor cells were with 10% serum rather than
the lower concentrations sometimes used to make cells more
susceptible to apoptosis.
[0608] More extensive studies of primary human tumor cells were
carried out utilizing confocal microscopy. Primary neoplastic cells
from metastatic carcinomas of the breast, prostate, lung (small
cell carcinoma), colon, and kidney were examined to determine if
these results were limited to neoplastic cell lines or whether MA
would induce morphological changes reflecting apoptosis in primary
human tumor cells.
[0609] With the exception of the kidney carcinoma, which was a
biopsy sample direct from the tumor, the samples were from pleural
effusions which developed after metastasis to the lung. The breast,
prostate, and colon carcinomas were freshly obtained and
immediately cultured, whereas the lung and kidney carcinomas went
through 22 and 18 passes respectively before they were used in
these studies. All cells were examined after 3 days culture in the
presence or absence of MA (200 ng/ml) or other treatments. Rapid
induction of apoptosis was observed with all the primary
carcinomas. FIG. 4, Panels J and K show representative examples
using prostate (PCANJ) and breast (BCAJR) carcinomas. pMA was also
effective in the same molar concentration range as MA (see Panels F
and I for examples).
[0610] Polypeptides with MA activity were also isolated from sera
and urine of mice and rats in early pregnancy. Though these
polypeptides have not yet been sequenced, they exhibit the full
range of activity as human MA. An illustrative example is shown
with rat MA (MA R) in the induction of apoptosis of the primary
human prostate (PCANJ) and breast carcinoma (BCAJR) cells (FIG. 4,
Panels J and K, last row). Taxol (5 ug/ml) was used as a positive
control (see Panels J and K, fourth row). A large number of other
control polypeptides were negative in these and all other assays.
Most importantly, no inhibition of growth or induction of apoptosis
was observed with equimolar or 5 to 10.times. greater
concentrations of purified native hCG (CR127), recombinant hCG,
r.beta.-hCG, .alpha.-hCG, or the native glycosylated .beta.-core
with any of the neoplastic cells. Other hCG synthetic peptides also
tested negative. These included: .beta.-hCG 21-52 (FIGS. 3 and 4),
6-39; and 9-119.
[0611] 9.2.3 MA Kills Human Tumor Cells In vivo
[0612] Both native and synthetic MA also inhibited growth (between
60% and 100%) and promoted apoptosis of tumors in vivo. In the
first set of experiments, MA was tested against tumors formed by
xenotransplantation of various human carcinoma and sarcoma cells in
immunodeficient mice. After allowing the tumors to develop for 7 to
10 days, treatment was initiated by I.P. injection with 200 ng
(about 60-100 pmoles)/day for seven days. FIG. 5, Panel A
illustrates a typical result with prostate carcinoma (PC3), and
Panel B shows results for KC cells (KS Y-1). The inhibitory effects
were dose-dependent and found with both native and synthetic MA and
pMA. At the dose range of 60-100 pmoles there was over 70%
inhibition of tumor size. Increasing the concentration to 300-500
ng/day (about 100-200 pmoles) and treating for 2 to 3 weeks lead to
complete tumor regression. Three different synthetic forms of MA
were used: full length (MA.sub.S3) and the previously described
MA.sub.S1 and MA.sub.S2 (Panel C). With equivalent and higher molar
concentrations, there was no effect of pure hCG (CR127) (150
pmoles), pure .beta.-hCG (rhCG) (140 pmoles), ahCG (200 pmoles), or
pure native glycosylated .beta.-core (150 to 400 pmoles) (FIG. 5,
Panel C), nor with a wide variety of control peptides derived from
.beta.- and .alpha.-hCG chains. However, inhibition was obtained
with some lots of crude preparations of hCG (APL, Wyeth-Ayrest) and
.beta.-hCG (CG10, Sigma) (FIG. 5, Panel C), as we have previously
reported,.sup.170 which is expected, since MA can be isolated from
these commercial preparations. The anti-tumor effects were observed
when therapy was initiated 1 week after the start of tumor
formation when tumor size had reached approximately 0.2.times.0 2
mm (about 30-50% of their maximum size in untreated animals).
Similar to the in vitro studies, the mechanism of tumor inhibition
was by rapid induction of apoptosis (FIG. 5, Panels A and B) as
determined by APO-TAG staining.
[0613] It is possible that an anti-angiogenic effect of MA also
contributes to tumor inhibition, since light microscopic
examination of the thin sections of the tumors revealed reduction
in blood vessel formation. However, whether this effect is direct,
and in turn facilitates tumor destruction by diminished blood
supply or is a consequence of primary tumor cell killing is not
settled because a rapid induction of tumor cell apoptosis may
prevent release of angiogenic factors such as VEGF.
[0614] Thus, all the anti-tumor activity may rest on the capacity
of MA to directly induce apoptosis of the tumor cells as observed
in vitro. In either event, MA has potent, broad, and specific
killing effects on human cancer cells in vitro and in vivo with
experimental models encompassing both mesenchymal derived tumors
(the sarcomas and some hematopoietic tumors) and epithelial
carcinomas (prostate, breast, lung, pancreas, colon, brain, and
renal), producing no evident cytotoxic effects on normal human
cells, even at higher concentrations. However, MA did not inhibit
growth and kill all kinds of tumor cells. For example, it had no
effect on HTLV-I induced adult T-cell leukemia nor some .beta.-cell
lymphomas.
[0615] 9.2.4 MA Inhibits Mouse and Rat Spontaneous
Tumorigenesis
[0616] As shown, MA killed a wide variety of human tumor cells in
vitro and in vivo killed xenotransplanted human tumors in immune
deficient mice. We wanted to be certain that the in vivo results
would not be limited to immune deficient mice. We were also curious
to see whether MA effected rodent tumors because we had evidence
that indicated that MA would cross species barriers in that human
MA could affect mouse cells and rodent MA (MA R) could affect
primate and rodent cells. Therefore, we tested the effects of MA
on: [0617] spontaneous fibrosarcomas which developed in three older
FVB/N mice; and [0618] spontaneous breast carcinomas which
developed in three older Sprague-Dawley rats.
[0619] The tumors were recognized and treated after different
stages of development. Samples of the tumor cells were grown in
short-term culture and when treated with MA underwent rapid
apoptosis. Treatment of the mouse fibrosarcomas with MA for 2 weeks
led to complete abolishment of the tumor in two of three animals
(tumor size less than 0.5 cm in diameter) (FIG. 6, Panel A).
Partial regression was observed in one animal first treated with MA
when the sarcoma progressed to about 1 cm. However, no further
growth was noted in the subsequent 8 weeks of the study.
[0620] Similar results were obtained with the spontaneous mammary
carcinomas of rats. Treatment of one rat with a tumor of
approximately 3.times.3 cm led to complete tumor regression (FIG.
6, Panel B). Two other rats with breast carcinomas of about
4.times.5 cm showed partial tumor regression after 2 weeks of
therapy with MA and no further growth during the subsequent 8 weeks
of the study.
[0621] 9.2.5 MA Inhibits Tumorigenesis in Standard Mouse Models
(B16 Melanoma and Lewis Lung Carcinoma).
[0622] B16 melanoma and Lewis lung (LL) carcinoma cells were first
shown to be susceptible to the pro-apoptotic effects of MA (FIG. 6,
Panels C and D) at the same dose range used with cultured human
tumor cells. The tumor cells grown in standard culture conditions
were used to generate metastatic cancers in pathogen-free 6-8 week
old C57/BL mice (Charles River). In one set of experiments, the
animals were treated with MA.sub.S1 or MA.sub.S2 at two doses, 0.1
ug or 1.0 ug either in a pre-treatment protocol (two subcutaneous
inoculations 24 and 48 hrs before transplanting the tumor cells).
In a second set of experiments after establishment of widespread
metastasis (day 7 and day 14), the animals (5 per group) were
treated daily for 14 days with the same low (0.1 ug) and high (1.0
ug) dose of MA.sub.S1 or MA.sub.S2. The results demonstrate a
potent pro-apoptotic effect of MA on neoplastic cells in vivo.
[0623] 9.3 Anti-HIV Effects of MA
[0624] The following sections describe empirical work demonstrating
the anti-HIV effects of MA peptides.
[0625] 9.3.1 MA Suppresses HIV-1 and SIV In Vitro
[0626] The effect of native and synthetic MA on HIV-1 infection in
vitro was evaluated using PHA and II-2 stimulated primary
peripheral blood cells (PBMC), CD4+ T-cells enriched fractions from
PBMC, and CD14+ macrophage enriched fractions. Infection was with
HIV-1 IIIB (PBMC and CD4+ T-cells), HIV-1 Ba-L (macrophages), and
two primary HIV-1 isolates not passaged in any cell line (PBMC). As
illustrated in FIG. 7, a dose dependent inhibition of virus (IIIB
and Ba-L) production was observed as determined by p24 levels in
the extracellular fluid. Virus production was inhibited 50% with
both native and synthetic MA at about 30 nM. Similar inhibition
curves were obtained with the primary HIV-1 isolates. No inhibition
of HIV replication occurred with pure native hCG (CR127), .beta.
hCG, native glycosylated .beta.-core or with .beta.-hCG synthetic
peptide AA 21-52 (FIG. 7) nor with scrambled MA peptides at
equimolar or higher concentrations. It is important to emphasize
that accurate in vitro assays of the effects of MA on chronic HIV
production in cell lines such as 119 or CEM are not possible
because of the cytotoxic effect of MA on these tumor cell lines. MA
also inhibited SIV.sub.MAC 251 infection of rhesus macaque
PBMCs.
[0627] Virus (MOI of 0.001) was added to 10.sup.6 cells in
RPMI-1640 with 10% FCS. One hour later the various polypeptides
were added once. The cells cultured for 3 days with 5 ug PHA and 20
i.m. of Il-2/ml at 37.degree., after which virus was estimated by
determining p24 (HIV-1) or p27 (SIV) (Organon-Teknika) Importantly,
no toxic side effects of MA occurred on the normal cells as
determined by trypan blue, MTT, and 3TdR incorporation assays. The
results are expressed as a mean of triplicate samples with a less
than 20% variation in the range of these values.
[0628] 9.3.2 MA Suppresses HIV-1 In vivo in HIV-1 Transgenic
Mice
[0629] In vivo effects of MA were also examined in: [0630] HIV-1
transgenic mice; [0631] a newly developed HIV-1 transgenic rat
model; and [0632] SIV.sub.MAC 251 infected rhesus macaques.
[0633] Methods used were previously described by Kestler et al. and
Letvin et al..sup.171
[0634] The transgenic mice contain 5.1 kb of the HIV-1 proviral
genome, lacking gag and pol genes and 2.3 kb of vector..sup.172 The
newly developed transgenic rat contains the same HIV-1 sequences
and vector. In both birth weights and features, the homozygous
animals are born with normal weight and gross appearance, but in
untreated animals shortly after birth, high levels of env and
regulatory genes are expressed promptly correlating with failure to
develop, cachexia, hyperkeratosis, immune abnormalities, kidney and
CNS lesions, and early mortality (death of >90% by 10 days of
age).
[0635] Twenty-four hours after birth the mothers were either
inoculated subcutaneously daily for 10 days or received an
implanted osmotic pump delivering 30-150 pmoles of either native or
synthetic MA or pMA. Various control polypeptides were used at
equimolar or vast molar excess. The newborn pups were then allowed
to feed from the foster mother breast milk over the entire period.
Since the animals are at risk from needle inoculation,
breastfeeding was the chosen route for MA administration.
[0636] Animals feeding from mothers inoculated with 30-150 pmoles
of MA, MA.sub.S or pMA survived (FIG. 8, Panels A and C),
quadrupled their weight over the ten day period (Panel A, and left
of Panel C), and when sacrificed for post-mortem examination their
tissues showed marked inhibition of viral expression.sup.173 (FIG.
8, Panel C, right side).
[0637] In these experiments total RNA was extracted from samples of
skin RNA.sub.206 10 days after birth of MA treated and untreated
mice. One ug of RNA was reverse transcribed into cDNA using random
hexamer primer and MMTV reverse transcriptase (Life Technologies,
Inc.) in a final vol. Of 30 ul. Three ul of each reaction was used
for PCR amplification of the HIV-1 gene sequences (env, tat, nef,
and rev) as described (ref), and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was amplified from each sample for
normalization. After 25 cycles, 10% of the products were resolved
by electrophoresis on 2% agarose gels, transferred to
nitrocellulose membranes by Southern blotting, and hybridized to
fluorescein isthiocyanate-labeled oligonucleotide probes
complementary to internal sequences of the amplicons (ref). Bound
probes were detected by chemiluminescence (Amersham) and relative
mRNA levels determined by densitrometry after normalization with
GAPDH mRNA levels.
[0638] The marked decrease in detectable HIV-1 RNA transcripts was
confirmed for HIV-1 proteins gp120 and Nef by in situ
histochemistry.
[0639] Similarly, treatment with 300 IU of crude preparations of
hCG (APL or CG-10), both of which also contain MA as an impurity,
were also protective. In contrast, mothers treated with an
irrelevant .beta.-hCG peptide (AA 21-52) or with purified native
(CR127) rhCG, pure .beta.-hCG, native glycosylated .beta.-core, or
control peptides (AA 21-52) had pups with full disease in that 10
of 10 mice were dead by day 10 (Panel B), and their tissues showed
a high expression of HIV-1 env and regulatory genes like untreated
controls. Animals reared from mothers receiving the low dose (30
pmoles) of MA had greater survival than control or untreated
animals, but the results were not as impressive in that about 50%
eventually died (after 2 mos). An extremely low amount of MA,
approximating only 100 pmoles, as an intact polypeptide or a
biologically active degradation product, is able to enter lactating
ducts of the mother, and cross the mucosal surface of the
gastrointestinal tract of the newborn pup, sufficiently distribute
throughout body tissues, and maintain adequate biological activity
to block HIV-1 gene expression and restore normal development of
the mice. An alternative possibility is that MA is able to induce a
factor in the mother mouse which accomplishes this feat.
[0640] In these experiments total RNA was extracted from samples of
skin RNA.sub.206 10 days after birth of MA treated and untreated
mice. One ug of RNA was reverse transcribed into cDNA using random
hexamer primer and MMTV reverse transcriptase (Life Technologies,
Inc.) in a final vol. Of 30 ul. Three ul of each reaction was used
for PCR amplification of the HIV-1 gene sequences (env, tat, nef,
and rev) as described (ref), and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was amplified from each sample for
normalization. After 25 cycles, 10% of the products were resolved
by electrophoresis on 2% agarose gels, transferred to
nitrocellulose membranes by Southern blotting, and hybridized to
fluorescein isthiocyanate-labeled oligonucleotide probes
complementary to internal sequences of the amplicons (ref). Bound
probes were detected by chemiluminescence (Amersham) and relative
mRNA levels determined by densitrometry after normalization with
GAPDH mRNA levels.
[0641] These results also indicate that the in vivo anti-HIV
effects involve mechanisms in addition to that involved in the
inhibition of HIV-1 infection in vitro, since the concentrations
required in vivo are less than those required in vitro in these
models. It is of interest that LH had a slight effect (FIG. 8). We
speculate that analogous to some hCG commercial preparations, the
activity of LH is due to a polypeptide present in preparations as
an impurity. It is likely that this hypothetical polypeptide will
be homologous to MA and accounts for this activity in sera and
urine of rodents in early pregnancy since rodents do not have CG
genes.
[0642] 9.3.3 MA Suppresses HIV-1 In vivo in HIV-1 Transgenic
Rats
[0643] Because of the extraordinary effects of MA in the mouse
modes, we were concerned that the results might be peculiar to the
model. Consequently, an HIV-1 transgenic rat model was developed.
An additional advantage of the rat model is the larger size of the
animals, enabling direct inoculation of MA into the developing pups
rather than therapy being indirect (the lactating mother) and
limited to the lactation phase. Thus, the transgenic rats can be
followed and treated much longer.
[0644] Like the transgenic mice, the transgenic rats contain HIV-1
provirus PNNL 4-3, again deleted in gag/pol, and also develop
hyperkeratosis. Additionally, they develop tissue fibrosis,
inflammatory changes, and some histopathology more similar to human
AIDS than in the mouse model, but unlike the HIV-1 transgenic mice
the effect is usually not lethal. The changes correlate with
expression of HIV-1 proteins. The transgenic rats are more fully
described in copending U.S. patent application Ser. No. 09/058,113,
by Bryant et al., filed
[0645] Apr. 9, 1998, entitled "HIV Transgenic Animals and Uses
Therefor," the entire disclosure of which is incorporated herein by
reference.
[0646] Inoculation (subcutaneous) of a 250-300 gm mother with 300
pmoles of MA for 10 days began after birth of the pups. As in the
mouse model, pups were breastfed during this period, but their
larger size and non-lethality of the provirus for these animals
allowed longer term direct subcutaneous inoculation with MA. The
MA-treated rats survived, developed normally, and had markedly
reduced transgene expression.
[0647] It is obvious that these HIV-1 transgenic animals do not
reflect an HIV natural infection in several major respects, such as
the lack of inclusion of any step in the infectious process prior
to provirus integration and the lack of specific human cellular
factors involved such as, for example, in the intranuclear
transcriptional events involving Tat. However, this model was
important for the following reasons: (1) though not all-inclusive
in providing targets for inhibition of HIV, nonetheless, the HIV-1
transgenics do contain the HIV-1 LTR elements, several of the HIV
protein biosynthetic events, and the proteolytic processing and
viral maturation steps in the HIV replication cycle, one or more of
which may have been targeted by MA; (2) independent from any
discussion of the utility of these models for exploring a new HIV
inhibitor, the transgenic studies clearly demonstrate that MA is
able to have profound in vivo effects at very low concentrations;
(3) the results show that MA is either able to survive harsh
conditions or induce other factors which achieve the biological
effects; and (4) the HIV-1 transgenic mouse assay proved to be the
most sensitive and accurate method for following the purification
of MA.
[0648] 9.3.4 SIV Infected Monkeys
[0649] Certain strains of SIV produce an AIDS-like illness in
susceptible monkeys. Among them SIV.sub.MAC 251 induces disease in
rhesus monkeys similar to AIDS in humans only with much greater
rapidity..sup.174 Therapy with purified MA of 3 monkeys with
end-stage disease infected 13-14 mos earlier was initiated when the
animals were losing weight, highly viremic (plasma virus 0.5 to
3.times.10.sup.6 copies of SIV RNA/ml by NASBA), and developing
pancytopenia. Methods used were as previously described by Kestler
et al. and Letvin et al..sup.175 Treatment of these monkeys with
end-stage AIDS with MA at 0.2 mg/kg dose 3 times weekly produced no
significant change in SIV titer over 6 mos. observation period.
However, in this period none of the 3 animals died. In contrast,
most untreated animals died by this period. Though the limitation
of animal number precludes strong conclusions related to survival.
The beneficial effect of MA in these end-stage animals was
indicated by the stabilization and in some instances even increase
in weight and T-cell number.
[0650] Far more impressive were experiments performed soon after
infection (3 weeks). In these experiments, crude urinary
preparations containing MA, but not crude fractions lacking MA,
were inoculated subcutaneously. Using a 10.sup.4.5 TCID.sub.50 of
cell free SIV.sub.mac 251, the characteristic rise in SIV p27,
reduction of CD4+ T-cells, and weight loss, which occurred in the
untreated animals, was prevented in MA treated animals. The
untreated animals died before 6 mos. The treated animals were
maintained for 7 mos. without weight loss, with normal CD4+ T-cell
counts, and barely detectable plasma p27 (less than 5 ng/ml in
contrast to over 200 ng/ml in control animals). However, stopping
therapy led to rapid onset of virus production and development of
AIDS-like disease.
[0651] 9.4 Pro-Hematopoietic Effects of MA
[0652] Preliminary experiments in monkeys treated with MA showed an
increase in peripheral blood T-cells..sup.176 Though no other blood
cells were measured in those experiments we speculated that MA
might have a pro-hematopoietic effect which could be a factor on
the very low concentrations required for some of its in vivo
effects, particularly the effects on HIV-1 transgenic rodents.
Consequently, we tested the effects of MA on hematopoiesis using
methods described by Lunardi-Iskandar et al..sup.177 The assays
first included in vitro effects of MA on various blood cell
precursors. Three types of in vivo tests were also carried out. The
in vivo tests were based on the effects of MA to rescue: [0653]
lethally irradiated rats and mice; [0654] rats treated with lethal
doses of a cancer chemotherapeutic agent (taxol) which suppresses
the bone marrow; and [0655] acutely bled but otherwise normal rats
and monkeys.
[0656] 9.4.1 MA Promotes Multi-Lineage Hematopoiesis In Vitro
[0657] Hematopoietic colony formation assays with bone marrow or
cord blood of normal human volunteers and bone marrow of monkeys,
rats, and mice were utilized. The assays are conventional
measurements of the number of progenitors for erythrocytes,
granulocytes, macrophages, platelets, and T-cells. The culture
conditions included growth factors that would give near optimum
growth for the particular lineage. These factors included II-3,
PHA-lymphocyte conditioned medium, erythropoietin (EPO), II-6, and
GM-CSF. The number of colonies developing in methylcellulose
reflects the number of progenitor cells, and the percent increase
is a measure of the effect of different polypeptides to increase
colony number above the maximum obtained with the indicated known
growth factors.
[0658] An illustrative example of a human bone marrow derived
culture is shown in FIG. 9. There was no significant effect above
background with equimolar or higher concentrations of r.beta.-hCG,
native glycosylated .beta.-core, and control peptides or pure hCG.
However, concentrations ranging from 10-500 ng/ml (approximately
3-150 nM) of both native MA and MA.sub.S1, MA.sub.S2, and MA.sub.S3
promoted an increase in colony number in a dose-dependent manner
reaching a maximum of 60-80% increase at 30-150 nM (FIG. 9) for all
hematopoietic progenitors. We observed similar results when the
source of hematopoietic precursors were: human cord blood, rhesus
macaque bone marrow, and rat and mouse bone marrow. Thus, like the
anti-tumor and anti-viral results, the multi-lineage
pro-hematopoietic effects of MA crosses several species. MA
promotes CFU-GEMM FIG. 9, Panel A) which includes precursors of
granulocytes, red blood cells, and macrophages; BFUOe, (FIG. 9,
Panel B) which are red blood cell precursors; CFU-GM (FIG. 9, Panel
C) which include myeloid (granulocyte) and macrophage precursors;
and T-CFC, (FIG. 9, Panel D) which are cells capable of forming
colonies of T-cells. The latter is a less conventional assay, and
details on the methods involved are described
elsewhere..sup.178
[0659] These in vitro results prompted us to determine whether MA
could produce significant effects in vivo on blood cell formation.
To this end we carried out several kinds of experiments which
utilized either lethally irradiated rats and mice, animals given
chemotherapeutic (anti-cancer drugs) agents which have toxic and at
high doses lethal side effects due to bone marrow suppression, and
severely acutely bled animals, and we determined the effects of
short-term (1 or 2 subcutaneous injections) pre-treatment with MA
or continuous (3.times./week) treatment for a few weeks beginning
after the traumatic event.
[0660] 9.4.2 Treatment with MA Rescues Lethally Irradiated Rats and
Mice
[0661] In order to determine in vivo relevance of the
prohematopoietic effects of MA treated rats and mice under several
conditions of bone marrow suppression. First, the effects of native
MA and pMA and of the synthetic 15-mer MA.sub.S1 were treated for
their capacity to reduce or prevent the effects of lethal doses of
gamma irradiation. Pre-experiments showed that 7Gy of total body
radiation were 100% lethal within 5 days with bone marrow
hypoplasia causing a rapid decline in PBMCs. MA, pMA, MA.sub.S1,
and various control polypeptides were given 48 and 24 hrs before,
during, or 24 hrs after radiation. The animals treated prior to
radiation received either 100 ng vs. 1 ug (rats) or 50 ng vs. 500
ng (mice) as 2 subcutaneous injections. Control animals were
untreated or treated with pure hCG (CR127), r.beta.-hCG, native
glycosylated .beta.-core, or .beta.-AA 21-52. Like the untreated
animals, rats (FIG. 10, Panel A) and mice treated 24 and 48 hrs
prior to radiation with hCG or with control polypeptides all died
before day 5. However, 100% of the pretreated animals survived at
least 25 days. Those treated with the higher dose of native MA
survived longest. A typical result is shown in FIG. 10, Panel A.
Survival varied from 60-95% at day 15.
[0662] Similar experiments were carried out in mice and rats
treated at the time of radiation with therapy maintained for 4
weeks by systemic treatment 3.times./week with either 100, 500, or
100 ng. All animals treated at the higher dose of MA and pMA (about
150 pmoles) or MA.sub.S1 (300 pmoles) survived, and 100% of these
animals continue to thrive (now over 100 days) (FIG. 10, Panel E).
Extraordinary promotion of survival was also seen at the lowest
doses of MA (30 pmoles) and MA.sub.S1 (about 50 pmoles) results in
80% survival. It is not yet possible to determine the precise molar
dose of pMA because of less certainty on its full-length sequence.
All control animals died within 5 days, including those treated
with the closely related native glycosylated .beta.-core (400
pmoles).
[0663] To ascertain that the MA effect did promote hematopoiesis in
these animals we examined the bone marrow for cellularity and
colony formation and determined peripheral blood counts. The bone
marrow of the radiated animals show severe hypocellularity and the
capacity to form colonies is reduced to near zero. As illustrated
in FIG. 10, Panels A-C, the blood of control non-irradiated rats
had 5 to 6 K/ul total lymphocytes, 7-8.times.10.sup.6 red blood
cells/ul and a hemoglobin content of 14-15 g/dL. After radiation
these were reduced to near zero (lymphocytes), about
3.times.10.sup.6/ul (red cells), and 6 g/dL (hemoglobin) by day 5.
MA treatment produced a rapid rise, presumably prior to the effect
of the radiation on hematopoiesis, followed by a precipitous fall
in these values but significantly less than in untreated animals.
Importantly, within one week the fall in blood cells was followed
by a hematopoietic recovery phase, and by days 20 to 30 the
recovery was complete. Similarly, bone marrow blood cell
progenitors returned to normal and histology showed marked
increased in cellularity.
[0664] These results suggest that MA may be useful in protection
against radiation injury and may allow higher doses of radiotherapy
for cancer. Although the rescue of lethally irradiated animals is
most likely mediated chiefly by the pro-hematopoietic effects of
MA, the MA treated animals also showed little or no
gastrointestinal distress. Thus, the effect of MA could be peculiar
to radiation injury (radioprotective) rather than a broad effect on
growth promotion of primitive progenitor cells that might extend
even to the gastrointestinal tract.
[0665] Therefore, we sought an in vivo pro-hematopoietic effect in
a different experimental setting. We chose a chemotherapy-induced
bone marrow suppression model because the information obtained
might also be clinically useful in that if MA protected against
these side effects, MA might reduce and even prevent side effects
of chemotherapy and as in the case of chemotherapy, it might allow
uses of higher doses in order to reach optimum cancer killing by
the chemotherapeutic agent. Additionally, MA itself is
anti-tumorigenic as shown.
[0666] 9.4.3 MA Protects Against the Side Effects of
Chemotherapy
[0667] We selected taxol as the chemotherapeutic agent because it
is one of the most effective and widely used anti-tumor agents and
because at high doses it can have serious toxic effects on bone
marrow. Sprague-Dawley rats were given taxol in large lethal doses,
4.5 mg in one I.P. inoculation. This amount lead to bone marrow
cytotoxicity, resultant blood cell deficiencies, and death of the
rats in less than 5 days. Bone marrow suppression can also be a
side effect of the clinical use of taxol..sup.179 As in the
radiation experiments, either MA, pMA, MA.sub.S1, or various
polypeptide controls were given subcutaneously only in two doses 24
and 48 hrs before taxol treatment or were given 3.times./week for 4
weeks. A representative experiment (FIG. 11, Panels A and B) shows
the rescue of the rats from the ultimate lethal effects of the high
dose paclitaxel (TAXOL.TM.). Sixty-percent (low dose) or 80-90%
(higher dose) of pretreated animals survived when given MA,
MA.sub.S1, or pMA. In animals in which therapy was maintained 28
days with low (100 ng) or high (1000 ng) doses of MA, MA.sub.S1, or
pMA, there was 100% survival on day 5 reducing to about 60% (low
dose) and 80% (high dose) maintained at this level with the animals
appearing healthy now at day 80, similar to the radiation
experiments. Again, no effect was obtained with pure hCG,
r.beta.-hCG, native glycosylated .beta.-core, control peptides, or
LH even at high doses, e.g., 5000 ng of native glycosylated
.beta.-core. Like untreated animals, these rats were all dead
before day 5.
[0668] Taxol treated rats showed a 70% to greater than 90%
reduction of their peripheral blood counts (FIG. 11, Panels C-F).
Total lymphocytes (Panel C), platelets (Panel D), hemoglobin (Panel
E), and red blood cells (Panel F) within a few days of receiving
the one inoculation of the high dose MA or MA.sub.S1 show striking
rise in number (blood counts were not performed on the pMA-treated
animals). The animals pretreated 2 days with MA or MA.sub.S1 but
not maintained longer rebounded by day 15 with platelets, RBC, and
hemoglobin returning to normal and above by day 20. Total
lymphocytes remain low somewhat longer. These animals treated with
MA or MA.sub.S1 after taxol but maintained on therapy (3 weekly
inoculations) recovered more rapidly, including total lymphocytes,
which also returned to normal levels by day 20. The radiation and
taxol experiments involved responses to MA after a highly traumatic
incident. We wished next to study its effects in a more
physiological setting.
[0669] 9.4.4 MA Peptides Rescue Acutely Bled Rats
[0670] To delineate the effects of MA on hematopoiesis in the
absence of any external bone marrow suppression, blood (35-40% of
total blood volume) was removed from five Sprague-Dawley rats by
intracardiac puncture. FIG. 12, Panels A-D shows the recovery in
rats untreated vs.
[0671] those given i.p. MA or MA.sub.S1 24 and 48 hrs after the
blood loss (each point represents a mean of 10 animals). MA and
MA.sub.S1 treated animals show a prompt recovery of all peripheral
blood cells. FIG. 12, Panels E-G shows blood counts in three
monkeys. Each panel illustrates the mean values for the three
animals treated with MA.sub.S1. Thus, MA and related derivatives
have potent pro-hematopoietic effects.
[0672] 9.5 Inactivity of .beta.-Core
[0673] For several reasons it was important to unambiguously
eliminate the native glycosylated hCG .beta.-core as a significant
contributor to the multiple effects described here for MA. First,
because of the relatedness of MA to a major part of the C-terminal
polypeptide of the native glycosylated .beta.-core there was a
possibility of copurification. Second, because one report claims an
in vitro inhibitory effect of native glycosylated .beta.-core on KS
tumor cells, though these effects were limited to cell culture
studies, and oddly the inhibitory effect on tumor cells was
reported to be specific for KS..sup.180 Third, because of the
interesting speculation that since the native glycosylated
.beta.-core of hCG is present in relative abundance in urine and
its N-polypeptide ((36-42) has some homology to PDGF it may have
biological function independent of hCG..sup.181 In other words,
there has been a search for some function of this relatively
abundant peptide, and good reasons to suspect it might have been
the factor accounting for these activities.
[0674] However, we have demonstrated that: [0675] homogenously
purified native glycosylated .beta.-core had none of the biological
activities ascribed here to MA; [0676] that MA was separated from
.beta.-core during purification; [0677] native glycosylated
.beta.-core is found in relative abundance in urine but only in
very low amounts in sera of early pregnancy,.sup.182 whereas MA is
demonstrable in both by bioassays, serological tests, and
purification; [0678] the hCG .beta.-core is not present in rodents,
whereas biological and serological activities of MA were found in
mice and rats; [0679] antibodies made against purified MA did not
bind pure native glycosylated .beta.-core but did bind MA and
neutralized MA biological activities both from purified and cruder
preparations derived from serum and urine of pregnant women as well
as from some commercial hCG preparations; [0680] three monoclonal
antibodies to native glycosylated .beta.-core did not react with MA
nor inhibit these biological activities; [0681] the PDGF related
sequences contained in the native glycosylated .beta.-core AA 6-42
N-terminal polypeptide are not part of MA, and despite the
interesting homology to PDGF, peptide 6-42 had none of the
activities described here; and most important, there was no
evidence of the complete native glycosylated .beta.-core in either
pMA or MA by AA sequencing and by mass spectroscopy.
[0682] 9.6 Summary of Active and Inactive Peptides
[0683] Table 2 summarizes active and inactive peptides according to
the work presented herein:
TABLE-US-00003 Active Peptides Non-Active Peptides Native MA
Sequence AA 55-89 from .beta.-hCG: .beta.-core: Full length native
glycosylated-AA MA: 1
----------------------------------------------- 6-40 & 55-92
VVCNYRDVRFESIRLPGCPRGVNPVVSYA MA .sub.S6: AAPGCPAA (SEQ ID NO: 32)
VALSCQ -----------35 (SEQ ID NO: 2) MA.sub.S1[MA synthetic peptide:
AA 8-22]: MA .sub.S7: PILP (SEQ ID NO: 33) VRFESIRLPGCPRGV (SEQ ID
NO: 4) MA.sub.S2[MA synthetic peptide: AA 4-33]: MA .sub.S8: LPGCRR
(SEQ ID NO: 34) NYRDVRFESIRLPGCPRGVNPVVSYAVALS (SEQ ID NO: 5)
MA.sub.23[MA synthetic peptide: AA 1-35]: MA .sub.S12: PGCP (SEQ ID
NO: 35) VVCNYRDVRFESIRLPGCPRGVNPVVSYA VALSCQ (SEQ ID NO: 6)
MA.sub.S5[MA synthetic peptide: AA 14-20]: MA .sub.S13: PALP (SEQ
ID NO: 36) RLPGCPR (SEQ ID NO: 7) MA.sub.S9: APGCPG (SEQ ID NO: 8)
MA .sub.S14: APGCPA (SEQ ID NO: 37) MA.sub.S10: LPGCPR (SEQ ID NO:
9) MA .sub.S15: AAGCAPR (SEQ ID NO: 38) MA.sub.S11: LPGCPQ (SEQ ID
NO: 10) MA .sub.S16: ALGCLR (SEQ ID NO: 39) SAT.sub.A2:
CLQGVLPALPQVVC (SEQ ID NO: 20) MA .sub.S17: LPAAPR (SEQ ID NO: 40)
SAT.sub.A3: CLQGRLPALPRVVC (SEQ ID NO: 21) MA .sub.S18: LPRRPR (SEQ
ID NO: 41) SAT.sub.A4: CRLPGLPRC (SEQ ID NO: 22) MA .sub.S19:
LPPPPR (SEQ ID NO: 42) MA .sub.S20: CPAAPC (SEQ ID NO: 43) MA
.sub.S21: NPGCPR (SEQ ID NO: 44) .beta.-hCG AA 1-20, AA 21-52, AA
6-16, AA 88-92, AA 8-34, AA 93-100, AA 74-95, AA 34-49, AA 125-
145, AA 100-110, AA 93-100, AA 134-144, AA 100- 112, AA 6-21 and AA
6-28 Scram 1: QCSLAVAYSVVPNVGRPCGPL- RISEFRVDRYNCVV (SEQ ID NO: 45)
Scram 2: SYAVALQRGVNPVVCAANYRD- VRFESIRL (SEQ ID NO: 46)
[0684] 9.7 Toxicity Studies
[0685] In the concentration ranges and over the time periods
reported here we observed no cytopathogenicity of MA on normal
cells in vitro. These cells included human, primate, and rodent
bone marrow, PBMCs, and human CD4+ T-cells, CD8+ T-cells, CD34+
cells, and macrophages enriched from normal PBMCs, as well as
normal human endothelial cells and fibroblasts. Cytotoxicity was
assessed by 3H-thymidine incorporation, trypan blue staining, cell
numbers, and morphological examination by light and confocal
microscopy. Hematopoiesis was not only unimpaired it was promoted.
Consequently, we studied the effects of higher concentrations (up
to 10.times. the effective concentrations) of MA and found no
evident cytotoxicity. Similarly, we observed no evident toxic side
effects in animal studies grossly or by blood counts. These studies
were chiefly performed with rats and mice, but also included rhesus
macaques treated with 0.2 mg/kg for several months. Like the in
vitro results, hematopoiesis was not impaired but instead
enhanced.
[0686] 9.8 Anti-MA Antibodies
[0687] Polyclonal and monoclonal antibodies to MA were raised
according to the methods of Harlow et al..sup.183 and de St Groth
et al.,.sup.184 respectively. Antigens were synthetic peptides
58-87 and 68-74.times.2 coupled to keyhole limpet hemocyanin
(KLH)..sup.185
TABLE-US-00004 58-87: (SEQ ID NO: 47)
NH.sub.2-NYRDVRFESIRLPGCPRGVNPVVSYAVALSC-COOH 68-74(2x): (SEQ ID
NO: 48) NH.sub.2-RLPGCPRRLPGCPRC-COOH
[0688] Antibodies to other MA peptides and other therapeutic
polypeptides of the invention may be prepared using the same
method. For example, the following antigens may be prepared by the
same method
TABLE-US-00005 (SEQ ID NO: 49) 77-88: NH.sub.2-NPVVSYAVALSC-COOH
(SEQ ID NO: 50) 62-81: NH.sub.2-VRFESIRLPGCPRGVNPVVS-COOH
[0689] 9.9 Characterization of Monoclonal Antibodies Raised Against
MA Peptides
[0690] Antigens listed are synthetic except for 55-92 (derived from
beta-core), .beta.-core (hCG .beta.-core isolated from pregnant
female urine), BSA (bovine serum albumin), PP2 (native pMA), Preg
(crude MA-enriched fraction from pregnant female urine). NT=not
tested.
TABLE-US-00006 Antibody Characterization ELISA (Antigen Coated on
plate) Western Antigen MA.sub.a 5887 6281 68742 55-92 .beta.core
BSA PP2 MA.sub.a PP2 Preg .beta.core Cone used (ELISA = ug/ml,
Western = ug/lane) Antibody Source M/P type Tit/C 1 1 2 5 0.2 0.5
20 mg 2 2.67 6.4 2 5887-1E7 1HV m-B/c G1-/2b 1 mg - - - - - - - NT
+/- + + + 5887-2D12 1HV m-B/c G1/k 1.5 mg - - - - - - - NT NT NT NT
NT 5887-13G6 1HV m-B/c G1/k 570 ug +/- + + - - +/- - - + + + +
5887-15E7 1HV m-B/c G3/k 400 ug - - - - - - - NT NT NT NT NT
5887-18G5 1HV m-B/c G1/k 860 ug - - - - - - - NT NT NT NT NT
5887-1B2 1HV m-BD G1/k 500 ug ++ ++ - - + + - ++ + + + + 5887-1H3
1HV m-BD G2b/1 1.5 mg ++ ++ - - + - - NT NT NT NT NT 5887-2D11 1HV
m-BD G1/k 840 ug + + + - - - - + + + + + 5887-3F3 1HV m-BD G2b/k
800 ug + + + - - - - +/- + + + + 5887-10F12 1HV m-BD G1/k 600 ug +
+ + - - - - +/- + +/- + + 5887-13A4 1HV m-BD G1/k 600 ug +/- + + -
- - - ++ + + + + 68742-18C9 1HV m/Bc G1/k 800 mg - - - - - - - NT -
+/- + +
TABLE-US-00007 Antibody Characterization ELISA (Antigen Coated on
plate) Western Antigen MA.sub.a 5887 6281 68742 55-92 .beta.core
BSA PP2 MA.sub.a PP2 Preg .beta.core Cone used (ELISA = ug/ml,
Western = ug/lane) Antibody Source M/P type Tit/C 1 1 2 5 0.2 0.5
20 mg 2 2.67 6.4 2 5887-1E7 1HV m-B/c G1-/2b 1 mg - - - - - - - NT
+/- + + + 5887-2D12 1HV m-B/c G1/k 1.5 mg - - - - - - - NT NT NT NT
NT 5887-13G6 1HV m-B/c G1/k 570 ug +/- + + - - +/- - - + + + +
5887-15E7 1HV m-B/c G3/k 400 ug - - - - - - - NT NT NT NT NT
5887-18G5 1HV m-B/c G1/k 860 ug - - - - - - - NT NT NT NT NT
5887-1B2 1HV m-BD G1/k 500 ug ++ ++ - - + + - ++ + + + + 5887-1H3
1HV m-BD G2b/1 1.5 mg ++ ++ - - + - - NT NT NT NT NT 5887-2D11 1HV
m-BD G1/k 840 ug + + + - - - - + + + + + 5887-3F3 1HV m-BD G2b/k
800 ug + + + - - - - +/- + + + + 5887-10F12 1HV m-BD G1/k 600 ug +
+ + - - - - +/- + +/- + + 5887-13A4 1HV m-BD G1/k 600 ug +/- + + -
- - - ++ + + + + 68742-18C9 1HV m-B/c G1/k 800 mg - - - - - - - NT
- +/- + +
[0691] 9.10 Immunopurification Using Antibodies
[0692] The inventors have successfully employed the antibodies of
the invention in the immunopurification of MA peptides from
biological fluids and for qualitative detection of MA peptides.
[0693] FIG. 13 shows silver stained 4-12% Bis-Tris NuPage SDS-PAGE
gel (Invitrogen/Novex) of column fractions derived from
non-pregnant female urine plus/minus a spike with MA.sub.s2 (100
ug/L). An MA.sub.s2-sized band is only seen in the plus spike acid
peak lane (4.sup.th from right) and MA.sub.s2 control (lane 6). Gel
is displayed upside down so low Mr material is at top. Lanes R to L
as displayed: (1) Novex Mark 12 Mr; (2) Urine+MAs2 column FT; (3)
Pk-1 (shoulder); (4) Pk-2 (acid peak), (5) Pk-3 (salt peak), (6)
MA.sub.s2 standard; (7) Urine-MA.sub.s2; (8) Pk-1 (acid peak); (9)
Pk-2 (salt peak); (10) Novex Mark 12 Mr.
[0694] FIG. 14 shows Western blot of SDS-PAGE showing MA-reactive
species only in acid peak (lane 4) and MA.sub.s2 control (lane 6).
Antibody used was a 1:500 dilution from a mix (1:1 ratio) of sera
from 2 rabbits both immunized with the 5887-KLH antigen. Rabbits
used were identified as AP-2032 and IHV-5887. Rabbit antibodies
were visualized with KPL Goat-anti-Rabbit-AP (1:1000) and BCIP/NBT.
Gel was loaded exactly as on previous page but is displayed right
side up. Lanes L to R as displayed: 1, Novex Mark 12 Mr, 2,
Urine+MAs2 column FT, 3, Pk-1 (shoulder), 4, Pk-2 (acid peak), 5,
Pk-3 (salt peak), 6, MAs2 std, 7, Urine-MAs2, 8, Pk-1 (acid peak),
9, Pk-2 (salt peak), 10, Novex Mark 12 Mr.
[0695] 9.11 Use of Anti-MA Monoclonal Antibodies to Neutralize the
Anti-HIV-I Effect of the MA Peptide In vitro
[0696] Anti-MA antibodies, or control (anti-MDC) antibodies were
added to an HIV-I infection assay to determine if they could
inhibit the anti-viral activity of the MA peptide. As shown in lane
2 of FIG. 15 anti-MA antibodies remove the anti-viral effect of
MA.sub.s2, resulting in elevated HIV-I p24 production. In
comparison, addition of a control antibody (lane 1) shows no effect
and results in continued full-inhibition of HIV-I p24 production as
seen in the MAs2 treated lane (3).
[0697] 9.12 Bacterial Production of MA Peptides
[0698] A bacterial-optimized DNA sequence encoding the MA.sub.s2
sequence was designed with 5' NcoI and 3' SapI restriction sites
and methionine on the N-terminal end. Complementary
oligonucleotides were constructed and a synthetic gene assembled in
vector pTYB3 (New England Bioloabs). N-terminal fused MA.sub.s2
sequence was DNA sequenced to confirm the sequence and integrity of
the intein fusion C-terminal to the MA sequences. Test expressions
revealed band of size desired by SDS-PAGE (silver stain) and
Western. Expression and cleavage of material, using NEB supplied
protocol (only changes were addition of 40 ug/ml pMSF, 5 mM
MgCl.sub.2 and 40 ug/ml Dnasel to lysis buffer), produced a band of
correct size (Silver stain of SDS-PAGE) and immunological
reactivity (Western vs pc-Ab)-data not shown.
[0699] Bacterial optimized MA sequence:
TABLE-US-00008 (SEQ ID NO: 51)
5'-CATGAAATACCGTGATGTGCGTTTTGAAAGCATTCGTCTGCCGGGTT
GTCCGCGCGGTGTG-AATCCGGTTGTGAGCTACGCGGTTGCGCTGAGCTG C-3'
[0700] The utilization of the intein cleavage system results in
production of a pure species without addition of external proteases
or detergents that could affect determinations of biological
activity if not 100% removed.
[0701] Optimized gene can be expressed as existing N-terminal
fusion or as a C-terminal fusion. CNBr can be used to cleave the
added N-terminal Met resulting in release of previously defined
MA.sub.s2 sequence.
[0702] This gene construct has been used here in a single
expression system (Impact T7, New England Biolabs) but has been
designed so that it can readily be moved into other systems. For
example, a suitable system could involve the use of a C-teminal
fusion protein to increase production levels (e.g., through
depoting in inclusion bodies or use of a stronger promoter) while
minimizing internal methionines. Fusion protein purified from such
as system could then be treated with CNBr to release the MA.sub.s2
peptide without the currently present Met.
[0703] FIG. 16, Panel A shows a Coomassie stained gel and Panel B
shows a Western blot of test expressions showing .alpha.-MA
reactive material of the expected size (58 kDa). Lanes: 1, vector
cntl (516.1), 2-6 induced lysates 517.1-517.5, 7, Novex Mk12 Mr, 8,
Pregnin, 9, MA.sub.s2 cntl, 10-11 buffer cntl, 12, Novex Mk12
Mr.
[0704] FIG. 17a shows silver stained SDS-PAGE and FIG. 17b a
Western blot showing column purification of clone 517.3 expressed
Bact:MA.sub.s2 and immunoreactivity with .alpha.-MA antibodies.
SDS-PAGE lanes: 1, Novex Mk12 Mr, 2, uninduced, 3, induced pre
column, 4, column FT, 5-10 column eluate fractions 1-6 (2 ml ea),
11, MAs2 control, 12, Novex Mk12 Mr. Western lanes: 1, Novex Mk12
Mr, 2, uninduced, 3, induced lysate, 4, column FT, 5-11 column
fractions 2-8 (2 ml ea), 12, MAs2 control.
[0705] 9.13 Extensive Proliferation Ex Vivo, Long Term Cultures
[LTC-IC] and Self Renewal of Human Primitive Hematopoietic Stem
Cells from Cord Blood/Bone Marrow or Peripheral Blood in the
Presence of MA Polypeptides
[0706] Hematopoietic tissues contain a small population of
primitive, stem cells capable of self-renewal and generating
committed progenitors of the different myeloid and lymphoid
compartments. In semisolid assays, hematopoietic progenitors in the
presence of specific growth factors proliferate and differentiate
to produce mature cells. In this system, depending on the
combination of growth factors, different classes of multipotent
committed progenitors CFU-GM, FRU-e, CFR-GEMM, T-CFC and CFU-Mk
with high differentiation but negligible self-renewal capacity are
used. Early progenitor/stem cells may also be used.
[0707] In the human system, a long-term culture-initiating assay
(LTC-IC) can be used to detect cells that can generate myeloid
clonogenic cells (CFC/colony forming cells) in long-term cultures
for a minimum of 5 weeks. Bone marrow stromal was produced by
culturing 107 fresh BM-MNC in a T25 flash for at least 2 weeks in 5
mL stromal medium; 12.5% horse [HS] and 12.5% fetal calf serum
[FCS]; both from hyclone [Logan, Utah], Iscove's modified
Dulbecco's medium [IMDM, GIBCO-BRL], 2-mercaptoethanol [Sigma, st.
Louis, Mo.], 10.sup.-6 mol/L hydrocortisone [Sigma] and
penicillin/streptomycine.
[0708] One or 2 days before co-cultures, bone marrow stroma was
irradiated with 15Gy and plated at 7.times.10.sup.3/cm.sup.2 in
24-well plates to form pre-established stromal layers for the
long-term cultures. Limiting dilution was 1 to 1000 CD34+ bone
marrow/cord blood cells and equivalent aliquots, i.e., limiting
dilutions of the cells grown in long-term cultures stroma-free
cultures after 2 to 20 weeks were seeded on top of the irradiated
bone marrow stroma in culture medium containing IMDM, 12.5% HS,
12.5% FCS, 2-mercptoethanol, and 10-6 mon-hydrocortisone for 5
weeks LTC-IC assay period. Non-adherent cells were removed from the
cultures, combined with the corresponding trypsinized adherent
cells, washed and assayed for colony forming units (CFU-Cs) in
methylcellulose medium containing 1.3% methylcellulose (Fluka
Chemilka Biochemika, buck\hs, Switzerland), 30% FCS, EPO (3 U/ml),
IL-3 (20 ng/ml), G-CSF (20 ng/ml), GM-CSF (20 ng/ml) and KL (c-kit,
50 ng/ml). These cultures were incubated at 37.degree. C. and
colonies were scored 2 to 3 weeks later. LTC-IC enumeration was
based on the number of CFU-C scored in the limiting assays [LDA]
and another technique.
[0709] Stroma-free long-term cultures were performed as follows. 2
to 10.times.10.sup.3 CD34+/CD34.sup.-CD38.sup.- from bone
marrow/CB/PBMC cells were cultured in quadrutriplicate flat
bottomed 24 well plates in 1 ml to of IMDM supplemented with 10%
FCS and the following cytokines: IL-3 (10 ng/ml), IL-6 (10 ng/ml),
KL [c-kit 50 ng/ml, FL (50 ng/ml) and TPO (10 U/ml), which added,
alone or in combination of two or more factors, to each series of
microwells twice a week. The wells were grown at 37.degree. C. and
share a number of features with in vivo murine long-term
repopulating cells. These include assay for cobblestone areas
colony forming cells (CAFC), high proliferative potentials
colony-forming cells (HPP-CFC), CFU-GEMM, CFU-Bl (blast colony
forming units).
[0710] Results were as shown in Table 2 (LTC-IC or stroma-free
culture after 3 mos):
TABLE-US-00009 CD34+, CD38- cells from human BMMC CFU-Mixed CFU-GM
BFU-e T-CFC 1. Control LTC 4 .+-. 2 64 .+-. 6 50 .+-. 7 64 .+-. 8
without stroma; untreated (n = 3) 2. LTC-IC 2 .+-. 1 28 .+-. 5 15
.+-. 6 19 .+-. 23 untreated (n = 3) 3. LTC stroma 13 .+-. 3 198
.+-. 11 161 .+-. 8 199 .+-. 10 free; treated with MA (n = 3)
[0711] Additional results were as follows for 1, 2, and 3
(respectively) as identified in the above table: [0712] CD34+
cells: 52, 42, and 77.71% [0713] CFU-GM: 25, 15, and 27% of CD14;
and 14, 12 and 22% of CD68 [0714] Rx MA.sub.S1: 35% of CD1 and 27%
of CD68 [0715] Rx MA: 30, 17 and 38% of CD14; and 21, 14 and 29% of
CD68 [0716] T colonies Rx MA [0717] CD3+/CD4+: 38, 28 and 46%
[0718] CD3+/CD8+: 32, 29 and 49%
[0719] These results demonstrate that the therapeutic polypeptides
of the invention are usefully applied with chemokines known to
cause differentiation of stem cells, in order to facilitate
improved yields of differentiated cells.
[0720] 9.14 MA Peptides Improve Engraftment of Ex vivo Expanded
Hematopoietic Progenitor Cells
[0721] The inventors also investigated the effect of MA on
engraftment of human bone marrow stem cells [CD34.sup.+, CD38.sup.-
cells, long term cultures after 4 mos, and stromal free cultures]
ex vivo expanded hematopoietic progenitor cells in Rats irradiated
[Sprague DAwley, 450 Rad] and immunodeficient mice [SCID or BNX-XID
mice].
[0722] Measurement of short-term hematopoietic reconstitution
kinetics and long term repopulating ability. Just prior to
implantation, SCID, BNX-XID-immunodeficient mice and Sprague Dawley
rats were exposed to 450 rad total body .gamma.-irradiation. The
aim was to determine whether human B, T, and myeloid cells will be
detected in the blood circulating after 10.sup.6 of human bone
marrow long term culture: CD34.sup.+, CD38.sup.-
(HBMLTC-CD34.sup..+-./APL: 68% are CD34.sup.+, CD38.sup.- cells but
HBMLTC-CD34.sup..+-./MA were implanted into the irradiated rats and
mice's spleen. One month after implantation, virtually all of the
circulating cells in the peripheral blood of rats or mice were
human cells. In contrast, the control immunodeficient mice [BNX-XID
or SCID mice] or rats irradiated and implanted with fresh untreated
human CD34+ cells did not produce human CD4.sup.+ T-cells: 0%,
CD14.sup.+ cells: 0% and CD34.sup.+ cells: 0% (N=3). But the SCID
and BNX-XID immunodeficient mice were repopulated by the cultured,
treated human cells.
[0723] The following table illustrates the effect on engraftment of
ex vivo expanded hematopoietic progenitor cells:
TABLE-US-00010 Rat S. Dawley CD34.sup.+, CD38.sup.+ cells from
[irradiated 450 BNX-XID human BMMC RAD (n = 3)] SCID Mice Mice 1.
LTC without stroma CD4 2% 0% 1% teated with APL 300 CD14 3% 5% 2%
IU (n = 3) CD34 48% 40% 39% 1. LTC stroma-free CD4 1% 2% 3% treated
with MA CD14 1% 2% 1% 500 ng/ml (n = 3) CD34 40% 38% 37%
[0724] 9.15 Discussion
[0725] Although rodents do not produce an exact homologue of CG, it
is likely that the active factor in sera of mice is derived from a
sequence similar to the MA peptides, since similar biological
activities were found in sera of mice and rats selectively during
early pregnancy. Partial purification showed that this murine
factor followed patterns similar to human MA. Moreover, specific
antibody to human MA bound to a factor from mouse and rat sera and
urine from early pregnancy but not proteins of non-pregnant female
rodents or male rodents.
[0726] The fact that murine .beta.-LH contains sequences 60%
homologous to MA, including some common structural motifs, is
consistent with a conclusion that murine MA is derived from their
.beta.-LH.
[0727] MA is a small cationic polypeptide which contains cysteine
residues at positions 3, 18 and 34 that could give rise to
intra-peptide disulfide bridges favoring its stabilization. As a
component of .beta.-hCG the MA sequence is a major portion of the
third .beta.-hairpin loop..sup.188 However, the free form is not
likely to retain secondary structure, and various truncated forms
with elimination of CYS-3 are as active as the full length form. MA
contains a polyproline II (PPII) sequence RL-PGCP-R (SEQ ID NO: 7)
which is related to a repeated PX.sup.2P motif present in five
places in .beta.-hCG.sup.189 and is found in LH, and as described
by Lapthorn et al.,.sup.199 comprises the .beta.-hairpin structure
in .beta.-hCG flanked by .beta.-strands. The sequence fits the
motif of 7-8 AAN SH3 binding PPII helix sequences found in adapter
molecules involved in amplification of tyrosine protein kinase
signaling pathways. Adjacent to the MA polyproline II sequence, the
N-terminal side is a PKC phosphylation site, SIR. The polyproline
II motif and phosphorylation sites may provide the most important
clues on the mechanisms of which MA exerts its effects. In view of
its pro-hematopoietic effects on normal blood cell precursors, the
possibilities of interactions with the SH3 sequences of Grb2 and
Vav merit noting, since Vav-Grb2 interactions are known to be
involved in embryonic development of the hematopoietic
system.sup.191 and Vav is expressed only in hematopoietic
cells..sup.192 Similarly, major hematopoietic effects, such as
marked enhancement of the capacity of erythropoietin and II-I to
induce activation of signal pathways, are mediated by Crk L by a
Ras-dependent mechanism..sup.193 Crk L is also an SH3 containing
adapter protein which could be affected by MA.
[0728] In view of MA's pro-apoptotic activity, the Ras-GAP
interaction is another possible target, since very recently Leblanc
et al. have reported that inhibition of their linkage in vitro with
antibodies directed against -SH3 promotes tumor cell
apoptosis..sup.194 An HIV gene product, Nef, contains three related
SH3 sequences. Its natural SH3 target was recently identified by
Renkema et al..sup.195 as a p21 activated kinase 2 (PAK2). Since
Nef promotes HIV replication, it is possible that the anti-HIY
effects of MA are mediated through interference with Nef-PAK2
interactions. Moreover, since PAK2 is activated by pro-apoptotic
caspases, it is possible that MA apoptotic and pro-hematopoietic
effects also involves interactions with SH3 of PAK2 or related
kinases. Finally, the 7-mer RLPGCPR (SEQ ID NO: 7) sequence of MA
is identical to the PPII sequence of inhibitors of the serine
proteinases, the acrosyn trypsin inhibitor precursor, also by
interactions with SH3 regions..sup.196 Inhibition of regulatory
proteinases could, of course, have major influences on cell growth
and differentiation.
[0729] Our results are consistent with the interpretation that MA
is a natural product rather than an hCG degradation form occurring
during laboratory purification, e.g.: [0730] consistency in the
isolation of very closely related polypeptides with the
bioactivities by different methods; [0731] consistency of finding
the multiple biological activities in the crudest preparations from
urine and sera; and [0732] immunoassays which distinguish MA from
.beta.-core, hCG, .alpha.- and .beta.-hCG, yet showing reactivity
with some crude hCG preparations and urinary concentrates, which
contain all the activities described here, and these materials are
only minimally handled in the laboratory virtually precluding their
genesis from laboratory manipulations.
[0733] Though the evidence suggests that MA is naturally occurring,
as opposed to a laboratory-derived degradation product, the
questions of its tissue of origin and mechanism by which it is
generated are not answered. Since several human tumors and cell
lines produce hCG and/or its subunits.sup.197 and even .beta.-core
in the absence of the hCG or its subunits,.sup.198 it is possible
that MA is produced by any number of tissues. However, it is far
more likely that MA is made by the trophoblast, since its
production is limited to early pregnancy and the trophoblast is the
source of hCG and both .alpha.- and .beta.-chains..sup.199 However,
some other tissues have been found to produce hCG and .beta.-hCG at
low levels and even different forms of .beta.-core with no known
function.
[0734] Several purifications of MA have generally led to the 35-mer
(.beta.-hCG 55-89) sequence with a molecular mass of 3,864.
However, in some instances MA peptides have been as small as
30-mer, as illustrated by the mass spectroscopy shown in FIG. 21,
corresponding to .beta.-hCG 55-84. The pMA sequence contains the
full length C-terminal polypeptide (.beta.-hCG 55-92) of
(.beta.-core. This 38-mer terminates at a known peptide cleavage
size and is routinely generated in the formation of the
.beta.-core. Thus pMA could be a precursor of MA. The most obvious
pathway is
hCG.fwdarw..beta.hCG.fwdarw..beta.-core.fwdarw.pMA.fwdarw.MA or
more immediately from .beta.-hCG, since the free subunit is also
independently produced by the trophoblast.sup.200 and by some human
tumors..sup.201 There is evidence that .beta.-core can be produced
in the absence of .beta.-hCG,.sup.202 which may suggest that
.beta.-core can arise from novel mRNAs, and alternate splicing has
been described from some .beta.-hCG RNAs..sup.203 Thus, it is
possible that MA could arise directly from .beta.-core or even from
its own alternate spliced transcript. Since MA inhibits growth of
tumor cells, the observations that some human tumors produce
.beta.-hCG and even .beta.-core and show no growth inhibition also
favor the interpretation of a more direct origin of MA. It is of
interest in this regard that among the several related glycoprotein
hormones and growth factors only the .beta.-chain of hCG is
polygenic. Six .beta.-hCG genes or related sequences have been
described which cluster on chromosome 19,.sup.204 and some are
known to produce novel transcripts by alternative splicing..sup.205
"Improperly" folded .beta.-hCG products are known to be
secreted..sup.206 MA could be one such polypeptide.
[0735] MA does not bind the hCG receptor. Saturation of cells with
pure hCG did not affect the biological activity of MA added later
to these cells. In this regard, unusual .beta.-hCG gene transcripts
have been known for several years,.sup.207 and as noted above,
among the several related heterodimeric glycoprotein hormones, only
.beta.-hCG has multiple genes. In addition, the .beta.-hCG subunit
is produced during early pregnancy..sup.208 These findings suggest
a role for one or more of these genes independent of hCG
function.
[0736] Although neither .beta.-hCG or any of its degradation
products significantly bind to the hCG receptor,.sup.209 several
alternative spliced transcripts for the receptor are known which
are developmentally timed,.sup.210 making it feasible that one or
more of these transcripts may encode receptors specific for certain
.beta.-hCG fragments. MA may be derived in this manner and may
utilize one of these receptors.
[0737] MA may have important physiological functions in the
developing embryo. This view is supported by that finding MA
activities are selectively expressed in an early embryonic period
in both rodents and humans. One function could be selective cell
killing, for instance in the known early selective killing of
syncytiotrophoblasts with concomitant cytotrophoblast
proliferation.sup.211 or in the subsequent molding of tissues,
while concomitantly promoting the development of blood forming
cells in the embryo. The finding that MA and smaller synthetic
forms have major biological effects in vivo, coupled with several
structural features such as its .beta.-hairpin structure, are
reminiscent of the in vitro hormone mimicry effects of created
small peptides with .beta.-hairpin structure recently described by
Wrighton et al and Linah et al..sup.212 These groups made peptide
libraries and screened them for binding to known receptors such as
the erythropoietin (EPO) receptor, and surprisingly found very
small peptides not homologous to EPO, which had remarkable
activities. On the other hand, we do not know whether there is an
MA receptor, and MA does not mimic the effects of hCG, its
parentally related hormone.
[0738] It is also possible that MA can enter cells in the absence
of specific receptors and directly mediate intracellular signaling
by SH3 interactions and/or protease inhibition as described above.
Alternatively MA could exert a direct effect on cell membranes
affecting channeling. In this context it is intriguing to view MA
as a possible component of the natural or innate defense mechanisms
helping to compensate for the reduction in the mother's adaptive
immune system in order to prevent fetal rejection. MA is a
proline-containing, small polypeptide with cysteine residues and
.beta.-hairpin-.beta.-strand configurations and can kill some cells
and inhibit an enveloped virus (HIV). These structural and
functional features are reminiscent of some selectively mammalian
defensins,.sup.213 which may have anti-microbial activity, kill
cells, or both by disrupting cell membranes. The N-terminal region
of MA also has perforin-related sequences, a molecule which, though
not homologous to defensins can similarly lead to membrane
disruption and cell death..sup.214
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Sequence CWU 1
1
521145PRTHomo sapiens 1Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro
Ile Asn Ala Thr Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val
Cys Ile Thr Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro
Thr Met Thr Arg Val Leu Gln Gly Val 35 40 45 Leu Pro Ala Leu Pro
Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile
Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val 65 70 75 80 Ser
Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser 85 90
95 Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp
100 105 110 Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro
Ser Leu 115 120 125 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr
Pro Ile Leu Pro 130 135 140 Gln 145 235PRTHomo sapiens 2Val Val Cys
Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu Pro 1 5 10 15 Gly
Cys Pro Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu 20 25
30 Ser Cys Gln 35 338PRTHomo sapiens 3Val Val Cys Asn Tyr Arg Asp
Val Arg Phe Glu Ser Ile Arg Leu Pro 1 5 10 15 Gly Cys Pro Arg Gly
Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu 20 25 30 Ser Cys Gln
Cys Ala Leu 35 415PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 4Val Arg Phe Glu Ser Ile Arg Leu Pro Gly
Cys Pro Arg Gly Val 1 5 10 15 530PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 5Asn Tyr Arg Asp Val
Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro 1 5 10 15 Arg Gly Val
Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser 20 25 30
635PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Val Val Cys Asn Tyr Arg Asp Val Arg Phe Glu
Ser Ile Arg Leu Pro 1 5 10 15 Gly Cys Pro Arg Gly Val Asn Pro Val
Val Ser Tyr Ala Val Ala Leu 20 25 30 Ser Cys Gln 35 77PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Arg
Leu Pro Gly Cys Pro Arg 1 5 86PRTHomo sapiens 8Ala Pro Gly Cys Pro
Gly 1 5 96PRTHomo sapiens 9Leu Pro Gly Cys Pro Arg 1 5 106PRTHomo
sapiens 10Leu Pro Gly Cys Pro Gln 1 5 1134PRTHomo sapiens 11Val Val
Cys Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu Pro 1 5 10 15
Gly Cys Pro Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu 20
25 30 Ser Cys 1236PRTHomo sapiens 12Val Val Cys Asn Tyr Arg Asp Val
Arg Phe Glu Ser Ile Arg Leu Pro 1 5 10 15 Gly Cys Pro Arg Gly Val
Asn Pro Val Val Ser Tyr Ala Val Ala Leu 20 25 30 Ser Cys Gln Cys 35
1337PRTHomo sapiens 13Val Val Cys Asn Tyr Arg Asp Val Arg Phe Glu
Ser Ile Arg Leu Pro 1 5 10 15 Gly Cys Pro Arg Gly Val Asn Pro Val
Val Ser Tyr Ala Val Ala Leu 20 25 30 Ser Cys Gln Cys Ala 35
1420PRTHomo sapiens 14Val Val Cys Asn Tyr Arg Asp Val Arg Phe Glu
Ser Ile Arg Leu Pro 1 5 10 15 Gly Cys Pro Arg 20 1532PRTHomo
sapiens 15Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu Ala Val Glu
Lys Glu 1 5 10 15 Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr Ile
Cys Ala Gly Tyr 20 25 30 1633PRTHomo sapiens 16Arg Pro Arg Cys Arg
Pro Ile Asn Ala Thr Leu Ala Val Glu Lys Glu 1 5 10 15 Gly Cys Pro
Val Cys Ile Thr Val Asn Thr Thr Ile Cys Ala Gly Tyr 20 25 30 Cys
1734PRTHomo sapiens 17Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu
Ala Val Glu Lys Glu 1 5 10 15 Gly Cys Pro Val Cys Ile Thr Val Asn
Thr Thr Ile Cys Ala Gly Tyr 20 25 30 Cys Pro 1835PRTHomo sapiens
18Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu Ala Val Glu Lys Glu 1
5 10 15 Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr Ile Cys Ala Gly
Tyr 20 25 30 Cys Pro Thr 35 1913PRTHomo sapiens 19Leu Gln Gly Val
Leu Pro Ala Leu Pro Gln Val Val Cys 1 5 10 2014PRTHomo sapiens
20Cys Leu Gln Gly Val Leu Pro Ala Leu Pro Gln Val Val Cys 1 5 10
2114PRTHomo sapiens 21Cys Leu Gln Gly Arg Leu Pro Ala Leu Pro Arg
Val Val Cys 1 5 10 229PRTHomo sapiens 22Cys Arg Leu Pro Gly Leu Pro
Arg Cys 1 5 2311PRTHomo sapiens 23Thr Cys Asp Asp Pro Arg Phe Gln
Asp Ser Ser 1 5 10 24169PRTEquine sp. 24Met Glu Thr Leu Gln Gly Leu
Leu Leu Trp Met Leu Leu Ser Val Gly 1 5 10 15 Gly Val Trp Ala Ser
Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro Ile 20 25 30 Asn Ala Thr
Leu Ala Ala Glu Lys Glu Ala Cys Pro Ile Cys Ile Thr 35 40 45 Phe
Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg Val 50 55
60 Met Pro Ala Ala Leu Pro Ala Ile Pro Gln Pro Val Cys Thr Tyr Arg
65 70 75 80 Glu Leu Arg Phe Ala Ser Ile Arg Leu Pro Gly Cys Pro Pro
Gly Val 85 90 95 Asp Pro Met Val Ser Phe Pro Val Ala Leu Ser Cys
His Cys Gly Pro 100 105 110 Cys Gln Ile Lys Thr Thr Asp Cys Gly Val
Phe Arg Asp Gln Pro Leu 115 120 125 Ala Cys Ala Pro Gln Ala Ser Ser
Ser Ser Lys Asp Pro Pro Ser Gln 130 135 140 Pro Leu Thr Ser Thr Ser
Thr Pro Thr Pro Gly Ala Ser Arg Arg Ser 145 150 155 160 Ser His Pro
Leu Pro Ile Lys Thr Ser 165 25141PRTOvis sp. 25Met Glu Met Leu Gln
Gly Leu Leu Leu Trp Leu Leu Leu Gly Val Ala 1 5 10 15 Gly Val Trp
Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln Pro Ile 20 25 30 Asn
Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val Cys Ile Thr 35 40
45 Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Leu Ser Met Lys Gln Val
50 55 60 Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg Val Cys Thr
Tyr His 65 70 75 80 Glu Leu Arg Phe Ala Ser Val Arg Leu Pro Gly Cys
Pro Pro Gly Val 85 90 95 Asp Pro Met Val Ser Phe Pro Val Ala Leu
Ser Cys His Cys Gly Pro 100 105 110 Cys Arg Leu Ser Ser Thr Asp Cys
Gly Gly Pro Arg Thr Gln Pro Leu 115 120 125 Ala Cys Asp His Pro Pro
Leu Pro Asp Ile Leu Phe Leu 130 135 140 26141PRTUnknownDescription
of Unknown Suidae (pig) family polypeptide 26Met Glu Met Leu Gln
Gly Leu Leu Leu Trp Leu Leu Leu Ser Val Ala 1 5 10 15 Gly Val Trp
Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro Ile 20 25 30 Asn
Ala Thr Leu Ala Ala Glu Asn Glu Ala Cys Pro Val Cys Ile Thr 35 40
45 Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg Val
50 55 60 Leu Pro Ala Ala Leu Pro Pro Val Pro Gln Pro Val Cys Thr
Tyr Arg 65 70 75 80 Glu Leu Ser Phe Ala Ser Ile Arg Leu Pro Gly Cys
Pro Pro Gly Val 85 90 95 Asp Pro Thr Val Ser Phe Pro Val Ala Leu
Ser Cys His Cys Gly Pro 100 105 110 Cys Arg Leu Ser Ser Ser Asp Cys
Gly Gly Pro Arg Ala Gln Pro Leu 115 120 125 Ala Cys Asp Arg Pro Leu
Leu Pro Gly Leu Leu Phe Leu 130 135 140 27138PRTCanis familiaris
27Leu Gln Gly Leu Leu Leu Trp Leu Leu Leu Ser Val Gly Gly Val Trp 1
5 10 15 Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro Ile Asn Ala
Thr 20 25 30 Leu Ala Ala Glu Asn Glu Ala Cys Pro Val Cys Ile Thr
Phe Thr Thr 35 40 45 Thr Ile Cys Ala Gly Tyr Cys Pro Ser Met Val
Arg Val Leu Pro Ala 50 55 60 Ala Leu Pro Pro Val Pro Gln Pro Val
Cys Thr Tyr His Glu Leu His 65 70 75 80 Phe Ala Ser Ile Arg Leu Pro
Gly Cys Pro Pro Gly Val Asp Pro Met 85 90 95 Val Ser Phe Pro Val
Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Leu 100 105 110 Ser Asn Ser
Asp Cys Gly Gly Pro Arg Ala Gln Ser Leu Ala Cys Asp 115 120 125 Arg
Pro Leu Leu Pro Gly Leu Leu Phe Leu 130 135
28141PRTUnknownDescription of Unknown Bovidae (bovine) family
polypeptide 28Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu
Gly Val Ala 1 5 10 15 Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro
Leu Cys Gln Pro Ile 20 25 30 Asn Ala Thr Leu Ala Ala Glu Lys Glu
Ala Cys Pro Val Cys Ile Thr 35 40 45 Phe Thr Thr Ser Ile Cys Ala
Gly Tyr Cys Pro Ser Met Lys Arg Val 50 55 60 Leu Pro Val Ile Leu
Pro Pro Met Pro Gln Arg Val Cys Thr Tyr His 65 70 75 80 Glu Leu Arg
Phe Ala Ser Val Arg Leu Pro Gly Cys Pro Pro Gly Val 85 90 95 Asp
Pro Met Val Ser Phe Pro Val Ala Leu Ser Cys His Cys Gly Pro 100 105
110 Cys Arg Leu Ser Ser Thr Asp Cys Gly Gly Pro Arg Thr Gln Pro Leu
115 120 125 Ala Cys Asp His Pro Pro Leu Pro Asp Ile Leu Phe Leu 130
135 140 29141PRTRattus norvegicus 29Met Glu Arg Leu Gln Gly Leu Leu
Leu Trp Leu Leu Leu Ser Pro Ser 1 5 10 15 Val Val Trp Ala Ser Arg
Gly Pro Leu Arg Pro Leu Cys Arg Pro Val 20 25 30 Asn Ala Thr Leu
Ala Ala Glu Asn Glu Phe Cys Pro Val Cys Ile Thr 35 40 45 Phe Thr
Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg Val 50 55 60
Leu Pro Ala Ala Leu Pro Pro Val Pro Gln Pro Val Cys Thr Tyr Arg 65
70 75 80 Glu Leu Arg Phe Ala Ser Val Arg Leu Pro Gly Cys Pro Pro
Gly Val 85 90 95 Asp Pro Ile Val Ser Phe Pro Val Ala Leu Ser Cys
Arg Cys Gly Pro 100 105 110 Cys Arg Leu Ser Ser Ser Asp Cys Gly Gly
Pro Arg Thr Gln Pro Met 115 120 125 Thr Cys Asp Leu Pro His Leu Pro
Gly Leu Leu Leu Phe 130 135 140 30143PRTFelis catus 30Met Glu Met
Leu Gln Gly Leu Leu Leu Leu Trp Leu Leu Leu Leu Asn 1 5 10 15 Val
Gly Gly Val Trp Thr Ser Arg Glu Pro Leu Arg Pro Leu Cys Arg 20 25
30 Pro Ile Asn Ala Thr Leu Ala Ala Glu Asn Glu Ala Cys Pro Val Cys
35 40 45 Val Thr Phe Thr Thr Thr Ile Cys Ala Gly Tyr Cys Pro Ser
Met Met 50 55 60 Arg Val Leu Pro Ala Ala Leu Pro Pro Val Pro Gln
Pro Val Cys Thr 65 70 75 80 Tyr Arg Glu Leu Arg Phe Ala Ser Val Arg
Leu Pro Gly Cys Pro Pro 85 90 95 Gly Val Asp Pro Val Val Ser Phe
Pro Val Ala Leu Ser Cys Arg Cys 100 105 110 Gly Pro Cys Arg Leu Ser
Ser Ser Asp Cys Gly Gly Pro Arg Ala Gln 115 120 125 Pro Leu Ala Cys
Asp Arg Pro Pro Leu Pro Gly Leu Leu Phe Leu 130 135 140
31141PRTHomo sapiens 31Met Glu Met Leu Gln Gly Leu Leu Leu Leu Leu
Leu Leu Ser Met Gly 1 5 10 15 Gly Ala Trp Ala Ser Arg Glu Pro Leu
Arg Pro Trp Cys His Pro Ile 20 25 30 Asn Ala Ile Leu Ala Val Glu
Lys Glu Gly Cys Pro Val Cys Ile Thr 35 40 45 Val Asn Thr Thr Ile
Cys Ala Gly Tyr Cys Pro Thr Met Met Arg Val 50 55 60 Leu Gln Ala
Val Leu Pro Pro Leu Pro Gln Val Val Cys Thr Tyr Arg 65 70 75 80 Asp
Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val 85 90
95 Asp Pro Val Val Ser Phe Pro Val Ala Leu Ser Cys Arg Cys Gly Pro
100 105 110 Cys Arg Arg Ser Thr Ser Asp Cys Gly Gly Pro Lys Asp His
Pro Leu 115 120 125 Thr Cys Asp His Pro Gln Leu Ser Gly Leu Leu Phe
Leu 130 135 140 328PRTHomo sapiens 32Ala Ala Pro Gly Cys Pro Ala
Ala 1 5 334PRTHomo sapiens 33Pro Ile Leu Pro 1 346PRTHomo sapiens
34Leu Pro Gly Cys Arg Arg 1 5 354PRTHomo sapiens 35Pro Gly Cys Pro
1 364PRTHomo sapiens 36Pro Ala Leu Pro 1 376PRTHomo sapiens 37Ala
Pro Gly Cys Pro Ala 1 5 387PRTHomo sapiens 38Ala Ala Gly Cys Ala
Pro Arg 1 5 396PRTHomo sapiens 39Ala Leu Gly Cys Leu Arg 1 5
406PRTHomo sapiens 40Leu Pro Ala Ala Pro Arg 1 5 416PRTHomo sapiens
41Leu Pro Arg Arg Pro Arg 1 5 426PRTHomo sapiens 42Leu Pro Pro Pro
Pro Arg 1 5 436PRTHomo sapiens 43Cys Pro Ala Ala Pro Cys 1 5
446PRTHomo sapiens 44Asn Pro Gly Cys Pro Arg 1 5 4535PRTHomo
sapiens 45Gln Cys Ser Leu Ala Val Ala Tyr Ser Val Val Pro Asn Val
Gly Arg 1 5 10 15 Pro Cys Gly Pro Leu Arg Ile Ser Glu Phe Arg Val
Asp Arg Tyr Asn 20 25 30 Cys Val Val 35 4629PRTHomo sapiens 46Ser
Tyr Ala Val Ala Leu Gln Arg Gly Val Asn Pro Val Val Cys Ala 1 5 10
15 Ala Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu 20 25
4731PRTHomo sapiens 47Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg
Leu Pro Gly Cys Pro 1 5 10 15 Arg Gly Val Asn Pro Val Val Ser Tyr
Ala Val Ala Leu Ser Cys 20 25 30 4815PRTHomo sapiens 48Arg Leu Pro
Gly Cys Pro Arg Arg Leu Pro Gly Cys Pro Arg Cys 1 5 10 15
4912PRTHomo sapiens 49Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser
Cys 1 5 10 5020PRTHomo sapiens 50Val Arg Phe Glu Ser Ile Arg Leu
Pro Gly Cys Pro Arg Gly Val Asn 1 5 10 15 Pro Val Val Ser 20
5197DNAHomo sapiens 51catgaaatac cgtgatgtgc gttttgaaag cattcgtctg
ccgggttgtc cgcgcggtgt 60gaatccggtt gtgagctacg cggttgcgct gagctgc
975213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Leu Gln Pro Val Leu Pro Pro Leu Pro Gln Val Val
Cys 1 5 10
* * * * *
References