U.S. patent application number 14/275754 was filed with the patent office on 2014-11-13 for novel double-stranded ribonucleic acids with rugged physico-chemical structure and highly specific biologic activity.
This patent application is currently assigned to Hemispherx Biopharma, Inc.. The applicant listed for this patent is Hemispherx Biopharma, Inc.. Invention is credited to William A. CARTER, David R. STRAYER.
Application Number | 20140335112 14/275754 |
Document ID | / |
Family ID | 45438740 |
Filed Date | 2014-11-13 |
United States Patent
Application |
20140335112 |
Kind Code |
A1 |
CARTER; William A. ; et
al. |
November 13, 2014 |
NOVEL DOUBLE-STRANDED RIBONUCLEIC ACIDS WITH RUGGED
PHYSICO-CHEMICAL STRUCTURE AND HIGHLY SPECIFIC BIOLOGIC
ACTIVITY
Abstract
A novel form of Rugged dsRNA with a unique composition and
physical characteristics was identified with high specificity of
binding to TLR3, which conveys an important range of therapeutic
opportunities. Unlike the previous known antiviral Ampligen.RTM.
(poly I, poly C12,U) the new and improved form (poly I, poly
C.sub.30,U) has a reduced tendency to form branched dsRNA which
results in increased bioactivity due to an increased ability to
bind TLR3 receptor. Pharmaceutical formulations containing the new
nucleic acid as active ingredients and methods of treatment are
also provided. The invention also provides a description of the
physicochemical properties of this novel form of Rugged dsRNA and a
method for its preparation in substantially pure form. DsRNAs
acting thru TLR3 receptor activation are potent antiviral compounds
as well as anticancer agents; also through secondary
immunomodulation they can enhance the bioactivity of vaccines and
also treat autoimmune disorders.
Inventors: |
CARTER; William A.;
(Birchrunville, PA) ; STRAYER; David R.; (Bryn
Mawr, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hemispherx Biopharma, Inc. |
Philadelphia |
PA |
US |
|
|
Assignee: |
Hemispherx Biopharma, Inc.
Philadelphia
PA
|
Family ID: |
45438740 |
Appl. No.: |
14/275754 |
Filed: |
May 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13077742 |
Mar 31, 2011 |
8722874 |
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14275754 |
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PCT/US2010/002970 |
Nov 12, 2010 |
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13077742 |
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12591270 |
Nov 13, 2009 |
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PCT/US2010/002970 |
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PCT/US2009/005797 |
Oct 23, 2009 |
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12591270 |
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61193030 |
Oct 23, 2008 |
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Current U.S.
Class: |
424/184.1 ;
424/278.1; 514/44A; 536/24.5 |
Current CPC
Class: |
A61P 35/02 20180101;
A61K 39/39 20130101; A61K 2039/55561 20130101; C12N 15/117
20130101; A61K 39/00 20130101; A61P 31/04 20180101; A61P 37/06
20180101; A61P 37/04 20180101; C12N 2320/31 20130101; C12N 2310/14
20130101; A61P 31/12 20180101; A61P 35/00 20180101; A61P 43/00
20180101; C12N 2320/52 20130101; C12N 15/1138 20130101; C12N
2310/17 20130101; A61K 31/713 20130101; C12N 15/111 20130101; C12N
2310/50 20130101; A61P 33/02 20180101; A61K 31/7105 20130101; C12N
2320/51 20130101 |
Class at
Publication: |
424/184.1 ;
536/24.5; 514/44.A; 424/278.1 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 39/39 20060101 A61K039/39; A61K 39/00 20060101
A61K039/00 |
Claims
1. An isolated double-stranded ribonucleic acid (dsRNA) which is
resistant to denaturation under conditions that are able to
separate hybridized poly(riboinosinic acid) and poly(ribocytosinic
acid) strands.
2. The isolated dsRNA of claim 1, wherein both strands of said
isolated dsRNA comprise one or more uracil or guanine bases that
are not based paired to an opposite strand.
3. An isolated double-stranded ribonucleic acid (dsRNA) which is
resistant to denaturation under conditions that are able to
separate hybridized poly(riboinosinic acid) and poly(ribocytidylic
acid) strands, wherein the isolated dsRNA: has an HPLC chromatogram
substantially the same as the 5 minute peak of FIG. 1; is stable to
exposure to thermal stress at 40.degree. C.; and has an increased
bioactivity as evidenced by binding to receptor TLR3-ECD as
compared to unimproved poly(I):poly(C.sub.12U).
4. An isolated double-stranded ribonucleic acid (dsRNA) which is
resistant to enzymatic degradation under conditions that are able
to degrade poly(riboinosinic acid) and poly(ribocytidylic acid)
strands, wherein the isolated dsRNA: has an HPLC chromatogram
substantially the same as the 5 minute peak of FIG. 1; has
increased stability to exposure to pancreatic ribonuclease A; and
has an increased bioactivity as evidenced by binding to receptor
TLR3-ECD as compared to unselected poly(I):poly(C.sub.12U).
5. A method of treating a subject with an immunological
dysfunction, said method comprising administration to the subject
of the isolated dsRNA defined in claim 1 in a therapeutic
amount.
6. The method according to claim 5 wherein said immunological
dysfunction is an autoimmune disorder.
7. A method of treating a subject with an incipient or established
microbial infection, said method comprising administration to the
subject of the isolated dsRNA defined in claim 1 in a therapeutic
amount.
8. The method according to claim 7 wherein said infection is a
bacterial, protozoan, or viral infection.
9. A method of treating a subject with chronic fatigue syndrome,
said method comprising administration to the subject of the
isolated dsRNA defined in claim 1 in a therapeutic amount.
10. A method of treating or preventing tumor or neoplasm formation
in a subject, said method comprising administration to the subject
of the isolated dsRNA defined in claim 1 in a therapeutic
amount.
11. The method according to claim 10 wherein said tumor or neoplasm
is a carcinoma, sarcoma, leukemia or lymphoma.
12. The method according to claim 10 wherein the tumor or neoplasm
is a glioma.
13. A method of inducing an immune enhancing effect in a subject,
said method comprising administration to the subject of the
isolated dsRNA defined in claim 1 in a therapeutic amount.
14. The method according to claim 13 wherein said isolated dsRNA is
administered as a vaccine adjuvant.
15. The method according to claim 14 wherein said isolated dsRNA is
administered sequentially or concurrently with the vaccine.
16. The method according to claim 13 wherein said subject is
administered an anti-tumor or anti-microbial vaccine.
17. The method according to claim 16 wherein the vaccine is an
anti-protozoan, anti-viral or anti-bacterial vaccine.
18. The method according to claim 5, wherein the therapeutic amount
of said isolated dsRNA is infused intravenously.
19. The method according to claim 5, wherein the therapeutic amount
is injected intradermally, subcutaneously, or intramuscularly;
inhaled or delivered intranasally or intratracheally; or applied
transdermally, transmucosally, intranasally, intratracheally,
oropharyngeally, or sublingually.
20. The method according to claim 5, wherein the therapeutic amount
is administered transocularly.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/077,742, filed Mar. 31, 2011, which is a
continuation-in-part of PCT/US2010/002970, filed Nov. 12, 2010,
which is a continuation-in-part of U.S. application Ser. No.
12/591,270, filed Nov. 13, 2009, which is a continuation-in-part of
International Application No. PCT/US2009/005797, filed Oct. 23,
2009 which claims priority benefit of U.S. provisional application
Ser. No. 61/193,030, filed Oct. 23, 2008 the disclosures of all of
which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to our discovery of a novel and
improved double-stranded ribo-nucleic acid (dsRNA) having specific
biological activities, which includes acting as a selective agonist
for activation of Toll-like receptor 3 (TLR3). Its smaller and
"rugged" molecular structure as measured by physico-chemical
techniques is resistant to molecular unfolding (i.e., denaturation)
and branching. This structure appears to be respon-sible for
increased efficacy of dsRNA in therapeutic applications and
improved biological activity (e.g., used as an immunoregulatory
agent).
BACKGROUND OF THE INVENTION
[0003] Ampligen.RTM. poly(I):poly(C.sub.12U) was developed as a
syn-thetic double-stranded ribonucleic acid (dsRNA) for therapeutic
applications based on an understanding of both the beneficial and
adverse effects induced by poly(I):poly(C) on the physiology of a
subject. Acting on the hypothesis that the nucleotide sequence
requirements for beneficial and adverse effects are different,
poly(I):poly(C.sub.12U) was developed by us to preserve the
beneficial aspects of dsRNA without the adverse effects of
poly(I):poly(C) by modifying the latter's structure with the
occasional introduction of uridylate into the poly(C) strand to
produce duplexes containing specifically-configured regions which
are not base paired (i.e., "mismatched") at the position of the
modification. These regions accelerate dsRNA hydrolysis and lessen
toxicity (Greene, 1984). On the other hand, the ability to induce
interferon synthesis was retained as long as the modified dsRNA
persisted for a half life of at least five minutes and the
frequency of random insertion into the poly(ribocytidylic acid)
strand was not greater than each 0.5 to 1.0 helical turn of
perfectly base-paired dsRNA (Brodsky, 1987).
[0004] While poly(I):poly(C.sub.12U) is stable in solution, it is
susceptible to hydrolysis like all other conventional nucleic
acids. The hydrolysis is highly dependent on nucleic acid
structure, as well as on the presence of nuclease and divalent
cations, pH, and temperature. RNA is more susceptible to hydrolysis
than DNA because of the 2'-OH group present in the former that
facilitates hydrolysis. Moreover, poly(I):poly(C.sub.12U) was
designed to degrade more rapidly than other dsRNA in a
nuclease-containing environment, such as blood and other tissue
fluids. Nucleic acids are initially stable in physiological salt
buffers at room temperature, but gradually begin to degrade with
time. This hydrolysis rate is temperature dependent, increasing
greatly at higher temperatures.
[0005] Properties of poly(I):poly(C.sub.12U) are characterized by
physico-chemical assays as shown in Table 1. Circular dichroism
(CD) (e.g., ellipticity, melting behavior) is used to characterize
the double-helical RNA structure, which is critical to potency.
Briefly, Toll-like receptor 3 (TLR3) is activated by dsRNA
(Alexopoulou, 2001), which leads to a host defense recruitment
sequence, ultimately producing type I interferons (Schroeder,
2005). Initiation of interferon production by dsRNA binding to TLR3
requires RNA helical structure (Bell, 2006). Although X-ray
diffraction and NMR alone are the definitive techniques to
determine RNA second-order structure, CD measurement with a
combination of scanning and thermal stress modes also can provide
precise characterization of the critical double-helical structure.
Indeed, minor changes in second-order structure of polynucleotides
have been measured by CD (Gray, 1995), including the effects of
ligand binding (Sumita, 2005).
TABLE-US-00001 TABLE 1 Biological Activity and Measured Attributes.
Measured Property Identity Attribute Activity Attribute
Conformation: Second Degree CD: Ellipticity Double-Stranded RNA:
binding to TLR3 integrity of helix CD: Melting Behavior:
Double-Stranded RNA: binding to TLR3 Melting Point 1/2 Width
integrity and binding to TLR3 uniformity of helix Composition and
Size Maximum Size No. of Repeat Units Tendency to form Branched
Structure C:U Ratio identity Tendency to form Branched
Structure
[0006] Therefore, circular dichroism can be employed to
characterize the therapeutic potency of specifically-configured
dsRNAs including poly(I):poly(C.sub.12U) and a new improved dsRNA
called Rugged dsRNA.
[0007] A problem of Ampligen.RTM., poly(I):poly(C12U), is its lower
than expected biological activity traced to a branching structure.
Our invention is the unexpected discovery of a new family of
improved dsRNAs having a specific physico-chemical structure and
highly specific biological activities, which includes acting as a
selective agonist for TLR3. This invention relates to the discovery
of this new and improved version of dsRNA with a superior
biological and therapeutic profile. The new and improved dsRNA,
called Rugged dsRNA, can be present in trace amounts within the
Ampligen.RTM. mixture. A method is disclosed to enrich the Rugged
dsRNA species so it becomes the dominant structure. Its rugged
structure as measured by physico-chemical techniques is resistant
to molecular unfolding (i.e., denaturation). Improvement in at
least one or more biological activities may result from the rugged
structure of this particular form of dsRNA. Other advantages and
improvements are described below, or would be appa-rent from the
disclosure herein.
[0008] The Eli Lilly and Company, U.S. Pat. No. RE 39,071E is an
example of a newly discovered biochemical/biological intermediate
in existing unimproved biochemical/biological mixtures of drugs
resulting in patentability. (See also U.S. Pat. No. 6,468,967 and
U.S. Pat. No. 6,852,689.)
SUMMARY OF THE INVENTION
[0009] It is an objective of the invention to provide new and
improved forms of double-stranded ribonucleic acid (dsRNA). Their
physico-chemical structure and biolo-gical activities are described
herein. A "rugged" dsRNA molecule resistant to unfolding (i.e.,
denaturation) of its helical structure and a reduced tendency to
form branched dsRNA molecular structures and having an improved
dsRNA activity as a selective agonist of Toll-like receptor 3
(TLR3). At least partial purification of Rugged dsRNA from other
dsRNA present after synthesis is expected to increase specificity
in its use as a medicament and thereby reduce adverse effects
attributable to the dsRNA that is not rugged.
[0010] Specifically-configured Ampligen.RTM. dsRNA mixture may be
of the general formula ribo(I.sub.n).ribo(C.sub.11-14U).sub.n, or
ribo(I.sub.n).ribo(C.sub.12U).sub.n, wherein the strands are
comprised of ribonucleotides (ribo) and n is an integer from about
500 to about 2,000 repeats. For example, a strand comprised of
poly(ribo-cytosinic.sub.11-14uracilic acid), or
poly(ribocytosinic.sub.12uracilic acid) may be partially hybridized
to an opposite strand comprised of poly(riboinosinic acid) such
that the two strands form an RNA double helix that is not paired at
the uracil base (i.e., mismatch).
[0011] After synthesis, Rugged dsRNA may be isolated from the
Ampligen.RTM. mixture by at least subjecting the partially
hybridized strands of a population of dsRNA to conditions that
denature most dsRNA (at least 50 mol %, at least 80 mol %, at least
90 mol %, or at least 95 mol %) in the population, and then
selection negatively or positively (or both) for dsRNA that remain
partially hybridized. The purity of Rugged dsRNA may thus be
increased from less than about 1-12 mol % (e.g., less than about 12
mol %) relative to all RNA in the population after synthesis. It is
preferred that the Rugged dsRNA be more than about 80-98 mol %
relative to all RNA present in the same mixture with the Rugged
dsRNA (at least 80 mol %, at least 90 mol %, at least 95 mol %, or
at least 98 mol %) after selection. The denaturing conditions to
unfold at least partially hybridized strands of dsRNA may comprise
appropriate choice of buffer salts, pH, solvent, temperature, or
any combination thereof. Conditions may be empirically determined
by observation of the unfolding or melting of the duplex strands of
ribonucleic acid. The yield of Rugged dsRNA may be improved by
partial hydrolysis of longer strands of ribonucleic acid, then
selection of (partially) hybridized stands of appropriate size and
resistance to denaturation.
[0012] The molecular weight of Rugged dsRNA may be from about 30
Kda to about 300 Kda, or from about 75 Kda to about 225 Kda.
Lengths of a single or both strands of Rugged dsRNA may be from
about 50 bases to about 500 bases, or from about 125 bases to about
375 bases. The number of helical turns made by duplexed RNA strands
of Rugged dsRNA may be from about 4.7 to about 46.7, or from about
11.7 to about 35 helical turns.
[0013] In another aspect, at least one or more different Rugged
dsRNA may be administered to a subject (e.g., human patient or
animal) in need of such treatment. Rugged dsRNA may be administered
at a dosage of from about 0.5 .mu.g to about 600 mg/dose. This
dosage may be administered once per week or month, or two or more
doses per week or month. Each dose (e.g., from about 0.5 .mu.g to
about 600 mg, from about 1 mg to about 100 mg, or from about 10 mg
to about 40 mg) may be provided to the subject without limitation
to the formulation of the pharmaceutical composition, or its route
of administration (although intravenous infusion is preferred). Use
of an effective amount of Rugged dsRNA to achieve a feeling of
improved health and may be continued until at least one symptom is
improved. The effective amount required to obtain such improve-ment
may be identical to or higher than the amount required for
maintenance of the effect(s).
[0014] The Rugged dsRNA may act specifically through a TLR3
receptor. The function and phenotype of dendritic cells may be
normalized in a subject (e.g., human patient or animal).
Administering at least an effective amount of one or more Rugged
dsRNA to a subject (e.g., human patient or animal) may thereby
decrease the number or reduce the severity of symptoms when the
subject is afflicted by a disease or other pathological condition.
Use of Rugged dsRNA may correct dendritic cell maturation
abnormalities in the subject without the hazard of inducing a
cytokine storm.
[0015] Antigen presenting cells (e.g., dendritic cells,
macrophages, B cells) and mucosal tissues (e.g., gastric or
respiratory epithelium) are preferred targets in the body for
Rugged dsRNA. One or more antigens may be presented to cells of the
immune system, and the antigen(s) should be susceptible to the
action of the Rugged dsRNA acting selectively as a TLR3 agonist.
Cells of the immune system, microbes, cancer cells, or other
transformed cells may be susceptible to specific cytokine response
patterns activated by Rugged dsRNA acting selec-tively as a TLR3
agonist. The Rugged dsRNA is preferably administered by intravenous
infusion; intradermal, subcutaneous, or intramuscular injection;
intranasal or intratracheal inhalation; or oropharyngeal or
sublingual application; or transocularly.
[0016] In another aspect, a medicament is provided as a
pharmaceutical composition. One or more different Rugged dsRNA may
be used for their beneficial effect(s) on a subject's health, as
selective TLR3 agonist(s), to treat a disease or other pathological
condition, or to manufacture medicaments or pharmaceutical
compositions to treat a disease or other pathological condition.
Optional inert ingredients of the composition include excipients
and a vehicle (e.g., saline buffer or water) as a single dose or a
multi-dose package (e.g., an injection vial or vials), and
instructions for their use. Processes for making and using the
pharmaceutical composition (medicament) are also provided. For
example, one or more different Rugged dsRNA may be formulated at a
concentration from about 0.05 mg/mL to about 0.25 mg/mL (e.g., 10
mg dissolved in 4 mL or 20 mg dissolved in 8 mL) in physiological
phosphate-buffered saline and stored at from 2.degree. C. to
8.degree. C. in a refrigerator under aseptic conditions.
[0017] Further aspects of the invention will be apparent from our
description of specific embodiments and the appended claims, and
generalizations thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A shows an HPLC chromatogram for
poly(I):poly(C.sub.12U). The minor peak (not integrated) centered
at a retention time of about 5.00 min is Rugged dsRNA. The first
major peak centered at a retention time of about 7.58 min is the
single-stranded poly(C.sub.12U). The second major peak centered at
a retention time of about 10.05 min is the single-stranded poly(I).
The molecular identity of each peak was determined by photodiode
array (PDA) analysis.
[0019] FIG. 1B is an HPLC chromatogram for lyophilized
poly(I):poly(C.sub.12U) showing aggregates lyophilization can
produce. Note that aggregation does not occur in the solution
process of FIG. 1C, which avoids lyophilization.
[0020] FIG. 1C shows a HPLC chromatogram of a sterile solution of
poly I:poly(C.sub.12U) also showing a novel 5 minute peak.
[0021] FIGS. 2A, 2B and 2C show PDA analyses of the three HPLC
peaks. Acetonitrile, which is used as a solvent, is responsible for
the strong absorbance at 230 nm. Absorbance at 245 nm indicates the
presence of poly(I); absorbance at 265 nm indicates the presence of
poly(C.sub.12U). FIG. 2A is PDA analysis of the peak centered at a
retention time of about 5.01 min, which contains both poly(I) and
poly(C.sub.12U) character. FIG. 2B is PDA analysis of the peak
centered at a retention time of about 7.58 min, which contains
poly(C.sub.12U). FIG. 2C is PDA analysis of the peak centered at a
retention time of about 10.05 min, which contains poly(I).
[0022] FIG. 3 is a circular dichroism (CD) of
poly(I):poly(C.sub.12U). The melting point of 64.degree. C.
represents the condition of 1/2 double stranded structure.
[0023] FIG. 4 is the CD wavelength scan of poly(I):poly(C.sub.12U).
The double stranded structure is characterized by two peaks at 245
nm and 278 nm, representing two chiral centers normally present in
fully double stranded poly I:poly(C.sub.12U). These centers
represent chirality due to base pair structure (278 nm) and the
base stacking which is associated with the formation of duplex
double helix.
[0024] FIG. 5 shows the circular dichroism of
poly(I):poly(C.sub.12U) with the characteristic chiral peaks at 245
nm and 278 nm
[0025] FIG. 6 shows a plot of the derivative of the thermal melt of
poly(I):poly(C12U). Integrity of the structure is characterized by
the melting point and the 1/2 width of this derivative profile,
both expressed as degrees C.
[0026] FIG. 7 shows by HPLC that preparation with heating abolishes
all double strand structure as reflected by loss of 245 nm peak
seen in FIG. 4 since the 245 nm peak is due to chiral base
stacking. However, analysis by circular dichroism shows that, as a
product of thermal stress, the 5 minute peak maintains both double
helix configuration and chiral centers in the backbone.
[0027] FIG. 8 shows a CD plot of a thermal melt of single stranded
poly(I). The chiral center for inosine provides a weak signal at
about 252 nm.
[0028] FIG. 9 shows a plot of the derivative of the thermal melt of
single stranded poly(I) There is no evidence of intra molecular
base stacking at thermal condition which would otherwise disrupt a
double helix.
[0029] FIG. 10 shows a CD plot of a thermal melt of single stranded
poly(C.sub.12U). A strong signal is apparent due to the chirality
of cytidine. However, the absence of a second peak at 245 nm shows
that intra molecular base stacking of Poly C12U does not occur.
[0030] FIG. 11 shows a plot of the derivative of the thermal melt
of single stranded poly(C.sub.12U). There is no evidence of intra
molecular base stacking at thermal condition which would otherwise
disrupt a double helix.
[0031] FIG. 12 shows a CD plot of a thermal melt of
poly(I):poly(C.sub.12U) and poly(I):poly(C). Base stacking is
evident in both compounds as indicated by the peak at 245 nm.
[0032] FIG. 13 shows a plot of the derivative of the thermal melt
of poly(I):poly(C.sub.12U) and poly(I):poly(C). Both compounds
exhibit the critical melting point for disruption of the double
helix. However, the lower melting point of Poly I:Poly C.sub.12U
illustrates a more labile character which in turn affords the
advantageous safety profile of the uridine substituted
compound.
[0033] FIG. 14 shows a CD wavelength scan of
poly(I):poly(C.sub.12U) and poly(A):poly(U). A very weak and
shifted single structure may be associated with the propensity for
chiral aggregation of poly(A):poly(U)
[0034] FIG. 15 shows a plot of the derivative of the thermal melt
of poly(I):poly(C.sub.12U) and poly(A):poly(U). Somewhat high
melting point is likely related to the aggregation tendency of
poly(A):poly(U) noted in FIG. 14.
[0035] FIG. 16 shows the derivative of a thermal melt of single
stranded poly(I):poly(C.sub.10U). The greater degree of Uridine
substitution (compare poly(I):poly(C.sub.12U, FIG. 6) has
compromised the double helical structure. The 1:12 ratio of U:C is
optimal, providing one interruption per helical turn.
[0036] FIG. 17 shows a CD plot of thermal melt of single stranded
poly(I):poly(C.sub.10U). Consistent with the lack of thermal melt
behavior (FIG. 16), the greater degree of Uridine substitution
(1:10 ratio of U:C, cf. 1:12 in poly(I):poly(C.sub.12U) has
abolished the base stacking signal at 245 nm.
[0037] FIGS. 18A, 18B and 18C show size exclusion chromatography of
complexes of TLR3-ECD and poly(I):poly(C.sub.12U) (FIG. 18A), the
receptor TLR3-ECD only (FIG. 18B), and the ligand
poly(I):poly(C.sub.12U) only (FIG. 18C).
[0038] FIG. 19 shows the effect of thermal stress (40.degree. C.)
on the size of dsRNA as measured by analytical centrifugation. The
decrease in sedimentation coefficient (S.sub.20,w) reflects a loss
of size due to hydrolysis.
[0039] FIG. 20 shows the effect of thermal stress (40.degree. C.)
upon the component strands of dsRNA (7 minute and 10 minute peaks)
and the Rugged dsRNA as measured by high performance liquid
chromatography (HPLC). Whereas the larger poly(I) and
poly(C.sub.12U) strands hydrolyze at 40.degree. C., the quantity of
Rugged dsRNA peak increases.
[0040] FIG. 21 shows the relative size of Ampligen.RTM. vs new
Rugged dsRNA (peak 5 minutes).
[0041] FIG. 22. Partial view of poly(I):poly(C.sub.12U) partially
hybridized strands and the interaction of bases of individual
poly(I) and the poly(C.sub.12U) strands. Molecular weight 1,100,000
da.
[0042] FIG. 23. Partial view of poly(I):poly(C.sub.12U) partially
hybridized strands and the interaction of bases of individual
poly(I) and the poly(C.sub.12U) strands. Molecular weight 286,000
da.
[0043] FIG. 24. is a lateral view of Rugged dsRNA (a minor
component in the unimproved Ampligen.RTM. mixture) bound to the
active site of the TLR3 homodimer (2 horseshoe shaped structures).
The C-terminal regions of each dimer face each other and bind to
the phosphate backbone of the dsRNA. The N-terminals of each TLR3
bind to opposite ends of the dsRNA with a minimum length of 50 bp
required for interaction with essential residues of TLR3 for
activation of intracellular signaling. Amino acids of TLR3 required
for binding of Rugged dsRNA are shown usingVan der Waals' radii
associated with the phosphate backbone.
[0044] FIG. 25. Illustrates the TLR3 homodimer complexed with
Rugged dsRNA (a minor component in the unimproved Ampligen.RTM.
mixture) as seen down the long axis of the dsRNA.
[0045] FIG. 26. Shows a typical example of a branched dsRNA
structure contained in the unimproved Ampligen.RTM. mixture.
[0046] FIG. 27 shows two typical examples of the branched dsRNA
structure contained in the unimproved Ampligen.RTM. mixture.
[0047] FIG. 28 shows a typical example of a more complexed branched
dsRNA structure contained in the unimproved Ampligen.RTM.
mixture.
[0048] FIG. 29 shows typical examples of unbranched dsRNA molecules
contained in the new/improved Rugged dsRNA.
DESCRIPTION OF SPECIFIC EMBODIMENTS
Definitions
[0049] dsRNA
[0050] Double-stranded (ds) RNA (ribonucleic acid) is chemically
very similar to DNA (deoxyribonucleic acid). It is also a long
molecule containing nucleotides linked together by 3'-5'
phoshodiester bonds. Two differences in its chemical groups
distinguish dsRNA from DNA. The first is a minor modification of
sugar component. The sugar of DNA is deoxyribose, where as RNA
contains ribose, which is identical to deoxyribose except for the
presences of an additional hydroxyl group. The second difference is
that RNA contains no thymine, but instead contains the closely
related pyrimidine, uracil. DsRNA forms from the hyridization of
two complementary polyribonucleotides forming a double helix
similar to that of DNA. The two strands of the double helix are
held together by hydrogen-bonded base pairs.
[0051] Ampligen.RTM.
[0052] Ampligen.RTM. is a particular dsRNA denoted Poly I: Poly
C.sub.12U, wherein one of the two polyribonucleotides is
polyriboinosinic acid and the other is polyribocytidylic 12,
uridylic acid. Thus, the pyrimidine building blocks of
Ampligen.RTM. are present in a ratio of 12 cytosines of each
uracil, while the complementary purine strand contains 13 inosine
residues. Within the double-stranded helical structure of
Ampligen.RTM. the pyrimidine, cytosine, hydrogen bonds with the
purine, inosine, while the pyrimidine, uracil, does not form any
hydrogen bonds. Therefore, a "mismatch" is created once for every
12 base pairs (bps) formed between the inosine and cytosine
residues. In contrast to Ampligen.RTM., Poly I:Poly C contains only
complementary inosine: cytosine base pairs. No uracil is present in
Poly I:Poly C and there are no mismatches.
##STR00001##
[0053] TLR3 (Toll-Like Receptor 3)
[0054] TLR3 is a receptor for a form of immunity called "innate
immunity" which recognizes double-stranded RNAs with a minimum size
of at least 50 base pairs. The size requirement or discrimination
of dsRNA by TLR3 prevents responses to non-microbial sources of
dsRNA micro (mi) RNA or transfer (t) RNA. TLR3 exists as a
horseshoe shaped monomer with a N-terminal, ligand-binding
extra-cytoplasmic domain (ECD), a transmembrane domain (TMD), and a
C-terminal cytoplasmic signaling domain (CSD). X-ray
crystallographic studies have provided structural data for the
TLR-3 ligand complex which consists of a TLR3 homo-dimer complexed
to dsRNA of at least about 50 consecutive base pairs. The formation
of the complex (FIG. 24) is believed to transmit a conformational
change in the CSD via the TMD connector that allows cytoplamic
signaling. Above 50 base pairs, binding affinity is a function of
size with a progressive increase in binding affinity with increased
length in linear non-branched dsRNA.
[0055] Rugged dsRNA
[0056] Rugged dsRNA is a novel form of dsRNA with a unique
composition and physical characteristics. Unlike the previously
known antiviral, Ampligen.RTM. (Poly I: Poly C.sub.12U), the new
and improved form of Rugged dsRNA (e.g., Poly I: Poly C.sub.30-35U
(preferably, Poly I: Poly C.sub.30U), wherein PolyC.sub.30-35U,
indicates a ratio, that is, that for every U there are 30-35 C's),
has an increased Ruggedness characterized by an increase resistance
to thermal denaturation and ribonuclease digestion. This improved
form of dsRNA also has a reduced tendency to form branched dsRNA
molecules which results in a increased bioactivity due to an
increased ability to bind TLR3 receptor. The minimal length of
Rugged dsRNA (termed the monomer unit) is about 50 base pairs
requiring about 4 to 5 (e.g., 4.7) helical turns (10.7 base pairs
are required for each complete turn of the helix) within its dsRNA
structure and represents the smallest or monomeric unit of Poly I:
Poly C.sub.30U, approximately 24,000 to 30,000 Daltons (a Dalton is
a unit of weight equal to the weight of a single hydrogen atom).
The maximal length of Rugged dsRNA is about 500 base pairs composed
of about 10 monomer units, requiring about 50 (e.g., 46.7) helical
turns and having a molecular weight of approximately 300,000
Daltons (e.g., about 225,000 Daltons). FIG. 21 shows the relative
size of the old unimproved Ampligen.RTM. vs. the New improved
Rugged dsRNA.
[0057] Branched dsRNA
[0058] Branching is seen as the major configuration of dsRNA
molecules within the Ampligen.RTM. unimproved mixture. FIGS. 26,
27, and 28 show typical examples of the branching molecules seen by
Transmission Electron Microscopy (TEM) in the unimproved
Ampligen.RTM. mixture of dsRNA molecules. The branching in the
molecules interferes with TLR3 binding. Ampligen.RTM. has 4-5 times
more molecules with .gtoreq.3 branched strands than Rugged dsRNA
and, therefore, Ampligen.RTM. has reduced bioactivity relative to
the new and improved Rugged dsRNA (e.g., Poly I: Poly C.sub.30-35U
(preferably, Poly I: Poly C.sub.30U)). This explains the increased
bioactivity of Rugged dsRNA compared to the unimproved
Ampligen.RTM. mixture. Typical examples of the major component of
Rugged dsRNA (unbranched dsRNA molecules) are shown in FIG. 29.
[0059] RNA Helix
[0060] A spiral structure of dsRNA with a repeating pattern
described by two simultaneous operations (rotation around the axis
and translation along the axis). DsRNA requires the translation of
10.7 base pairs to complete one complete rotation around the axis
(i.e. one helical turn).
[0061] Dalton
[0062] A unit of weight equal to the weight of a single hydrogen
atom. One Kda=1000 Daltons
[0063] Rugged dsRNA Monomer
[0064] The minimum active size of Rugged dsRNA comprised of about
40-50 base pairs, requiring about 4-5 (e.g., 4.7) helical turns and
having a molecular weight of approximately 24,000 to 30,000
Daltons.
[0065] Rugged dsRNA Polymeric Units
[0066] Rugged dsRNA composed of multiple monomeric units (or
fractional units thereof) of Rugged dsRNA up to a maximum of about
10 monomeric units held together by covalent phosphodiester bonds
in a linear structure. The maximum Rugged dsRNA molecular size
contains about 500 base pairs, requiring about 50 (e.g., 46.7)
helical turns and having a molecular weight of approximately
300,000 Daltons (e.g., about 225,000 Daltons).
[0067] Many uses of double-stranded ribonucleic acid (dsRNA) are
known. Efficacy of such treatments, which includes a decrease in
the number and/or a reduction in the severity of adverse effects of
nonselected populations of dsRNA, is improved by the use of at
least partially purified, Rugged dsRNA. The invention may be used
to treat a subject (e.g., human or animal, especially birds,
fishes, or mammals) with an incipient or established microbial
infection, to treat a subject for other pathological conditions
marked by abnormal cell proliferation (e.g., neoplasm or tumor), or
for use as an immunostimulant to treat the subject for a disease or
other pathological condition caused by at least infection, abnormal
cell proliferation, chronic fatigue syndrome, or cell damage from
autoimmunity or neurodegeneration. It is preferred that the amount
of Rugged dsRNA used is sufficient to bind Toll-Like Receptor 3
(TLR3) on immune cells of the subject. Innate or adaptive immunity
may be triggered thereby. Preferably, Rugged dsRNA may be used to
activate TLR3 selectively without activating other Toll-like
receptors like TLR4 or an RNA helicase like RIG-I or mda-5, or
without inducing an excessive pro-inflammatory response as seen
with the nonselective TLR3 agonist poly(I):poly(C) in a phenomenon
known as "cytokine storm" in the art.
[0068] The subject may be infected with at least one or more
bacteria, protozoa, or viruses. A pharmaceutical composition which
is comprised of Rugged dsRNA in an amount sufficient to bind to
TLR3 is administered to the subject. Infection of the subject is
reduced or eliminated thereby as assayed by decreased reco-very
time, increased immunity (e.g., increase in antibody titer,
lymphocyte proliferation, killing of infected cells, or natural
killer cell activity), decreased division or growth of the microbe,
or any combination thereof as compared to the subject not treated
with the Rugged dsRNA. The immunity induced by treat-ment is
preferably specific for the microbe, although inducing innate
immunity may also be efficacious.
[0069] An infection by a microbe may be treated. The microbe may
infect a human or animal subject. The infection may be incipient or
established. The microbe may be a bacterium, protozoan, or virus;
especially those that cause disease (i.e., pathogenic microbes).
Here, the terms "microbe" and "microorganism" are used
interchangeably.
[0070] The bacterium may be a species of the genus Bacillus (e.g.,
B. anthracis, B. cereus), Bartonella (B. henselae), Bordetella
(e.g., B. pertussis), Borrelia (e.g., B. burgdorferi), Brucella
(e.g., B. abortus), Campylobacter (e.g., C. jejuni), Chlamydia
(e.g., C. pneumoniae), Clostridium (e.g., C. botulinum, C.
difficile, C. perfringens, C. tetani), Corynbacterium (e.g., C.
amycolatum, C. diphtheriae), Escherichia (e.g., E. coli O175:H7),
Haemophilus (e.g., H. influenzae), Heliobacter (e.g., H. pylori),
Klebsiella (K. pneumoniae), Legionella (e.g., L. pneumophila),
Listeria (e.g., L. monocytogenes), Mycobacterium (e.g., M. avium,
M. bovis, M. branderi, M. leprae, M. tuberculosis), Mycoplasma
(e.g., M. genitalium, M. pneumoniae), Neisseria (e.g., N.
gonorrheae, N. meningitidis), Pneumocystis (e.g., P. carinii),
Pseudomonas (P. aeruginosa), Rickettsia, (e.g., R. rickettsia, R.
typhi), Salmonella (e.g., S. enterica), Shigella (e.g., S.
dysenteriae), Staphylococcus (e.g., S. aureus, S. epidermidis),
Streptococcus (e.g., S. pneumoniae, S. pyogenes), Treponema (e.g.,
T. pallidum), Vibrio (e.g., V. cholerae, V. vulnificus), or
Yersinia (e.g., Y. pestis). These include Gram-negative or
Gram-positive bacteria, chlamydia, spirochetes, mycobacteria, and
mycoplasmas.
[0071] The protozoan may be a species of the genus Cryptosporidium
(e.g., C. hominis, C. parvum), Entamoeba (e.g., E. histolytica),
Giardia (e.g., G. intestinalis, G. lamblia), Leishmania (e.g., L.
amazonensis, L. braziliensi, L. donovani, L. mexicana, L. tropica),
Plasmodium (e.g., P. falciparum, P. vivax), Toxoplasma (e.g., T.
gondii), or Trypanosoma (e.g., T. bruci, T. cruzi).
[0072] The virus may be a DNA or RNA virus that infects humans and
animals. DNA viruses include those belonging to the Adenoviridae,
Iridoviridae, Papillomaviridae, Polyomavirididae, and Poxyiridae
families (Group I double-stranded DNA viruses); the Parvoviridae
family (Group II single-stranded DNA viruses). RNA viruses include
those belonging to the Birnaviridae and Reoviridae families (Group
III double-stranded RNA viruses); the Arteriviridae, Astroviridae,
Caliciviridae, Hepeviridae, and Roniviridae families (Group IV
positive single-stranded RNA viruses); and the Arenaviridae,
Bornaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, and
Rhabdoviridae families (Group V negative single-stranded RNA
viruses). Rugged dsRNA may also be used to treat infection by DNA
viruses from the Herpesviridae family and RNA viruses from the
Flaviviridae, Hepadnaviridae, Orthomyxoviridae, Picornaviridae,
Retroviridae, and Togaviridae families.
[0073] The subject may be afflicted by a disease or pathological
condition that is characterized by abnormal cell proliferation
(e.g., neoplasm or tumor, other transformed cells). A
pharmaceutical composition which is comprised of Rugged dsRNA in an
amount sufficient to bind to TLR3 is administered to the subject.
Disease, symptoms thereof, their number, or their severity in the
subject may be reduced or eliminated thereby as assayed by improved
morbidity or morta-lity, increased immunity (e.g., increase in
antibody titer, lymphocyte proliferation, killing proliferating or
transformed cells, or NK cell activity), decreased division or
growth of proliferating or transformed cells, or any combination
thereof as compared to the condition of a subject not treated with
Rugged dsRNA.
[0074] The subject's cells undergoing the abnormal proliferation
may be a neoplasm or tumor (e.g., carcinoma, sarcoma, leukemia,
lymphoma, glioma), especially cells transformed by a tumor virus
(e.g., DNA or RNA virus carrying a trans-forming gene or oncogene)
or otherwise infected by a virus associated with cancer. For
example, Epstein-Barr virus is associated with nasopharyngeal
cancer, Hodgkin's lymphoma, Burkitt's lymphoma, and other B
lymphomas; human hepatitis B and C viruses (HBV and HCV) are
associated with liver cancer; human herpesvirus 8 (HHV8) is
associated with Kaposi's sarcoma; human papillomaviruses (e.g.,
HPV6, HPV11, HPV16, HPV18, or combination thereof) are associated
with cervical cancer, anal cancer, and genital warts; and human
T-lymphotrophic virus (HTLV) is associated with T-cell leukemia and
lymphoma. Cancers include those originating from the
gastrointestinal (e.g., esophagus, colon, intestine, ileum, rectum,
anus, liver, pancreas, stomach), genitourinary (e.g., bladder,
kidney, prostate), musculoskeletal, nervous, pulmonary (e.g.,
lung), or reproductive (e.g., cervix, ovary, testicle) organ
systems.
[0075] Dendritic cell maturation may be induced in the subject.
Immature dendritic cells, which are capable of antigen uptake, may
be induced to differentiate into more mature dendritic cells, which
are capable of antigen presentation and priming an adaptive immune
response (e.g., antigen-specific T cells). During their conversion
from immature to mature dendritic cells, they may at least change
cell-surface expression of major histocompatibility complex (MHC)
molecules, costimulatory molecules, adhesion molecules, or
chemokine receptors; decrease antigen uptake; increase secretion of
chemokines, cytokines, or proteases; grow dendritic processes;
reorganize their cytoskeleton; or any combination thereof. They may
be induced to migrate to sites of inflammation or lymphoid tissue
through blood or lymph to bring microbes, neoplastic or tumor
cells, or other transformed cells into proximity.
[0076] The subject may be vaccinated against at least infection
(e.g., microbial infection) or cancer. The vaccine can be, for
example, an anti-viral anti-protozoan or anti-bacterial vaccine. In
some cases, e.g., virus-induced cancers, both infection and cancer
may be treated. Immediately before, during, or immediately after
vaccination (e.g., within 10 days of vaccination), a medicament or
pharmaceutical composition which is comprised of Rugged dsRNA in an
amount sufficient to bind to TLR3 is administered to the subject.
The immune response to a vaccine or dendritic cell preparation is
stimulated thereby. The vaccine or dendritic cell preparation may
be comprised of killed, fixed, or attenuated whole microbes or
cells (e.g., proli-ferating or transformed); a lysate or purified
fraction of microbes or cells (e.g., proliferating or transformed);
one or more isolated microbial antigens (e.g., native, chemically
synthesized, or recombinantly produced); or one or more isolated
tumor antigens (e.g., native, chemically synthesized, or
recombinantly produced). In situ vaccination may be accomplished by
the subject's production of antigen at a site or circulation
thereto (e.g., produced in a natural infection or cell growth, or
shed antigen), and Rugged dsRNA acting as an adjuvant thereon. The
dsRNA can be administered sequentially or concurrently with the
vaccine.
[0077] Rugged dsRNARugged dsRNA
[0078] Rugged dsRNA as at least a portion of a medicament or
formulated with other compatible components in a pharmaceutical
composition may be administered to a subject (e.g., human patient
or animal, especially birds, fishes, or mammals) by any local or
systemic route known in the art including enteral (e.g., oral,
feeding tube, enema), topical (e.g., device such as a nebulizer for
inhalation through the respiratory system, skin patch acting
epicutaneously or transdermally, suppository acting in the rectum
or vagina), and parenteral (e.g., subcutaneous, intravenous,
intramuscular, intradermal, or intraperitoneal injection; buccal,
sublingual, or transmucosal; inhalation or instillation
intranasally or intratracheally). The Rugged dsRNA may be
micronized by milling or grinding solid material, dissolved in a
vehicle (e.g., sterile buffered saline or water) for injection or
instillation (e.g., spray), topically applied, or encapsulated in a
liposome or other carrier for targeted delivery. Dissolving the
Rugged dsRNA in water for injection (WFI) and injection of the
composition into the subject are preferred. A carrier may be used
to target the Rugged dsRNA to the TLR3 receptor on antigen
presenting cells and epithelium. For example, immature dendritic
cells may be contacted in skin, mucosa, or lymphoid tissues. It
will be appreciated that the preferred route may vary with the age,
condition, gender, or health status of the subject; the nature of
disease or other pathological condition, including the number and
severity of symptoms; and the chosen active ingredient.
[0079] Formulations for administration (i.e., pharmaceutical
compositions) may include aqueous solutions, syrups, elixirs,
powders, granules, tablets, and capsules which typically contain
conventional excipients such as binding agents, fillers,
lubricants, disintegrants, wetting agents, suspending agents,
emulsifying agents, preservatives, buffer salts, flavoring,
coloring, and/or sweetening agents. It will be appreciated that the
preferred formulation may vary with the age, condition, gender, or
health status of the subject; the nature of disease or other
pathological condition, including the number and severity of
symptoms; and the chosen active ingredient.
[0080] The recommended dosage of Rugged dsRNA will depend on the
clinical status of the subject and the physician's or
veterinarian's experience treating the disease or other
pathological condition. Rugged dsRNA may be dosed at from about 0.5
.mu.g to about 600 mg, from about 1 mg to about 100 mg, or from
about 10 mg to about 40 mg in a subject (e.g., body mass of about
70-80 Kg for a human patient) on a schedule of once to thrice
weekly (preferably twice weekly), albeit the dose amount and/or
frequency may be varied by the physiccian or veterinarian in
response to the subject's symptoms. Nucleic acid in solid form may
be dissolved in physiological phosphate-buffered saline and then
infused intravenously. Cells or tissues that express TLR3 are
preferred sites in the subject for delivering the nucleic acid,
especially antigen presenting cells (e.g., dendritic cells,
macrophages, B lymphocytes) and endothelium (e.g., endothelial
cells of the respiratory and gastric systems). It will be
appreciated that the preferred dosage may vary with the age,
condition, gender, or health status of the subject; the nature of
disease or other pathological condition, including the number and
severity of symptoms; and the chosen active ingredient.
[0081] Dendritic cells which act as sentinel cells possess
molecular surface structures that recognize pathogen-associated
molecular patterns (PAMPs). These PAMPs include a set of Toll-like
receptors (TLRs) that specifically recognize all dsRNA. In
particular, dsRNA is a selective agonist of TLR3. Rugged dsRNA may
be used as a selective agent for activation of TLR3. Dysfunction in
co-stimulatory molecule (e.g., CD80, CD83, CD86) signaling in
dendritic cells may be associated with the disease or other
pathological condition to be treated. This abnormality may be
normalized by using Rugged dsRNA as a selective TLR3 agonist. The
effects of Rugged dsRNA may be inhibited or blocked by mutation of
the TLR3 gene (e.g., deletion), down regulating its expression
(e.g., siRNA), binding with a competitor for TLR3's ligand-binding
site (e.g., neutralizing antibody) or a receptor antagonist, or
interfering with a downstream component of the TLR3 signaling
pathway (e.g., MyD88 or TRIF).
[0082] Circular dichroism (CD) is a physico-chemical technique for
characterizing the conformation of specifically-configured dsRNA.
It can also be used as a surrogate for binding of
Ampligen.RTM.dsRNA as a receptor agonist to its receptor TLR3.
Furthermore, the helical structure of Rugged dsRNA and the
structural requirements for binding of dsRNA to TLR3 can be
precisely characterized by CD.
[0083] Other physico-chemical techniques that may be used to
characterize Rugged dsRNA are reverse phase chromatography, PDA
(photodiode array) analysis, gas pressure chromatography (GPC),
specific ligand binding to TLR3 receptor, and sedimentation
velocity measured by ultracentrifugation.
[0084] Rugged dsRNA provides a selective agent for dissecting out
the effects of TLR3 activation on the immune system that was not
previously available with such potency. Other agents like TLR
adapters MyD88 and TRIF mediate signaling by all TLR or TLR3/TLR4,
respectively. Thus, activation or inhibition of signaling through
MyD88 or TRIF would not restrict the biological effects to those
mediated by TLR3. Since the presence of TLR3 and its signaling is a
requirement for Ampligen.RTM. poly(I):poly(C.sub.12U) to act as a
receptor agonist, one could assay for the absence of TLR3
mutations, the presence of TLR3 protein, intact TLR3-mediated
signaling, or any combination thereof in the cell or tissue of a
subject prior to administration of the agonist. Such confirmation
of TLR3 activity can be performed before, during, or after
administration of the agonist. The agonist can be used to restrict
the immune response to activation of TLR3 without activating other
Toll-like receptors or RNA helicases. For example, abnormal
cytokine (e.g., IFN-.alpha., IFN-.beta., IFN-.gamma., TNF-.alpha.,
IL-6, IL-10, IL-12) production or co-stimulatory molecule (e.g.,
CD80, CD83, CD86) signaling may have resulted from at least
infection by the microbe, abnormal cell proliferation, autoimmune
damage, or neurodegene-ration. This abnormality may be remodulated
by using Rugged dsRNA as a selective agonist of TLR3. Antigen
presentation may be improved by conjuga-ting the antigen (or a
peptide analog thereof) to a ligand (or a receptor) that
specifically binds to the cell surface (especially a component of
the endosome-phagosome internalizing pathway) of one or more
antigen presenting cells. The specific binding molecule may be an
antibody to a cell surface molecule, or a derivative thereof (e.g.,
Fab, scFv).
[0085] Expression of CD80, CD83, and CD86 may be analyzed by flow
cytometry using fluorescently-labeled antibodies. Following
overnight shipment, blood samples are stained within one hour of
receipt. Conventional techniques are used for lysis of red blood
cells and cell marker analyses by flow cytometry. Dendritic cells
are identified based on low level expression of lymphocyte,
monocyte, and NK cell markers along with high HLA-DR expression.
Dendritic cells may also characterized according to CD11c and CD123
expression. Monocytes are identified by side scatter analysis and
expression of a monocyte lineage marker. Analyses of CD80, CD83,
and CD86 expression are performed after cell type identification.
Measurements from healthy volunteers serve as controls, and they
would indicate normal distribution and levels of marker expression
for mature dendritic cells such as CD80, CD83, and CD86.
[0086] Rugged dsRNA can be prepared by chromatographic separation,
wherein Rugged dsRNA is separated from the majority of unimproved
dsRNA.
[0087] An exemplary chromatographic procedure involves the
following steps:
[0088] 1. Binding of all forms of unimproved dsRNA to a reversed
phase chromatography resin. The resin contains hydrophobic
functional groups. In the current example the resin is Phenomenex,
Polymerx, RP-1, but may alternately be selected from a range of
commercially available hydrophobic resins as directed by
conventional practice. Resin particle size is 10 microns in the
current example, but may be varied widely to afford optimal
separations or to produce Rugged dsRNA at differing scales of
operation.
[0089] 2. The dsRNA is injected as a solution of 2.5 mg/ml in
phosphate buffered saline, pH 7.0, in the current example. The
concentration and pH range can be varied to include alternative
appropriate buffering systems utilized by those familiar with the
art. The diluent can also contain stabilizing elements such as
magnesium. The ionic strength, 200 mM in the current example, can
be varied to achieve optimal loading and separation performance
conditions as directed by conventional practices.
[0090] 3. The mobile phase composition contains a relatively polar
organic solvent to modulate binding during loading and the
subsequent, gradient elution. In the current example, the mobile
phase contains acetonitrile at an initial loading concentration of
6-8 vol % which produces enriched Rugged dsRNA fractions during the
gradient elution to 20 volume %. Alternative solvent systems can be
selected having optimal solvent concentration ranges as directed by
conventional practice.
[0091] 4. The sample loading is 13 mg unimproved dsRNA/ml column.
Loading can be decreased to afford tighter fractionation of Rugged
dsRNA. Alternative combinations of solvent and ionic strength will
require individually determined optimal loading conditions as
directed by conventional practice.
[0092] 5. The mobile phase flow rate for sample loading and elution
range is 5 ml/min in the current example (3 column volumes/hr). The
flow rate can be varied to achieve optimal conditions for differing
scales of operation and resin particle size as directed by
conventional practice.
[0093] 6. Elution is achieved by imposing a solvent gradient to the
composition of the mobile phase. In the current example, the
gradient of acetonitrile composition is increased from the loading
condition of 6-8% to 20%, over a period of 14 minutes. The type of
solvent and the gradient profile can be altered based upon the
character of the hydrophobic functionality as determined by
conventional practices.
[0094] 7. In the current example, improved Rugged dsRNA is
collected at 10-12 minutes or 0.25-0.30 column volumes. The peak
location can vary depending upon alternative choices of solvent,
flow rate, column type as provided above.
[0095] One skilled in the art will appreciate that separations
suitable for isolation of Rugged dsRNA can be scaled up for
commercial purposes.
EXAMPLES
[0096] Synthesis of single-stranded poly(I) and poly(C.sub.12U)
began with enzymatic polynucleotide synthesis of the
polynucleotides from the respective mononucleotide starting
materials: inosine for poly(I); cytidine (C) and uridine (U) for
poly(C.sub.12U). Then repetitive extraction and precipitation steps
were used to remove residual impurities. The reaction solutions
containing the products were concentrated by ultrafiltration and
extracted with phenol four times. The concentrated and extracted
solutions were precipitated, dissolved, and re-precipitated from
aqueous ethanol (50:50). Whereas precipitated poly(I) was separated
by centrifugation, the supernatant (waste) liquid phase of adherent
poly(C.sub.12U) was simply removed by aspiration. The precipitated
pastes were redissolved, then concentrated, diafiltered, and
further concentrated. The final bulk solutions containing
polynucleotide was filtered. The filtered solution was freeze dried
and the raw materials were stored frozen.
[0097] Enzymatic Synthesis.
[0098] The enzymatic synthesis used in the manufacturing process is
dependent on the enzyme polynucleotide phosphorylase to synthesize
polyinosinic acid and polycytidilic.sub.12uridilic acid from their
respective starting materials: cytidine 5'-diphosphate, trisodium
salt (CDP.Na.sub.3), uridine 5'-diphosphate, disodium salt
(UDP.Na.sub.2) and inosine 5' diphosphate, trisodium salt
(IDP.Na.sub.3).
[0099] The enzyme catalyzes polynucleotide formation in a
reversible reaction using Mg.sup.++ as a co-factor and ATP as a
source of energy. Polynucleotides were synthesized in the 5' to 3'
direction with concurrent liberation of inorganic phosphate.
Maximum yield was limited by the equilibrium between synthesis and
reverse rates, degradative reaction (phosphorolysis). The progress
of the reaction was followed by measuring the consumption of CDP or
IDP. Viscosity of the reaction solution was also monitored.
Purified water was filtered into the tank. The following
ingredients were added to the tank one at a time with mixing: TRIS
(hydroxymethyl) aminomethane, urea, magnesium chloride hexahydrate
(MgCl.6H.sub.20), and ethylenediaminetetraacetic acid (edetate),
disodium salt (EDTA.Na.sub.2). Raw material mononucleotides were
also added.
[0100] Each ingredient was dissolved before the next one was added.
After all of the ingredients were added, the solution was mixed for
a minimum of 10 minutes. The mixture was then adjusted and purified
water was added to obtain a final batch volume. This pre-enzyme
reaction mixture was sampled for initial CDP or IDP concentration.
The enzyme polynucleotide phosphorylase was added with mixing,
whereupon the synthesis of polynucleotide commenced. Also, the
viscosity profile at the optimal enzyme concentration must exhibit
the usual increase in viscosity over time without significant
decrease at the conclusion of the batch reaction; significant
decrease in viscosity would indicate undesired degradation of
polynucleotide. After the optimized amount of enzyme was added to
the production batch, enzymatic synthesis progressed under
constant, controlled agitation. The consumption of CDP or IDP was
monitored approximately every hour. The reaction was terminated by
the addition of a stop solution. Viscosity was also monitored, for
information only, during the process.
[0101] Concentration of Reaction Solution.
[0102] To minimize the required volume of phenol for extractions,
the reaction product solution was concentrated.
[0103] Extraction of dsRNA Mixture.
[0104] Residual enzyme was removed predominately by phenol
extraction. The concentrated single stranded RNA reaction product
solutions were transferred into seperate extraction tanks and 2M
TRIS and sodium dodecyl sulfate (SDS) were added. After at least 5
minutes of mixing, liquefied phenol was added and the two phase
solution was mixed to disperse the phenol phase in the aqueous
phase. SDS was employed as a surface-active agent to facilitate
dissolution of denatured protein into the phenol phase; TRIS was
required to buffer the solution at an optimal pH for
polynucle-otide stability. The extraction mixture stands without
mixing for predetermined settling times to afford coalescence of
the phases. The lower phenol waste phase is then pumped into
containers for disposal. The location of the phenol cut was
important in order to effectively separate phenol and protein from
the upper, product phase, which contains single stranded RNAs. The
phenol phase and an intermediate "rag" layer, which contains
denatured protein solids, were discarded by visually observing the
liquid flowing through the site glass at the tank outlet. When the
phenol and rag layer disappeared and only product phase was
observed, the outlet valve was closed and the phenol cut is
consi-dered complete.
[0105] Precipitation of Single Stranded RNAs.
[0106] Contaminating phenol, SDS, and other salts remaining in
solution were removed by precipitation with denatured ethyl
alcohol. The single stranded RNA concentrated solution was pumped
into the precipitation tank. The denatured alcohol was added and
after mixing the precipitate was separated.
[0107] Concentration and Diafiltration.
[0108] Remaining bulk salts, a small amount of unreacted
mononucleotide, and phenol were removed by diafiltration against
water. The precipitate was dissolved in the original precipitation
vessel with gentle mixing and heating. After dissolving, the
solution was then concentrated and diafiltered against water for
injection (WFI). The solution was filtered prior to freeze
drying.
[0109] Freeze Drying.
[0110] The filtered single stranded RNA material was loaded into a
freeze drier. The material was frozen, and a vacuum was then
applied. The product was considered dry when the programmed cycle
was complete.
[0111] Manufacture of dsRNA, Sterile Solution, for Intravenous
Infusion.
[0112] The single stranded RNAs were dissolved in
phosphate-buffered saline. Equal molar amounts were mixed in an
annealing step, and cooled to room temperature. The solutions were
sterile filtered.
[0113] Preparation of Buffer Vehicle, Excipient Solution.
[0114] WFI was added to the tank. The excipients were added to the
tank, and mixed. After mixing, the batch was sampled for pH and
osmolality. Quality control must be within in-process limits prior
to use for formulating the solutions.
[0115] Formulating Poly(I) and Poly(C.sub.12U) Solutions.
[0116] An initial quantity of buffer solution was subdivided
according to the batch formula and was filtered into the tank. The
single stranded RNAs were added to the buffer solution, and
dissolved by mixing. The temperature of the solution was increased
and maintained with mixing. The solution is then recirculated.
[0117] Annealing of Poly I:Poly C.sub.12U Strands.
[0118] Equivalent quantities of poly(I) and poly(C.sub.12U) were
transferred to the tank. With continual mixing, the temperature of
the solution was increased. Samples were removed and tested for
potency, and pH.
[0119] Sterile Filtration. The formulated bulk was sterile filtered
in-line into a steam sterilized surge vessel.
[0120] Filling Operations.
[0121] The filling operation was performed. After each vial was
filled, a sterile stopper is used to stopper the vial. Stoppered
vials were then conveyed from the aseptic processing area where
they were sealed.
[0122] Rugged dsRNA was isolated from the annealed
poly(I):poly(C.sub.12U) mixture, which was prepared according to
the above, by either analytical or preparative high performance
liquid chromatography (HPLC) as a substantially purified and
pharmaceutically-active molecule. Its molecular weight is from
about 30 Kda to 300 Kda and is about 50 to 500 base pairs in length
with about 4.7 to 46.7 complete turns of the RNA helix. It is only
from about 4 mol % to about 16 mol % of an unfractionated
Ampligen.RTM. composition. Most dsRNA (over 80 mol % molecules)
after synthesis have a molecular weight of about 1.2 Mda and are
about 2000 base pairs in length with about 187 complete turns of
the RNA helix. The Rugged dsRNA in the 5 min HPLC peak is about 4
to 40 times smaller than the bulk of the dsRNA, and more closely
fits the ligand binding site of its cell surface receptor
(TLR3).
[0123] Due to its structure, Rugged dsRNA is unusually resistant to
disruption of its RNA double helix and molecular unfolding. Thus,
Rugged dsRNA under the assay conditions described herein has about
100- to about 1,000% greater bioactivity than the same weight of
unimproved Ampligen.RTM. poly(I):poly(C.sub.12U).
(a) Protection by Mismatched dsRNA is by Selective Activation of
TLR3
[0124] TLR3 Activation is Linked to Expression of
IFN-.alpha./.beta., IL-6, or IL-12.
[0125] The relationship between IFN expression through TLR3
activation by dsRNA was established by Alexopoulou (2001) using
293T cells that express different Toll-like receptors (human TLR1,
TLR2, TLR3, TLR4, TLR5, TLR6, or TLR9). Only those cells containing
human TLR3 showed marked expression of IFN-.alpha./.beta., IL-6 or
IL-12 when stimulated with poly(I):poly(C).
[0126] Mismatched dsRNA Induces Host Defense Gene Modulation
through Highly Selective Activation of TLR3. To understand the
relationship of the TLR3-dependent innate immune response to viral
protection, Gowen (2007), subjected TLR3-deficient mice to dsRNA
and measured expression of IFN-.alpha./.beta., IL-6, and IL-12. The
mice were also subsequently challenged by exposure to Punta Toro
virus (PTV). Protection from the viral challenge was exquisitely
sensitive to treatment with mismatched dsRNA. Viral protection
conferred by mismatched dsRNA was completely abolished for the case
of TLR3-deficient mice. When contrasted to the partial but
significant effectiveness of mismatched dsRNA in TLR3.sup.-/- mice,
it is clear that the structural substitutions of uridine in the
cytidine strand of mismatched dsRNA are responsible for the highly
specific, TLR3-dependent pathway. Furthermore, measurements of
IFN-.alpha./.beta. and IL-6 directly link PTV protection or lack
thereof to the modulation of these cytokines.
[0127] This selective targeting of the TLR3 signaling pathway
represents a significant advantage for therapeutic applications of
mismatched dsRNA as compared to other possible cytosolic mechanisms
such as, for example, the use of unsubstituted dsRNA
poly(I):poly(C) to stimulate cytokine production through RNA
helicases such as MDA-5 and RIG-1 (Pichlmair, 2006).
(b) Binding of dsRNA to TLR3 Requires a Helical Non-Branching
[0128] Conformation of dsRNA
[0129] Molecular Model of the human TLR3 dimer ecodomain and its
Rugged dsRNA ligand. The X-ray crystallographic structure of the
mouse TLR3 dimer/dsRNA complex (3CIY) and the human TLR3 monomer
(1ZIW) provided the coordinates for the model of human TLR3 with
Rugged dsRNAbound to its active site (Liu, 2008; Bell, 2005).
Molecular modeling used Accelrys' Discovery Suite software (version
2.5.5). The dsRNA structure of 3CIY was mutated in situ to the
respective poly I and poly C.sub.30U chains (Rugged dsRNA)
maintaining the phosphate backbone linear translational coordinates
of the X-ray crystallographic structure that is represented as
lines for its structure. A uridine in the poly C.sub.30U strand is
at position U23. The coordinates of the human TLR3 monomer were
used to replace each homodimer. Several unacceptable close van der
Waals contacts for the human TLR3 crystal coordinates were resolved
by an alternate rotamer conformation of the individual amino acid R
group. The TLR3 homodimers are represented as structural elements
with the blue arrows signifying direction of .beta.-sheets and the
red cylinders signifying .alpha.-helices. FIG. 24 is viewed from a
lateral view of Rugged dsRNA bound to the active site of the TLR3
homodimer. The C-terminal regions of each dimer face each other and
bind to the phosphate backbone of the dsRNA. The N-terminals of
each TLR3 bind to opposite ends of the dsRNA with a minimum length
of 50 bp required for interaction with essential residues of TLR3
for activation of intracellular signaling. Amino acids of TLR3
required for binding of Rugged dsRNA are shown as Van der Waals'
radii associated with the phosphate backbone. FIG. 25 illustrates
the TLR3 homodimer complexed with Rugged dsRNAas seen down the long
axis of the dsRNA.
[0130] TLR3 Binding Site.
[0131] Studying the structure of native TLR3 crystals, Choe (2005)
found that TLR3 is a large horseshoe-shaped, right-handed, solenoid
structure comprised of 23 leucine-rich repeats. The glycosylated,
convex surface and negatively-charged concave surfaces are unlikely
binding sites for dsRNA. Consequently, they proposed that dsRNA
binding occurs at positively-charged patches located on the lateral
face.
[0132] Using mutational analysis, Bell (2005, 2006) modified
putative TLR3 binding sites in the positively-charged patches and
observed formation of a dsRNA/TLR3 complex by size-exclusion
chromatography. Despite the presence of numerous positively-charged
residues, only two amino acids N541 and H539 were required for
binding. The amido group of H539 could interact with dsRNA by
hydrogen binding. Proximity of the second positively-charged
residue N541 was also important, albeit the role of this amino acid
was not as clear. Mutation to negatively-charged aspirate prevented
binding by dsRNA, however conversion to a neutral alanine residue
had no effect on binding by dsRNA.
[0133] Binding to TLR3 Requires Helical Conformation of dsRNA.
[0134] Following the structural determination of most likely dsRNA
binding surfaces on TLR3, Choe (2005) further proposed that the
helical symmetry of dsRNA structure is necessary for the creation
of the symmetric dimer form of activated, membrane-associated TLR3.
In the ternary complex at the membrane surface, two symmetrically
opposed TLR3 molecules are linked to either side of the helix of
the common dsRNA.
[0135] As discussed above, using mutational analysis, Bell (2005,
2006) defined two highly conserved residues (N541 and H539) that
are necessary for binding of dsRNA to TLR3. Moreover, the
constraining requirement for ligand binding to both of these
residues of TLR3 is satisfied only by the minor groove architecture
of the (helical) conformation of dsRNA. When dsRNA phosphate binds
in proximity to the charge sensitive H539, then the amide side
(H541) becomes aligned with hydrogen bonding site of a 2' dsRNA
hydroxyl only when helical dimensions are utilized.
(c) Helical Conformation of dsRNA and Alteration Thereto
Accompanying Ligand Interactions are Precisely Characterized by
Circular Dichroism
[0136] Circular dichroism provides detailed information concerning
the secondary, helical structures of dsRNA or alterations thereof
which accompany ligand binding; as well as structural changes
caused by enzymatic hydrolysis and addition of metal ions. Also, in
the thermal stress mode, conformational information imparted by CD
provides valuable insights to explain RNA stability.
[0137] dsRNA Characterization.
[0138] Gray (1995) showed that CD, applied in the mixing curve
protocol, complemented ultraviolet absorption measurements to
determine the stoichiometry of duplex RNA (A-G:C-T(U)). In this
approach, the optical property is analyzed as a function of the
added ratios of individual strands. The magnitudes of CD difference
plots were maximal for 50:50 mixtures. Further, isodichroic
behavior correlated with the formation of higher ordered or intra
strand structures.
[0139] Ligand Interactions.
[0140] Ghazaryan (2006) studied the ligand interaction of dsRNA
with a family of positively charged pyridinium porphyrins. From CD
measurements they found that minor modifications of porphyrin
structure led to profound differences in mode of their attachment
to the double helical structure. Whereas TEtOHPyP4 associated by
intercalation, TMetAlPyP4 attached by forming an external,
self-stacking assembly.
[0141] Using circular dichroism, Brown (2002) showed that ADAR1, a
human dsRNA, (chimeric) converted from the A to Za form upon
binding to adenosine deaminase. Corroboration was provided by
crystallization of the complex and Raman spectroscopy. Sorrentino
(2003) studied the powerful enzymatic degradation of dsRNA by human
pancreateic ribonuclease (HP-RNase). Circular dichroism of the
RNA/enzyme complex revealed that multi-site attachment of the dsRNA
to HP-RNase was responsible for the destabilization of the RNA
helix.
[0142] Stability of dsRNA.
[0143] Studying the rRNA component of the 70S ribosomal complex,
Sumita (2005) showed that psuedouridine substitutions stabilized
the dsRNA helix based upon structural information provided by
circular dichroism (CD). Specifically, pseudouridine substitutions
created duplex regions with closing base pairs and water-mediated
hydrogen bonds. Stabilization by Mg.sup.++ was also characterized
by CD in this study. Investigating the stability of RNADNA hybrids
with variants in base composition, Lesnik (1995) showed that more
stable hybrids retain ellipticity at 210 nm, a wavelength
characteristic of the single component RNA band (A-form hybrid). In
contrast, less stable hybrids showed lowered 210 nm ellipticity,
values which were intermediate between the RNA and DNA
components.
[0144] A double-stranded RNA composition may be analyzed by high
performance liquid chromatography (HPLC) as shown in FIGS. 1A, 1B
and 1C. Analysis of a representative lot of Ampligen.RTM.
poly(I):poly(C.sub.12U) mixture resulted in two distinct peaks: one
with retention times from 9.85 to 10.35 min corresponding to the
poly(I) strand and from 7.30 to 7.80 min corresponding to the
poly(C.sub.12U) strand. Rugged dsRNA is found at a retention time
of about 5 min representing a molecular species uniquely resistant
to denaturation and unfolding. Denaturing conditions would
eliminate biological activity exclusively due to TLR3 receptor
binding. This analytical method may also be used as a stability
indicating assay and, in particular, it may be used to show that
the Rugged dsRNA is unusually resistant to disruption of its double
helix and to molecular unfolding.
[0145] For the lyophilized (freeze-dried) preparation (FIG. 1B),
aggregates maybe present and elute at 13 and 15 min. A small
fraction of individual nucleosides, inosine, cytosine and uridine,
elute at 1 min. The overall purity of poly I:polyC.sub.12U
(Ampligen.RTM.) determined by HPLC is represented by the sum of the
7.4, 8.7 and 10 minute peaks and is 96-99%. "New dsRNA" comprises
1-4% and is different from Ampligen.RTM. by its size and
physico-chemical properties as discussed herein.
[0146] The identity of each peak is determined by analysis with a
photodiode array (PDA) detector as shown in FIGS. 2A, 2B and 2C. At
each selected retention time, a UV absorption scan of wavelengths
from 200 nm to 360 nm was obtained. Duplex poly(I):poly(C.sub.12U)
and individual poly(I) and poly(C.sub.12U) strands have their own
specific peak absorption wavelengths. Absorption peaks centered at
both 248 nm and 265 nm indicate the presence of Rugged dsRNA (about
286,000 daltons) having poly(I) and poly(C.sub.12U), respectively
(FIG. 2A). Peak absorption centered at about 265 nm indicates the
presence of the poly(C.sub.12U) strand (FIG. 2B). Peak absorption
centered at about 248 nm indicates the presence of the poly(I)
strand (FIG. 2C). Absorption centered at about 230 nm is due to
acetonitrile used as solvent. Because of the relative scarcity of
Rugged dsRNA, the signal at 230 nm was subtracted from FIG. 2A.
[0147] FIG. 21 shows the relative size of Ampligen.RTM. vs new
Rugged dsRNA (peak 5 minutes)
[0148] Shown in FIG. 22 is a partial view of
poly(I):poly(C.sub.12U) partially hybridized strands and the
interaction of bases of individual poly(I) and the poly(C.sub.12U)
strands. Single inosine bases bind to cytosine bases, but not to
the uridine base. In this structure, the poly(inosinic acid) is
hydrogen bonded (dashed lines between bases) to poly(cytidylic
acid), with uridylic acid substitution occurring on an average of
every 12-13 bases.
(13C.sub.10H.sub.11N.sub.4O.sub.7P).sub.n:((12C.sub.9H.sub.12N.sub.3O.su-
b.7P)(C.sub.9H.sub.11N.sub.2O.sub.8P)).sub.n Molecular formula:
[0149] Molecular size: about 1,200,000 daltons
The number of repeat units (n) corresponding to the size of
poly(I):poly(C.sub.12U) of approximately 1.2 Mda is 2000 base pairs
or 187 full helical turns.
TABLE-US-00002 TABLE 2 Molecular Weight (MW) of Unimproved Ampligen
.RTM. Mixture Components. Common name: poly(I):poly(C.sub.12U)
(1,200,000 daltons) Chemical name: poly(inosinic
acid):poly((cytidylic acid).sub.12(uridylic acid)) MW Unit Unit MW*
Inosine 5' mono- 330 13 4056 phosphate Cytidine 3' mono- 305 12
3444 phosphate Uridylic acid 306 1 288 Overall Average: 318 Sum:
7788 *Note: One molecule of H.sub.2O(mw = 18)is lost for each
phosphodiester bond formed
[0150] Shown in FIG. 23 is a partial view of Rugged dsRNA,
poly(I):poly(C.sub.30U), partially hybridized strands and the
interaction of bases of individual poly(I) and the poly(C.sub.30U)
strands. Single inosine bases bind to cytosine bases, but not to
the uridine base. In this structure, the poly(inosinic acid) is
hydrogen bonded (dashed lines between bases) to poly(cytidylic
acid), with uridylic acid substitution occurring on an average of
every 30-31 bases. This is "rugged" dsRNA.
(31C.sub.10H.sub.11N.sub.4O.sub.7P).sub.n:((30C.sub.9H.sub.12N.sub.3O.su-
b.7P)(C.sub.9H.sub.11N.sub.2O.sub.8P)).sub.n Molecular formula:
[0151] Molecular size: about 300,000 daltons
[0152] The number of repeat units (n) corresponding to the size
range of new variant, also termed Rugged dsRNA (also termed peak 5
min on HPLC) is about 30-300 Kda having about 50-500 base pairs
representing 4.7-46.7 complete turns of RNA helix and is resistant
to disassembly of hydrogen-bonded strands under elevated thermal or
abnormal ionic conditions.
TABLE-US-00003 TABLE 3 Molecular Weight (MW) of Novel Rugged dsRNA
Components. MW Unit Unit MW* Inosine 5' mono- 330 31 9,672
phosphate Cytidine 3' mono- 305 30 8,610 phosphate Uridylic acid
306 1 288 Overall Average: 318 Sum: 18,570 *Note: One molecule of
H.sub.2O(mw = 18)is lost for each phosphodiester bond formed
[0153] Circular dichroism (CD) has been used to measure secondary
structure (duplexed helices) of biological and synthetic polymers,
including proteins and nucleic acids. CD is the measurement of
absorption of right- or left-circular polarized light, at a
specific wavelength, by chiral molecules. Chemical chirality is the
property of a molecule being nonsuperimposable on its mirror image.
An atom that makes its molecule chiral is called a chiral atom or,
more commonly, a chiral center. Rugged dsRNA and
Poly(I):poly(C.sub.12U) have a number of chiral centers because of
their primary and secondary structures. Chiral centers are found in
the nucleotide bases, which form the two primary structures for the
two individual RNA strands (ssRNA) of Rugged dsRNA and
poly(I):poly(C.sub.12U). Additional chiral centers come from
hybridizing each ssRNA to the other through hydrogen bonding of
their complementary bases. Hydrophobic bonding between adjacent
bases of dsRNA is known as base stacking and produces a flexible,
linear symmetrical, helical secondary structure of defined shape
and size. CD spectra for Rugged dsRNA and Ampligen.RTM., which are
dependent on the wavelength, are observed to be a function
reflecting the Gaussian absorption for each chiral center.
Therefore, the CD spectrum for a dsRNA such as Rugged dsRNA is
dependent on the complementary base pairing of double-stranded
structures and the complex chirality of the resultant helical
structure.
[0154] It has been demonstrated by UV and CD spectroscopy that the
biological activity of dsRNA is dependent on these specific spatial
and steric configurations. Since perturbation of helical structure
results in loss of the chiral centers characteristic of the
secondary structure, the analysis and monitoring of secondary
structure by CD provides a method to characterize the
physico-chemical properties of Rugged dsRNA and
poly(I):poly(C.sub.12U) that are associated with their
bioactivity.
[0155] The specific ellipticity measured in a wavelength scan
provides a quantitative parameter, which is calculated as the
ellipticity ratio at certain "critical" wavelengths. The value of
this structural parameter, the ratio CD.sub.278/CD.sub.245, is a
characteristic of Rugged dsRNA or the unimproved Ampligen.RTM.
mixture. In a second CD analysis, ellipticity is measured during
heating. As poly(I):poly(C.sub.12U) is heated and thermally
denatured, the individual poly(I) and poly(C.sub.12U) strands
unwind due to the breakdown of hydrogen bonding between
complementary base pairs. When the temperature derivative of
ellipticity is plotted, the minimum derivative value corresponds to
melting temperature, defined as the point where 50% of the
double-stranded conformation is unwound. The width at half-height
of the peak, a measure of structural uniformity, also becomes an
indication of its integrity. Taken together, these thermal indices
provide a measure of the strength of the dsRNA helixes.
[0156] The wavelength scan detects two peaks: a first peak at 245
nm corresponding to the doubled stranded helix of the
poly(I):poly(C.sub.12U) and a second peak at 278 nm corresponding
to the stacking of the nucleic acid's base pairs. As shown
previously in FIGS. 4 and 5, dsRNA affords separate peaks in the CD
wavelength scan, at 245 and 278 nm, the former peak associated with
base stacking attribute of helical structure. Accordingly, the
ratio of peak heights at 278/245 is typically within 0.69-0.79 for
dsRNA but much higher in the absence of double helical
structure.
Table 4 summarizes CD wavelength scans obtained by isolation of
fractions of the three reversed phase HPLC peaks previously
discussed in FIGS. 1B and 1C. The reversed phase HPLC assay is
utilized to distinguish "rugged" dsRNA (5.0 minute peak) from the
separated, component strands of poly(I):poly(C.sub.12U):7.0 (poly
C.sub.12U) and 10.0 minute peaks (poly I). It is clear from the
278/245 ratio that only the 5.0 minute, Rugged dsRNA fraction
retains helical structure, in contrast to the separated, component
strands of Poly I:Poly C.sub.12U. This result underscores the
greater stability of Rugged dsRNA during the reversed phase
isolation, in which all polynucleotides experience binding and
elution. Whereas poly(I):poly(C.sub.12U) is separated into the
component 7 and 10 minute polynucleotide strands, the 5 minute,
Rugged dsRNA, retains the double stranded conformation.
TABLE-US-00004 TABLE 4 PREPARATIVE HPLC*: Peak Analysis by Circular
Dichroism Peak Circular Dichroism Lot (min) 278/245 nm response
requirement (0.69-0.79) 0303SD 5 Min 0.78 7 min 12.3 10 min 1.74
0301SD 5 Min 0.79 7 min 2.49 10 min N/A *Preparative HPLC
Methodology: HPLC Equipment: Beckman Coulter Preparative HPLC
(System Gold 126P Solvent Module), Beckman coulter (System Gold 168
Detector) Column: Phenomenex, Polymerx, 10.mu., RP-1, 100.degree.
A, 250 .times. 21.20 mm Mobile Phase: 200 mM of Triethylamine
Acetate buffer, pH 8.7 Flow Rate: 5.0 mL/minute Injection Volume: 5
mL Wavelength: 255 nm and photodiode array detection.
TABLE-US-00005 TABLE 5 Gradient Condition: Time (Minute)
Acetonitrile (%) Buffer (%) 0 8 92 3 10 90 6 12 88 9 14 86 12 16 84
14 20 80 30 20 80
The column was equilibrated with 8% acetonitrile and 92% buffer for
at least 30 minutes. Peaks were collected at 10-12 minutes and
22-27 minutes which corresponded to differing gradient acetonitrile
compositions indicated in Table 5 above. The injection process was
repeated 20-30 times and fractions from the first peak (10-12
minutes) were pooled for subsequent analysis. The pooled fractions
were concentrated and solvent was displaced with a water wash,
using Amicon Ultra Centrifugal Filters (Amicon Ultra, Cat UFC
50104). The concentrated samples were analyzed for concentration by
UV, based upon averaging of the concentration responses which were
separately calculated at the wavelength maximum for each
polynucleotide chain: .epsilon.=5.2.times.10 3 at .lamda.=265 nm
and .epsilon..sub.2=4.9.times.10 3 at .lamda.=249 nm. Only 5 minute
exhibits double stranded base stacking character: [0157]
*Significant 245 nm response reflecting double strand helix (245
nm) [0158] *Acceptable 245/278 Ratio reflecting base pairs=278 nm
(chiral centers in backbone).
[0159] Precision.
[0160] Ampligen.RTM. poly(I):poly(C.sub.12U), lot 9807CD, at a
concentration of 2.5 mg/mL was repeatedly assayed to investigate
the preci-sion of the CD assay. The percent relative standard
deviations (% RSD) for the melting temperature (T.sub.M), for the
width at half-height for the first derivative of the melting curve
and for the ratio of measurements of the CD peaks at 278 nm and 245
nm were calculated as 0.76%, 9.09%, and 1.41%, respectively. This
demonstrated that CD assay of Ampligen.RTM. poly(I):poly(C.sub.12U)
acts in a precise manner during thermal analysis for the
determination of T.sub.M and width at half height of the first
derivative of the thermal melt curve and during the CD scan
analysis for determination of the ratio of CD at 278 nm to CD at
245 nm.
[0161] Specificity.
[0162] This CD method for characterizing poly(I):poly(C.sub.12U) is
also specific because it can between differentiate duplexed nucleic
acids and single-stranded nucleic acids, or other similar
double-stranded nucleic acids that do not meet the manufacturing
and release specifications for Ampligen.RTM.
poly(I):poly(C.sub.12U). The specificity of this method, in regards
to analysis of single versus double-stranded nucleic acids, was
demonstrated by comparing scanning profiles and melting temperature
curves. See FIG. 7. The scans of double-stranded molecules such as
poly(I):poly(C.sub.12U), poly(I):poly(C), and poly(A):poly(U)
differed significantly from those obtained during analysis of
single-stranded molecules such as poly(I) and poly(C.sub.12U). See
FIGS. 8-17. Furthermore, each of the CD scans was unique for the
molecular species being assayed.
[0163] The specificity of the assay was also investigated to
assess, unequivocally, the ability to detect compounds of closely
related structure.
(a) Double-stranded ribonucleic acids of different nucleotide base
composition, such as poly(I):poly(C.sub.12U), poly(I):poly(C), and
poly(A):poly(U). (FIGS. 10, 12 and 14). (b) Ampligen.RTM.
poly(I):poly(C.sub.12U) that meets the polymer size specification.
(c) Double-stranded ribonucleic acid formulated from poly(I) and
poly(C.sub.xU.sub.y) strands with a cytidine to uridine base ratio
of 11-14 to 1 (FIGS. 16 and 17) (C:U ratio=11:1 to 14:1).
[0164] The specificity of assays for dsRNA that differed in their
nucleotide base composition was evidenced by comparison of CD scans
and melting curves of similar, but different, double-stranded
molecules, such as poly(I):poly(C.sub.12U), poly(I):poly(C), and
poly(A):poly(U). CD scanning profiles appear to be similar, as seen
with the scans of Ampligen.RTM. poly(I):poly(C.sub.12U) and
poly(I):poly(C). But calculations of the ratios obtained at 278 nm
and 245 nm, and subsequent t-test statistical analysis for equal
means showed that the CD scan of Ampligen.RTM. differs
significantly from similar dsRNA having different nucleotide base
compositions. Specificity for the dsRNA of different nucleic acid
base composition was also demonstrated by their thermal melting
curves. Thermal melt curves for dsRNA differed significantly from
each other. Statistical analysis (t-test for equal means) of data
from the plots of the first derivative of the melting curves
confirmed that the results obtained for their respective T.sub.M
and width at half-height are significantly different. Therefore,
specificity of the CD method differentiates Ampligen.RTM. from
other dsRNA mole-cules by parameters of both the scan and the
thermal melt profiles.
[0165] The CD method is specific for detection of
poly(I):poly(C.sub.12U) formulated from polymers not meeting the
aforementioned specifications for size. When one or both polymers
of the poly(I):poly(C.sub.12U) molecule is outside the 4-8S size
specification, the results from the CD analysis of these molecules
do not meet specifications for Ampligen.RTM. in regards to T.sub.M
and width at half-height of the first derivative of the thermal
melt curve. The failure to meet speci-fications for these CD
parameters is observed with these formulations even when the
.+-.1.5S size differential specification is satisfied. Relative to
the data obtained from the thermal melt analyses of Ampligen.RTM.
formula-tions, the CD.sub.278/CD.sub.245 ratio determinations were
less specific. CD scans alone did not differentiate between
poly(I):poly(C.sub.12U) and non-poly(I):poly(C.sub.12U)
formulations that did not meet manufacturing and/or release
specifications for polymer size.
[0166] As discussed above, the specificity of CD analysis is
sensitive to the size of the single-stranded polymer strands. In
addition, when the size difference between the complementary
single-stranded polymer components, poly(I) and poly(C.sub.12U), is
2.4S or greater, the CD thermal melt analyses will differentiate
poly(I):poly(C.sub.12U) from similar molecules not meeting the
specification for the complementary polymer size differential.
[0167] CD analysis can distinguish between poly(I):poly(C.sub.12U)
and similar molecules that do not meet specifications for the
amount of double strandedness or base pairing between the
complementary poly(I) and poly(C.sub.12U) strands. The amount of
base pairing is dependent on the relative proportion of cytidylic
acid to uridylic acid (C:U ratio) of the poly(C).sub.xU.sub.y)
polymer. The ratio of cytidine to uridine in the
poly(C).sub.xU.sub.y) polymer affects the melting temperature
(T.sub.M) as well as the width at half height of the first
derivative of the melting curve. When the ratio of cytidine to
uridine is less than 11:1, there is less double strandedness or
base pairing (between polyinosinic acid and polycytidylic acid
complementary strands of the duplex RNA helix) than that for
Ampligen.RTM.. This results in lower observed T.sub.m's and larger
widths at half-height for the first derivative of the thermal melt
curves relative to those observed for poly(I):poly(C.sub.12U).
Increasing the cytidine to uridine ratio of the
poly(C.sub.xU.sub.y) strand increases the base pairing between the
complementary strands of the helix and, therefore, increases the
observed T.sub.M and decreases the observed width at half-height of
the first derivative of the thermal curve. The
CD.sub.278/CD.sub.245 ratio determinations were demonstrated to be
less sensitive to differences in the C:U ratio in Ampligen.RTM.
formulations.
[0168] Both the size of the complementary polymer strands and the
C:U ratio of the poly(C.sub.12U) strand contribute to double
strandedness of a poly(I):poly(C.sub.12U) molecule. The double
strandedness, in turn, contributes to the efficacy of the drug
product as discussed in the introduction. Therefore, CD method is
an important analytical tool for characterization of
poly(I):poly(C.sub.12U). Although CD scans and determinations of
the CD.sub.278/CD.sub.245 ratio are less specific than the thermal
melt analysis determinations of T.sub.M and width at half-height of
the first derivative of the melt curve, all three CD parameters may
be used in combination for the thorough characterization and
identification of poly(I):poly(C.sub.12U).
Bioactivity and Stability of Rugged dsRNA
[0169] Bioactivity of dsRNA and poly(I):poly(C.sub.12U) were
measured, and then compared utilizing a ligand-binding assay.
Stability was measured using the product release test, reverse
phase HPLC assay.
[0170] A summary of the results is presented below, followed by
more detailed discussion. The combination of enhanced bioactivity
and much greater stability under the thermal stress of 40.degree.
C. illustrate the "ruggedness" of this novel variant dsRNA (i.e.,
Rugged dsRNA) and suggest that it will be more bioavailable than
most of the dsRNA molecules in a formulation of Ampligen.RTM..
1. Bioactivity of Rugged dsRNA Shows Two-Fold Greater Binding
Affinity as Compared to Unselected dsRNA
[0171] Rugged dsRNA binding sites become unsaturated at a ratio of
0.50:1 (TLR3:Rugged dsRNA) or higher. But binding sites for
Ampligen.RTM. poly(I):poly(C.sub.12U) become unsaturated at a ratio
of 0.20:1 (TLR3:unselected dsRNA) or higher.
2. Stability of Rugged dsRNA is Four-Fold Greater than Unselected
dsRNA
[0172] Ampligen.RTM. poly(I):poly(C.sub.12U) is stable (i.e.,
S.sub.w,20>10.0) for less than 90 days when subjected to
hydrolysis under thermal stress of 40.degree. C. By contrast,
Rugged dsRNA is stable for greater than 360 days under the same
conditions. Rugged dsRNA also has an increased resistance to
ribonuclease digestion.
3. Stability and Bioactivity Data Show that Rugged dsRNA is More
Bioavailable than Unselected dsRNA
[0173] From these stability and bioactivity considerations, Rugged
dsRNA is more bioavailable for the relevant signaling receptor that
conveys the therapeutic benefit. The Rugged dsRNA has the
additional benefit of maintaining long-term stability at ambient
temperatures, which has important clinical implications for
treating populations in regions of the world without adequate
refrigeration capabilities.
Bioactivity Background
[0174] Toll-like receptors (TLR) are signaling molecules
recognizing pathogen-associated molecular patterns (PAMP) and
activating innate immune defense mechanisms. TLR3 recognizes dsRNA,
the genomic structure of some viruses, and also an intermediate
generated during viral RNA replication. dsRNA is also produced
intracellularly by stem-loop forming or with siRNA-aligned mRNAs.
Ampligen.RTM. is comprised of dsRNA molecules that act through TLR3
binding and downstream signaling events. While poly(I):poly(C)
signaling has alternate routes, the poly(I):poly(C.sub.12U) pathway
acts exclusively through TLR3 binding as Ampligen.RTM. treatment
protects TLR3.sup.+/+ but not TLR3.sup.-/- mice from Punta Toro
virus infection. TLR3.sup.-/- cells do not produce IFN upon
poly(I):poly(C.sub.12U) treatment while IFN is induced by
poly(I):poly(C) in TLR3 knockout cells.
[0175] The TLR3 molecule ectodomain (ECD) conformation and its
relation to binding of dsRNA is well characterized, including the
prospective binding site. Amino acids H539 and N541 are involved in
the interaction with the double helix. Mutational analysis of these
amino acids at the binding site further strengthens the
argument.
[0176] The effect of length and structure of dsRNA on TLR3 binding
and IFN induction is known. Inosine.sub.30 (I.sub.30):poly(C) or
poly(I):Cytosine.sub.30 (C.sub.30) induced interferon (IFN), but
shorter dsRNA stretches do not induce IFN. Compared to them,
however, IFN induction by poly(I):poly(C) was always superior.
I.sub.20:C.sub.20, I.sub.30:C.sub.30, and I.sub.40:C.sub.40 were
ineffective IFN inducers. Therefore, characterizing Ampligen.RTM.
by its TLR3 binding capacity is a biomarker to predict its
biological activity.
Bioactivity Method
[0177] A range of ratios of TLR3-ECD to unselected Ampligen.RTM. or
Rugged dsRNA are reacted by the method of Leonard (2008). The
components are separated by the size-exclusion chromatographic
method described below. From the peak quantities of free TLR3-ECD
and the ligand-receptor complex, the ratio of TLR3-ECD that is
required for saturation of either Ampligen.RTM. or Rugged dsRNA is
determined. This threshold TLR3-ECD/dsRNA ratio provides a direct
indication of the strength of the ligand-receptor binding and,
therefore, of bioactivity.
[0178] The following method is an adaptation of the experimental
procedures used to characterize TLR3 ligand binding at a molecular
level. Since TLR3-ECD (1.12.times.10.sup.2 Kda) and
poly(I):poly(C.sub.12U) (0.2-2.times.10.sup.3 Kda) have different
elution patterns, they can be separated from each other by
size-exclusion chromatography (SEC). According to results obtained
from poly(I):poly(C) using a SUPERDEX 200 PC 3.2/30 column and
collecting 80 .mu.l fractions, most of the poly(I):poly(C) appears
in fractions 3-5 while TLR3-ECD is eluted in fractions 9-12 (Bell,
2005).
[0179] The binding of TLR3-ECD to poly(I):poly(C) or
poly(I):poly(C.sub.12U) creates a complex that is larger in size
than either of the initial components. The later eluting free
TLR3-ECD is separated from the complex. Optimization of the
separation identified that the SUPEROSE 200 PC column afforded
superior binding by reducing tailing, due to absence of nonspecific
interactions with dsRNA.
[0180] FIGS. 18A, 18B and 18C show the resulting chromatograms
obtained from the reacted mixture of
TLR3-ECD/poly(I):poly(C.sub.12U) compared to component injections
of TLR3-ECD and poly(I):poly(C.sub.12U) alone, respectively.
[0181] Characterization of Peaks.
[0182] Identification and quantitation of TLR3-ECD in
size-exclusion chromatography fractions is possible in an ELISA
format. The commercially-available TLR3-ECD is a His tag-containing
recombinant protein. A capture anti-His tag antibody immobilizes
TLR3-ECD in a microplate well. A second, biotinylated primary
antibody quantitatively binds to the immobilized TLR3-ECD. This
secondary antibody is selected to have an epitope distal from the
dsRNA binding site on the TLR3-ECD molecule and also from the
epitope recognized by the capture antibody. HRP-conjugated
streptavidin recognizes the biotinylated second primary antibody.
The appropriate substrate metabolized by HRP produces a soluble
color suitable for quantitative measurement of TLR3-ECD.
[0183] Ampligen.RTM. concentration in the size-exclusion
chromatography fractions is measured by fluorescence using standard
dilutions and chromatography fractions in a quantitative riboGreen
test. This assay permits testing of Ampligen.RTM. out-of-the-bottle
(i.e., not selected for Rugged dsRNA) without further processing,
preparation, or extraction, thereby maintaining its condition as a
pharmaceutical.
[0184] Bioactivity Results.
[0185] Results in Table 6 show the percentage of free TLR3-ECD that
remains in a series of reactions using different ratios of TLR3-ECD
to dsRNA. These studies were conducted with either unimproved
Ampligen.RTM. as well as Rugged dsRNA.
[0186] Binding of TLR3-ECD to Rugged dsRNA is more effective than
binding of TLR3-ECD to unimproved Ampligen.RTM.. An approximately
2-fold greater ratio of TLR3-ECD is required to "unsaturate" Rugged
dsRNA (-0.50:1) as compared to Ampligen.RTM. (0.25:1). Also, the
binding profile at various ratios shows a much sharper endpoint for
saturation for the case of Rugged dsRNA which may reflect greater
structural uniformity for this more compact dsRNA. Table 6.
Bioactivity Measurements of Unimproved Ampligen.RTM. vs. Rugged
dsRNA.
TABLE-US-00006 Unimproved/Old Ampligen .RTM., Molar Lot # 0701HE
New Rugged dsRNA Ratio of dsRNA/TLR3 Free dsRNA/TLR3 Free TLR3 to
Complex TLR3 Complex TLR3 dsRNA Area % Area % Area % Area % 0.20:1
99.0 0.978 99.4 0.577 0.25:1 78.4 21.6 99.1 0.880 0.33:1 20.9 79.1
92.9 7.086 0.50:1 58.9 41.1 60.3 39.723 0.67:1 15.4 84.6 11.3
88.660
[0187] The TLR3 binding of Rugged dsRNA is 100% superior inin
receptor binding than the unimproved/old Ampligen.RTM. preparation.
As shown in Table 6, Free TLR3 (area>10%) appears at a
TLR3:dsRNA ratio of 0.25:1 for unimproved Ampligen.RTM. as compared
to a 0.50:1 for Rugged dsRNA
[0188] Stability of Rugged dsRNA.
[0189] Stability of poly(I):poly(C.sub.12U) was measured at an
accelerated temperature condition of 40.degree. C. as compared to
the long-term storage temperature of from 2.degree. C. to 8.degree.
C. As shown in FIG. 19, the size of poly(I):poly(C.sub.12U) decays
at this temperature as measured by analytical ultracentrifugation
(S.sub.20,w). Decrease in size is due to unfolding of the double
helix (loss of hydrogen bonds) and concurrent hydrolysis of the
phosphodiester bonds. For bioactivity unimproved Ampligen.RTM.
(poly(I):poly(C.sub.12U) requires a sedimentation coefficient from
about 10.0 to about 15.0 S(.sub.20,w), whereas the size of
poly(I):poly(C.sub.12U) at more than 180 days is about 8.0
S(.sub.20,w) and indicates a loss of bioactivity.
[0190] FIG. 20 shows the results of a second stability indicating
parameter, the reversed phase HPLC assay, previously described,
that separates poly(I):poly(C.sub.12U) into its individual strands
(7 minute and 10 minute peaks). It is clearly evident that
hydrolysis begins with the poly(I) strand (10 minute peak) followed
by the poly(C.sub.12U) strand (7 minute peak). HPLC results show
that loss of size does not begin until commencement of the
hydrolysis of the second strand poly(C.sub.12U); the RNA molecule
retains double-stranded structure when only one of the strands
undergoes hydrolysis. This loss of size at about 90 days occurs
with the hydrolysis of both poly(I) and poly(C.sub.12U)
strands.
[0191] Importantly, the Rugged dsRNA (5 min) peak is entirely
unaffected by thermal stress. In fact, it increases in relation to
the poly(I) and poly(C.sub.12U) strands. This conclusively shows
that Rugged dsRNA is not only "rugged" but can form spontaneously
from smaller strands of degraded poly(I):poly(C12U).
Structure of Novel Improved Rugged dsRNA Compared to Old Unimproved
Ampligen.RTM. Mixture Transmission Electron Microscopy (TEM) was
used to compare the structure of the new improved Rugged dsRNA vs.
the old unimproved Ampligen.RTM. mixture of dsRNA molecules. As
shown in FIGS. 26, 27, and 28 the unimproved Ampligen.RTM. mixture
contains molecules with a high degree of branching compared to the
new improved Rugged dsRNA (FIG. 29). In fact the majority of
molecules in the Ampligen.RTM. mixture are branching, while the
majority of dsRNA molecules in the improved Rugged dsRNA are
unbranched. Also, the unimproved Ampligen.RTM. mixture contains 4-5
times more molecules with 3 or more branches than the new improved
Rugged dsRNA. Therefore, not only does Rugged dsRNA contain a
higher percentage of non-branched molecules, the small percentage
of branched molecules present contain primarily a single branch as
compared to the unimproved Ampligen.RTM. mixture which contains
many more highly branched variant molecules with .gtoreq.3 branched
strands. The binding affinity of dsRNA to TLR3 is a function of the
length of the linear non-branched dsRNA. This explains the
increased bioactivity of Rugged dsRNA compared to the old
unimproved Ampligen.RTM. mixture.
[0192] Patents, patent applications, books, and other publications
and information sources cited herein are incorporated by reference
in their entirety.
[0193] In stating a numerical range, it should be understood that
all values within the range are also described (e.g., one to ten
also includes every integer value between one and ten as well as
all intermediate ranges such as two to ten, one to five, and three
to eight). The term "about" may refer to the statistical
uncertainty associated with a measurement or the variability in a
numerical quantity which a person skilled in the art would
understand does not affect operation of the invention or its
patentability.
[0194] All modifications and substitutions that come within the
meaning of the claims and the range of their legal equivalents are
to be embraced within their scope. A claim which recites
"comprising" allows the inclusion of other elements to be within
the scope of the claim; the invention is also described by such
claims reciting the transitional phrases "consisting essentially
of" (i.e., allowing the inclusion of other elements to be within
the scope of the claim if they do not materially affect operation
of the invention) or "consisting of" (i.e., allowing only the
elements listed in the claim other than impurities or
inconsequential activities which are ordinarily associated with the
invention) instead of the "comprising" term. Any of these three
transitions can be used to claim the invention.
[0195] It should be understood that an element described in this
specification should not be construed as a limitation of the
claimed invention unless it is explicitly recited in the claims.
Thus, the granted claims are the basis for determining the scope of
legal protection instead of a limitation from the specification
which is read into the claims. In contradistinction, the prior art
is explicitly excluded from the invention to the extent of specific
embodiments that would anticipate the claimed invention or destroy
novelty.
[0196] Moreover, no particular relationship between or among
limitations of a claim is intended unless such relationship is
explicitly recited in the claim (e.g., the arrange-ment of
components in a product claim or order of steps in a method claim
is not a limitation of the claim unless explicitly stated to be
so). All possible combinations and permutations of individual
elements disclosed herein are considered to be aspects of the
invention. Similarly, generalizations of the invention's
description are considered to be part of the invention.
[0197] From the foregoing, it would be apparent to a person of
skill in this art that the invention can be embodied in other
specific forms without departing from its spirit or essential
characteristics. The described embodiments should be considered
only as illustrative, not restrictive, because the scope of the
legal protection provided for the invention will be indicated by
the appended claims rather than by this specification.
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