U.S. patent application number 13/560925 was filed with the patent office on 2013-02-14 for control of antibody responses to synthetic nanocarriers.
This patent application is currently assigned to Selecta Biosciences, Inc.. The applicant listed for this patent is David H. Altreuter, Yun Gao, Petr Ilyinskii, William Kuhlman, Lynnelle Ann McNamee Pittet. Invention is credited to David H. Altreuter, Yun Gao, Petr Ilyinskii, William Kuhlman, Lynnelle Ann McNamee Pittet.
Application Number | 20130039954 13/560925 |
Document ID | / |
Family ID | 47597380 |
Filed Date | 2013-02-14 |
United States Patent
Application |
20130039954 |
Kind Code |
A1 |
Pittet; Lynnelle Ann McNamee ;
et al. |
February 14, 2013 |
CONTROL OF ANTIBODY RESPONSES TO SYNTHETIC NANOCARRIERS
Abstract
Disclosed are synthetic nanocarrier compositions that comprise B
cell antigen for desired antibody production and an off-target
response attenuating polymeric coating as well as related
methods.
Inventors: |
Pittet; Lynnelle Ann McNamee;
(Brighton, MA) ; Altreuter; David H.; (Wayland,
MA) ; Gao; Yun; (Southborough, MA) ;
Ilyinskii; Petr; (Cambridge, MA) ; Kuhlman;
William; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pittet; Lynnelle Ann McNamee
Altreuter; David H.
Gao; Yun
Ilyinskii; Petr
Kuhlman; William |
Brighton
Wayland
Southborough
Cambridge
Somerville |
MA
MA
MA
MA
MA |
US
US
US
US
US |
|
|
Assignee: |
Selecta Biosciences, Inc.
Watertown
MA
|
Family ID: |
47597380 |
Appl. No.: |
13/560925 |
Filed: |
July 27, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61513496 |
Jul 29, 2011 |
|
|
|
61513526 |
Jul 29, 2011 |
|
|
|
61513527 |
Jul 29, 2011 |
|
|
|
Current U.S.
Class: |
424/400 ;
424/193.1; 424/78.3; 977/700; 977/906 |
Current CPC
Class: |
A61P 33/02 20180101;
A61P 31/00 20180101; A61P 31/14 20180101; A61P 37/02 20180101; A61P
31/20 20180101; A61K 47/593 20170801; A61K 2039/55555 20130101;
A61K 47/59 20170801; A61K 39/39 20130101; Y02A 50/386 20180101;
Y02A 50/30 20180101; A61P 33/06 20180101; A61P 31/16 20180101; Y10T
428/2982 20150115; A61P 37/04 20180101; Y02A 50/41 20180101; A61P
31/12 20180101; A61P 31/18 20180101; Y02A 50/412 20180101; A61K
39/385 20130101; A61P 35/00 20180101; A61K 47/645 20170801 |
Class at
Publication: |
424/400 ;
424/193.1; 424/78.3; 977/700; 977/906 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61P 37/04 20060101 A61P037/04; A61K 39/385 20060101
A61K039/385 |
Claims
1. A composition comprising: a population of synthetic
nanocarriers, wherein the synthetic nanocarriers comprise (i) a B
cell antigen and (ii) an off-target response attenuating polymeric
coating, wherein the B cell antigen is coupled to the synthetic
nanocarrier.
2. The composition of claim 1, wherein the coating comprises one or
more polymers present at at least a portion of the surface of the
synthetic nanocarriers.
3. The composition of claim 1, wherein the B cell antigen is
coupled to the off-target response attenuating polymeric
coating.
4. The composition of claim 1, wherein the off-target response
attenuating polymeric coating comprises a polymer with a weight
average or number average molecular weight of greater than 2000
g/mole, of greater than 3000 g/mole, of greater than 4000 g/mole,
of greater than 5000 g/mole, of between 3500 g/mole and 5000
g/mole, or of 5000 g/mole.
5.-9. (canceled)
10. The composition of claim 1, wherein the B cell antigen is
coupled to the polymer.
11. The composition of claim 1, wherein the off-target response
attenuating polymeric coating comprises another polymer.
12. The composition of claim 11, wherein the B cell antigen is
coupled to the other polymer.
13.-18. (canceled)
19. The composition of claim 11, wherein the polymer and other
polymer are the same type of polymer.
20. The composition of claim 11, wherein the polymer and other
polymer are not the same type of polymer.
21. The composition of claim 1, wherein the ratio of the average
number of polymers coupled to the B cell antigen across the
population of synthetic nanocarriers to the average number of
polymers not coupled to the B cell antigen across the population of
synthetic nanocarriers, the ratio of the average number of polymers
coupled to the B cell antigen across the population of synthetic
nanocarriers to the average number of polymers coupled to the B
cell antigen across the population of synthetic nanocarriers plus
the average number of polymers not coupled to the B cell antigen
across the population of synthetic nanocarriers, or the ratio of
the average number of polymers not coupled to the B cell antigen
across the population of synthetic nanocarriers to the average
number of polymers coupled to the B cell antigen across the
population of synthetic nanocarriers plus the average number of
polymers not coupled to the B cell antigen across the population of
synthetic nanocarriers is between 0.001 and 1, between 0.01 and 1,
between 0.1 and 1, between 0.25 and 1, between 0.5 and 1, or
between 0.75 and 1.
22.-26. (canceled)
27. The composition of claim 21, wherein the ratio is based on the
polymeric coating across the population of synthetic nanocarriers,
or on the synthetic nanocarrier as a whole across the population of
synthetic nanocarriers.
28. (canceled)
29. The composition of claim 1, wherein the ratio by weight
averaged across the population of synthetic nanocarriers of polymer
coupled to the B cell antigen nanocarriers to polymer not coupled
to the B cell antigen, the ratio by weight averaged across the
population of synthetic nanocarriers of polymer coupled to the B
cell antigen nanocarriers to polymer coupled to the B cell antigen
plus polymer not coupled to the B cell antigen, or the ratio by
weight averaged across the population of synthetic nanocarriers of
polymer not coupled to the B cell antigen nanocarriers to polymer
coupled to the B cell antigen plus polymer not coupled to the B
cell antigen is between 0.1 and 1, between 0.25 and 1, between 0.5
and 1, between 0.1 and 0.5, or 0.5.
30.-35. (canceled)
36. The composition of claim 1 or claim 11, wherein the polymer
and/or other polymer comprises polyethylene glycol, a
polyethyloxazoline, a polyamino acid, polycarbonate, hydrophilic
polyacetal, hydrophilic polyketal, saccharide polypropylene, or
polyethyleneimine.
37.-38. (canceled)
39. The composition of claim 1, wherein the B cell antigen
comprises a protein, peptide, small molecule or
oligosaccharide.
40. (canceled)
41. The composition of claim 1, wherein the composition further
comprises a T cell antigen.
42. (canceled)
43. The composition of claim 1, wherein the composition further
comprises an adjuvant and/or a pharmaceutically acceptable
excipient.
44. (canceled)
45. A dosage form comprising the composition of claim 1.
46. A vaccine comprising the dosage form of claim 45.
47. A method comprising administering the dosage form of claim 45
to a subject in need thereof.
48.-49. (canceled)
50. A process for producing a synthetic nanocarrier comprising an
off-target response attenuating polymeric coating, comprising the
steps of: (a) providing a composition comprising one or more
polymers present at at least a portion of the surface of a
synthetic nanocarrier; (b) coupling a B cell antigen to said
synthetic nanocarrier under conditions where: (i) the molecular
weight of the polymers (as weight average or number average
molecular weight); and/or (ii) the ratio of the average number of
polymers coupled to the B cell antigen across the population of
synthetic nanocarriers to the average number of polymers not
coupled to the B cell antigen across the population of synthetic
nanocarriers; and/or (iii) the ratio by weight averaged across the
population of synthetic nanocarriers of polymer coupled to the B
cell antigen nanocarriers to polymer not coupled to the B cell
antigen; and/or (iv) the ratio of the average number of polymers
coupled to the B cell antigen across the population of synthetic
nanocarriers to the average number of polymers coupled to the B
cell antigen plus the average number of polymers not coupled to the
B cell antigen across the population of synthetic nanocarriers;
and/or (v) the ratio by weight averaged across the population of
synthetic nanocarriers of polymer coupled to the B cell antigen
nanocarriers to polymer coupled to the B cell antigen plus polymer
not coupled to the B cell antigen; and/or (vi) the ratio of the
average number of polymers not coupled to the B cell antigen across
the population of synthetic nanocarriers to the average number of
polymers coupled to the B cell antigen plus the average number of
polymers not coupled to the B cell antigen across the population of
synthetic nanocarriers; and/or (vii) the ratio by weight averaged
across the population of synthetic nanocarriers of polymer not
coupled to the B cell antigen nanocarriers to polymer coupled to
the B cell antigen plus polymer not coupled to the B cell antigen;
are selected such that an antibody response against the B cell
antigen is at least two-fold greater than an off-target antibody
response.
51.-59. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119 of U.S. provisional application 61/513,496, 61/513,526
and 61/513,527, each filed Jul. 29, 2011, the entire contents of
each of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to synthetic nanocarrier compositions
that comprise an off-target response attenuating polymeric coating,
and related methods, such as for treating diseases or conditions in
which generating an immune response against a B cell antigen is
desirable.
BACKGROUND OF THE INVENTION
[0003] Anti-carrier antibody generation by a nanocarrier vaccine is
an off-target side effect that may have direct unintended or
undesirable impacts on pharmaceutical or biomedical formulations of
related compositions, and may interfere with the generation of
desired anti-B cell antigen antibodies. Therefore, improved
compositions and therapeutic methods to avoid or minimize
undesirable anti-carrier effects are needed to provide improved
therapies for diseases and conditions in which generating an immune
response against a B cell antigen is desirable.
SUMMARY OF THE INVENTION
[0004] In one aspect, a composition comprising a population of
synthetic nanocarriers, wherein the synthetic nanocarriers comprise
a B cell antigen and an off-target response attenuating polymeric
coating is provided. In one embodiment, the B cell antigen is
coupled to the synthetic nanocarrier.
[0005] In another aspect, a composition comprising a population of
synthetic nanocarriers, wherein the synthetic nanocarriers comprise
(i) a B cell antigen and (ii) a coating comprising one or more
polymers present at at least a portion of the surface of the
synthetic nanocarriers is provided. In one embodiment, the B cell
antigen is coupled to the synthetic nanocarrier.
[0006] In an embodiment of any of the compositions provided, the
synthetic nanocarriers generate on average across the population of
synthetic nanocarriers an antibody response against the B cell
antigen that is at least two-fold greater than an off-target
antibody response. In another embodiment, the antibody response
against the B cell antigen is at least five-fold greater than the
off-target antibody response. In another embodiment, the antibody
response against the B cell antigen is at least ten-fold greater
than the off-target antibody response. In another embodiment, the
antibody response against the B cell antigen is at least 25-fold
greater than the off-target antibody response. In another
embodiment, the antibody response against the B cell antigen is at
least 50-fold greater than the off-target antibody response. In
another embodiment, the antibody response against the B cell
antigen is at least 100-fold greater than the off-target antibody
response. In one embodiment, the off-target antibody response is an
undesired antibody response not specific to the B cell antigen. In
another embodiment, the off-target antibody response is an antibody
response again the synthetic nanocarrier. In another embodiment,
the off-target antibody response is an antibody response again the
coating. In another embodiment, the off-target antibody response is
an antibody response against a polymer of the coating. In another
embodiment, the off-target antibody response is an IgG or IgA
antibody response. In another embodiment, the desired antibody
response is also an IgG or IgA antibody response, respectively. In
another embodiment, the off-target antibody response is an IgG
antibody response and the desired antibody response is also an IgG
antibody response. In another embodiment, the off-target antibody
response is an IgA antibody response and the desired antibody
response is also an IgA antibody response. In another embodiment,
the antibody responses are each measured as an antibody titer with
an ELISA. In another embodiment, the antibody titer is an IgG or
IgA titer (EC50).
[0007] In one embodiment, the B cell antigen is coupled to the
coating. In another embodiment, the B cell antigen is coupled to
one or more polymers of the coating. In another embodiment, the B
cell antigen is coupled to another part of the synthetic
nanocarriers.
[0008] In yet another embodiment, the off-target antibody response
is an undesired antibody response not specific to the B cell
antigen. In another embodiment, the off-target antibody response is
an antibody response against a polymer (or portion thereof) of the
nanocarrier or its coating.
[0009] In one embodiment, the antibody response against the B cell
antigen is at least five-fold greater than the antibody response
against a polymer of the off-target response attenuating polymeric
coating. In another embodiment, the antibody response is at least
ten-fold greater. In still another embodiment, the antibody
response is at least 25-fold greater. In yet another embodiment,
the antibody response is at least 50-fold greater. In a further
embodiment, the antibody response is at least 100-fold greater.
[0010] In one embodiment, the off-target response attenuating
polymeric coating comprises a polymer with a molecular weight of
greater than 2000 g/mole. In another embodiment, the off-target
response attenuating polymeric coating comprises a polymer with a
molecular weight of greater than 3000 g/mole. In yet another
embodiment, the off-target response attenuating polymeric coating
comprises a polymer with a molecular weight of greater than 4000
g/mole. In still another embodiment, the off-target response
attenuating polymeric coating comprises a polymer with a molecular
weight of greater than 5000 g/mole. In another embodiment, the
off-target response attenuating polymeric coating comprises a
polymer with a weight average or number average molecular weight of
between 3500 g/mole and 5000 g/mole In a further embodiment, the
off-target response attenuating polymeric coating comprises a
polymer with a molecular weight of 5000 g/mole. In another
embodiment, the B cell antigen is coupled to the polymer. In one
embodiment of any of the foregoing embodiments, the molecular
weight is the weight average molecular weight. In another
embodiment of any of the foregoing embodiments, the molecular
weight is the number average molecular weight. In still another
embodiment of any of the foregoing embodiments where the polymer
does not comprise polyethylene glycol, the molecular weight is the
weight average molecular weight. In yet another embodiment of any
of the foregoing embodiments where the polymer does comprise
polyethylene glycol, the molecular weight is the number average
molecular weight.
[0011] In still another embodiment, the off-target response
attenuating polymeric coating comprises another polymer. This other
polymer may be the same type of polymer as the aforementioned
polymer or it may be a different type of polymer. In one
embodiment, this other polymer has a molecular weight of greater
than 2000 g/mole. In another embodiment, this other polymer has a
molecular weight of greater than 3000 g/mole. In still another
embodiment, this other polymer has a molecular weight of greater
than 4000 g/mole. In yet another embodiment, this other polymer has
a molecular weight of greater than 5000 g/mole. In still another
embodiment, this other polymer has a molecular weight of between
3500 g/mole and 5000 g/mole. In yet another embodiment, this other
polymer has a molecular weight of 5000 g/mole. In one embodiment,
the B cell antigen is coupled to this other polymer. In another
embodiment, the B cell antigen is coupled to this other polymer and
the aforementioned polymer. In one embodiment of any of the
foregoing embodiments, the molecular weight is the weight average
molecular weight. In another embodiment of any of the foregoing
embodiments, the molecular weight is the number average molecular
weight. In still another embodiment of any of the foregoing
embodiments where the polymer does not comprise polyethylene
glycol, the molecular weight is the weight average molecular
weight. In yet another embodiment of any of the foregoing
embodiments where the polymer does comprise polyethylene glycol,
the molecular weight is the number average molecular weight.
[0012] In one embodiment, the ratio of the average number of
polymers coupled to the B cell antigen across the population of
synthetic nanocarriers to the average number of polymers not
coupled to the B cell antigen across the population of synthetic
nanocarriers is between 0.001 and 1. In another embodiment, the
ratio is between 0.01 and 1. In still another embodiment, the ratio
is between 0.1 and 1. In yet another embodiment, the ratio is
between 0.25 and 1. In a further embodiment, the ratio is between
0.5 and 1. In still a further embodiment, the ratio is between 0.75
and 1. In yet another embodiment, the ratio is between 0.1 and 0.5.
In a further embodiment, the ratio is 0.5.
[0013] In one embodiment, the ratio is based on the polymeric
coating across the population of synthetic nanocarriers. In another
embodiment, the ratio is based on the synthetic nanocarrier as a
whole across the population of synthetic nanocarriers.
[0014] In another embodiment, the ratio of the average number of
polymers coupled to the B cell antigen across the population of
synthetic nanocarriers to the average number of polymers coupled to
the B cell antigen across the populariton of synthetic nanocarriers
plus the average number of polymers not coupled to the B cell
antigen across the population of synthetic nanocarriers is between
0.001 and 1. In another embodiment, the ratio is between 0.01 and
1. In still another embodiment, the ratio is between 0.1 and 1. In
yet another embodiment, the ratio is between 0.25 and 1. In a
further embodiment, the ratio is between 0.5 and 1. In still a
further embodiment, the ratio is between 0.75 and 1. In yet another
embodiment, the ratio is between 0.1 and 0.5. In a further
embodiment, the ratio is 0.5.
[0015] In one embodiment, the ratio is based on the polymeric
coating across the population of synthetic nanocarriers. In another
embodiment, the ratio is based on the synthetic nanocarrier as a
whole across the population of synthetic nanocarriers.
[0016] In still another embodiment, the ratio of the average number
of polymers not coupled to the B cell antigen across the population
of synthetic nanocarriers to the average number of polymers coupled
to the B cell antigen across the populariton of synthetic
nanocarriers plus the average number of polymers not coupled to the
B cell antigen across the population of synthetic nanocarriers is
between 0.001 and 1. In another embodiment, the ratio is between
0.01 and 1. In still another embodiment, the ratio is between 0.1
and 1. In yet another embodiment, the ratio is between 0.25 and 1.
In a further embodiment, the ratio is between 0.5 and 1. In still a
further embodiment, the ratio is between 0.75 and 1. In yet another
embodiment, the ratio is between 0.1 and 0.5. In a further
embodiment, the ratio is 0.5.
[0017] In one embodiment, the ratio is based on the polymeric
coating across the population of synthetic nanocarriers. In another
embodiment, the ratio is based on the synthetic nanocarrier as a
whole across the population of synthetic nanocarriers.
[0018] In one embodiment, the polymer and/or other polymer
comprises polyethylene glycol. In another embodiment, the polymer
and/or other polymer comprises a polyethyloxazoline. In still
another embodiment, the polymer and/or other polymer comprises a
polyamino acid, polycarbonate, hydrophilic polyacetal, hydrophilic
polyketal, polysaccharide, polypropylene or polyethyleneimine.
[0019] In one embodiment, the B cell antigen comprises a protein,
peptide, small molecule or oligosaccharide. In another embodiment,
the B cell antigen comprises a cancer antigen, an infection or
infectious disease antigen, a non-autoimmune or degenerative
disease antigen or an addiction antigen.
[0020] In yet another embodiment, the composition and/or B cell
antigen further comprises an additional antigen. In one embodiment,
the additional antigen is a T cell antigen. In another embodiment,
the T cell antigen is a T helper cell antigen. In yet a further
embodiment, the T cell antigen is a T helper cell antigen. In still
a further embodiment, the additional antigen is another B cell
antigen. In still another embodiment, the one or more, two or more,
three or more, four or more, five or more, six or more, seven or
more, eight or more, nine or more, ten or more, 11 or more, 12 or
more, 13 or more, 14 or more, 15 or more, 20 or more, etc.
additional antigens are comprised in the compositions provided
herein. In one embodiment, the additional antigens are B cell or T
cell antigens or some combination thereof. In another embodiment,
all of the additional antigens are B cell antigens.
[0021] In another embodiment, the additional antigen is also
coupled to the synthetic nanocarriers. In a further embodiment, the
additional antigen is also coupled to the off-target response
attenuating polymeric coating of the synthetic nanocarriers. In yet
another embodiment, the additional antigen is coupled to another
population of synthetic nanocarriers. In still another embodiment,
the additional antigen is not coupled to any synthetic
nanocarriers.
[0022] In one embodiment, the composition further comprises one or
more adjuvants.
[0023] In another embodiment, the composition further comprises one
or more pharmaceutically acceptable excipients.
[0024] In another aspect, a dosage form comprising any of the
compositions provided is provided.
[0025] In yet another aspect, a vaccine comprising any of the
dosage forms provided is provided.
[0026] In still another aspect, a method comprising administering
any of the compositions provided herein to a subject in need
thereof is provided. In one embodiment, the subject is a human. In
another embodiment, the subject has or is at risk of having cancer.
In still another embodiment, the subject has or is at risk of
having an infection or infectious disease. In yet another
embodiment, the subject has or is at risk of having a
non-autoimmune or degenerative disease. In a further embodiment,
the subject has or is at risk of having an addiction.
[0027] In another embodiment, any of the compositions provided
herein is administered by oral, subcutaneous, pulmonary,
intranasal, intradermal, intravenous, transmucosal, intramucosal or
intramuscular administration.
[0028] In another aspect, a method comprising producing synthetic
nanocarriers that comprise a B cell antigen and an off-target
response attenuating polymeric coating and determining the level of
antibody response against the B cell antigen and the level of
off-target antibody response is provided. In one embodiment, the
method further comprises comparing the antibody response against
the B cell antigen and the off-target antibody response. The
antibody response against the B cell antigen and the off-target
antibody response can be determined with any of the methods
provided herein. The synthetic nanocarriers may be any of the
synthetic nanocarriers described herein.
[0029] In another aspect, a process for producing an off-target
response attenuating polymeric coating, comprising the steps of:
(a) providing a composition comprising one or more polymers present
at at least a portion of the surface of a synthetic nanocarrier;
(b) coupling a B cell antigen to said synthetic nanocarrier under
conditions where: (i) the molecular weight of the polymers; and/or
(ii) the ratio of the average number of polymers coupled to the B
cell antigen across the population of synthetic nanocarriers to the
average number of polymers not coupled to the B cell antigen across
the population of synthetic nanocarriers; and/or (iii) the ratio by
weight averaged across the population of synthetic nanocarriers of
polymer coupled to the B cell antigen nanocarriers to polymer not
coupled to the B cell antigen; and/or (iv) the ratio of the average
number of polymers coupled to the B cell antigen across the
population of synthetic nanocarriers to the average number of
polymers coupled to the B cell antigen plus the average number of
polymers not coupled to the B cell antigen across the population of
synthetic nanocarriers; and/or (v) the ratio by weight averaged
across the population of synthetic nanocarriers of polymer coupled
to the B cell antigen nanocarriers to polymer coupled to the B cell
antigen plus polymer not coupled to the B cell antigen; and/or (vi)
the ratio of the average number of polymers not coupled to the B
cell antigen across the population of synthetic nanocarriers to the
average number of polymers coupled to the B cell antigen plus the
average number of polymers not coupled to the B cell antigen across
the population of synthetic nanocarriers; and/or (vii) the ratio by
weight averaged across the population of synthetic nanocarriers of
polymer not coupled to the B cell antigen nanocarriers to polymer
coupled to the B cell antigen plus polymer not coupled to the B
cell antigen; are selected such that an antibody response against
the B cell antigen is at least two-fold greater than an off-target
antibody response. In one embodiment, the antibody response against
the B cell antigen and the off-target antibody response are each
IgG antibody responses. In another embodiment, they are each IgA
antibody responses. In another embodiment, these antibody responses
are measured as antibody titers (EC50) with an ELISA.
[0030] In one embodiment, the ratio is based on the polymeric
coating across the population of synthetic nanocarriers. In another
embodiment, the ratio is based on the synthetic nanocarrier as a
whole across the population of synthetic nanocarriers.
[0031] In one embodiment, the molecular weight, ratio of average
number and/or ratio by weight of the one or more polymers is as
defined herein. In another embodiment, the molecular weight is the
weight average molecular weight. In another embodiment of any of
the foregoing embodiments, the molecular weight is the number
average molecular weight. In still another embodiment of any of the
foregoing embodiments where the polymer does not comprise
polyethylene glycol, the molecular weight is the weight average
molecular weight. In yet another embodiment of any of the foregoing
embodiments where the polymer does comprise polyethylene glycol,
the molecular weight is the number average molecular weight.
[0032] In another aspect, any of the compositions, dosage forms or
vaccines provided herein can be used for therapy or
prophylaxis.
[0033] In still another aspect, any of the compositions, dosage
forms or vaccines provided herein can be used for any of the
methods provided herein.
[0034] In yet another aspect, any of the compositions, dosage forms
or vaccines provided herein can be used in vaccination.
[0035] In a further aspect, any of the compositions, dosage forms
or vaccines provided herein can be for use in a method of therapy
or prophylaxis of cancer, an infection or infectious disease, a
non-autoimmune or degenerative disease or an addiction.
[0036] In still a further aspect, any of the compositions, dosage
forms or vaccines provided herein can be for use in a method of
therapy or prophylaxis comprising administration by oral,
subcutaneous, pulmonary, intranasal, intradermal, intravenous,
transmucosal, intramucosal or intramuscular administration.
[0037] In another aspect, a use of any of the compositions provided
herein for the manufacture of a medicament, for example a vaccine,
for use in any of the methods provided herein is provided.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 shows the anti-nicotine antibodies (target or desired
antibodies) and anti-PEG antibodies (off-target or undesired
antibodies) at day 40 after inoculation.
[0039] FIG. 2 shows the anti-nicotine antibody titers and anti-PEG
antibody titers following a prime and two-boost inoculation
schedule.
DETAILED DESCRIPTION OF THE INVENTION
[0040] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particularly
exemplified materials or process parameters as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments of the
invention only, and is not intended to be limiting of the use of
alternative terminology to describe the present invention.
[0041] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety for all purposes.
[0042] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the content clearly dictates otherwise. For example, reference to
"a polymer" includes a mixture of two or more such molecules or a
mixture of differing molecular weights of a single polymer species,
reference to "a synthetic nanocarrier" includes a mixture of two or
more such synthetic nanocarriers or a plurality of such synthetic
nanocarriers, reference to "a DNA molecule" includes a mixture of
two or more such DNA molecules or a plurality of such DNA
molecules, reference to "an adjuvant" includes mixture of two or
more such adjuvant molecules or a plurality of such adjuvant
molecules, and the like.
[0043] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited integer (e.g. a feature, element,
characteristic, property, method/process step or limitation) or
group of integers (e.g. features, elements, characteristics,
properties, method/process steps or limitations) but not the
exclusion of any other integer or group of integers. Thus, as used
herein, the term "comprising" is inclusive and does not exclude
additional, unrecited integers or method/process steps.
[0044] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of". The phrase "consisting
essentially of" is used herein to require the specified integer(s)
or steps as well as those which do not materially affect the
character or function of the claimed invention. As used herein, the
term "consisting" is used to indicate the presence of the recited
integer (e.g. a feature, element, characteristic, property,
method/process step or limitation) or group of integers (e.g.
features, elements, characteristics, properties, method/process
steps or limitations) alone.
A. INTRODUCTION
[0045] Hapten-carrier conjugates are commonly employed constructs
for vaccine formulation. A well-known phenomenon related to their
use is the creation, or augmentation, of an immune response to the
carrier (e.g., anti-carrier antibodies, which are also referred to
herein as undesired or off-target antibodies). The anti-carrier
response is often of concern as it is not the intended effect of
the vaccine and it may relate to undesirable side effects. In the
similar case of nanocarrier vaccine formulations, such as
biocompatible synthetic nanocarriers presenting an antigen, an
anti-carrier effect may also be observed, such as initiated or
enhanced undesired antibody generation to the synthetic components
of the nanocarrier. In the case of nanocarriers which contain
synthetic components, even those with an extended history of safe
medical use in humans (e.g., PLGA, PLA, or PEG), anti-carrier
effects can result and attenuate the intended vaccine response or
alter the vaccine's immune response to those components in other
medical applications. It is, therefore, valuable to identify means
to formulate nanocarrier vaccines such that the anti-carrier effect
is attenuated or absent.
[0046] The inventors have unexpectedly and surprisingly discovered
that the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. The inventors believe
that the invention provided herein is the first of its kind to
offer the ability to optimize a target antibody response specific
for a B cell antigen of a synthetic nanocarrier composition while
attenuating an off-target anti-carrier antibody response. Prior
studies have not addressed the design of synthetic nanocarriers
relative to optimizing humoral immune responses. Specifically, the
inventors have discovered that nanocarriers can be rationally
designed as a function of B cell antigen content and/or polymer
molecular weights or composition to optimize target antibody
generation to the B cell antigen and minimize or eliminate
off-target antibody generation. In particular, the inventors have
unexpectedly discovered that it is possible to provide compositions
with improved target antibody response versus off-target antibody
response, and related methods, that comprise a population of
synthetic nanocarriers, wherein the synthetic nanocarriers comprise
(i) a B cell antigen and (ii) an off-target response attenuating
polymeric coating, wherein the synthetic nanocarriers generate on
average across the population of synthetic nanocarriers an antibody
response against the B cell antigen that is at least two-fold
greater than an off-target antibody response. In one embodiment,
the respective antibody responses are measured as an antibody titer
(e.g., IgG or IgA EC50) with ELISA. In one embodiment, the
off-target antibody response is an undesired antibody response
against the synthetic nanocarrier or a component thereof not
specific to the B cell antigen. In another embodiment, the
off-target antibody response is an antibody response against a
polymer (or portion thereof) of the synthetic nanocarrier, such as
a polymer (or portion thereof) of the coating. The B cell antigen
may be coupled to the off-target response attenuating polymeric
coating. In another embodiment, the B cell antigen is not coupled
to the off-target response attenuating polymeric coating but is
coupled to the synthetic nanocarrier. In embodiments, the B cell
antigen or portion thereof is present at the surface of the
synthetic nanocarrier.
[0047] Preferably, in one embodiment, the off-target response
attenuating polymeric coating comprises a polymer with a molecular
weight of greater than 2000 g/mole, 3000 g/mole, 4000 g/mole or
5000 g/mole given as the weight average molecular weight or number
average molecular weight. In another embodiment, the off-target
response attenuating polymeric coating comprises a polymer with a
molecular weight of between 2000-5000 g/mole, between 2500-5000
g/mole, between 3000-5000 g/mole, between 3500-5000 g/mole or
between 4000-5000 g/mole. The B cell antigen may be coupled to the
polymer, another polymer or to another portion of the synthetic
nanocarrier that is not the coating. In another embodiment, wherein
the B cell antigen is coupled to the other polymer, the other
polymer has a molecular weight of greater than 2000 g/mole, 3000
g/mole, 4000 g/mole or 5000 g/mole given as a weight average
molecular weight or as a number average molecular weight. In
another embodiment, the other polymer has a molecular weight of
between 2000-5000 g/mole, between 2500-5000 g/mole, between
3000-5000 g/mole, between 3500-5000 g/mole or between 4000-5000
g/mole. In a certain preferred embodiment, the polymer and other
polymer both have a molecular weight of 5000 g/mole given as a
weight average molecular weight or a number average molecular
weight. In still another embodiment of any of the foregoing
embodiments where the polymer does not comprise polyethylene
glycol, the molecular weight is the weight average molecular
weight. In yet another embodiment of any of the foregoing
embodiments where the polymer does comprise polyethylene glycol,
the molecular weight is the number average molecular weight.
[0048] In another preferred embodiment, the ratio of the average
number of polymers coupled to the B cell antigen across the
population of synthetic nanocarriers to the average number of
polymers not coupled to the B cell antigen across the population of
synthetic nanocarriers, the ratio of the average number of polymers
coupled to the B cell antigen across the population of synthetic
nanocarriers to the average number of polymers coupled to the B
cell antigen across the population of synthetic nanocarriers plus
the average number of polymers not coupled to the B cell antigen
across the population of synthetic nanocarriers, or the ratio of
the average number of polymers not coupled to the B cell antigen
across the population of synthetic nanocarriers to the average
number of polymers coupled to the B cell antigen across the
population of synthetic nanocarriers plus the average number of
polymers not coupled to the B cell antigen across the population of
synthetic nanocarriers is between 0.001 and 1, 0.01 and 1, 0.1 and
1, 0.25 and 1, 0.5 and 1, 0.75 and 1, 0.01 and 0.75, 0.01 and 0.5,
0.01 and 0.25, 0.1 and 0.75, 0.1 and 0.5 or 0.1 and 0.25. In
embodiments, this ratio may be calculated for the polymeric coating
of the synthetic nanocarriers. In other embodiments, this ratio is
calculated for the synthetic nanocarriers as a whole. The polymers
coupled to the B cell antigen and the polymers not coupled to the B
cell antigen may be the same type of polymer or may be different
types of polymers. In one embodiment, the polymers coupled to the B
cell antigen and/or the polymers not coupled to the B cell antigen
have a molecular weight of greater than 2000 g/mole, 3000 g/mole,
4000 g/mole or 5000 g/mole. In another embodiment, the polymers
coupled to the B cell antigen and/or the polymers not coupled to
the B cell antigen have a molecular weight of between 2000-5000
g/mole, between 2500-5000 g/mole, between 3000-5000 g/mole, between
3500-5000 g/mole or between 4000-5000 g/mole. Again, in a certain
preferred embodiment, the polymer and other polymer both have a
molecular weight of 5000 g/mole. As above the molecular weight may
be the weight average molecular weight or it may be the number
average molecular weight.
[0049] In yet another preferred embodiment, the ratio by weight
averaged across the population of synthetic nanocarriers of polymer
coupled to the B cell antigen nanocarriers to polymer not coupled
to the B cell antigen, polymer coupled to the B cell antigen
nanocarriers to polymer coupled to the B cell antigen plus polymer
not coupled to the B cell antigen, or polymer not coupled to the B
cell antigen nanocarriers to polymer coupled to the B cell antigen
plus polymer not coupled to the B cell antigen is greater than 0.1,
0.25 or 0.5 and less than 1. Again, this ratio may be calculated
based on the polymeric coating of the synthetic nanocarriers. In
other embodiments, this ratio is calculated based on the synthetic
nanocarriers as a whole. Again, the polymers coupled to the B cell
antigen and the polymers not coupled to the B cell antigen may be
the same type of polymer or may be different types of polymers. In
one embodiment, the polymers coupled to the B cell antigen and/or
the polymers not coupled to the B cell antigen have a molecular
weight of greater than 2000 g/mole, 3000 g/mole, 4000 g/mole or
5000 g/mole. In another embodiment, the polymers coupled to the B
cell antigen and/or the polymers not coupled to the B cell antigen
have a molecular weight of between 2000-5000 g/mole, between
2500-5000 g/mole, between 3000-5000 g/mole, between 3500-5000
g/mole or between 4000-5000 g/mole. Again, in a certain preferred
embodiment, the polymer and other polymer both have a molecular
weight of 5000 g/mole. The molecular weight may be a weight average
molecular weight or it may be a number average molecular
weight.
[0050] In one embodiment, any of the ratios referred to herein can
be based on the polymeric coating across the population of
synthetic nanocarriers. In another embodiment, the ratio is based
on the synthetic nanocarrier as a whole across the population of
synthetic nanocarriers.
[0051] In another aspect, dosage forms and vaccines comprising any
of the compositions provided herein are provided.
[0052] In still another aspect, any of the compositions may be
administered to a subject in need thereof. The subject may have or
be at risk of having cancer, an infection or infectious disease, a
non-autoimmune or degenerative disease or an addiction.
[0053] The invention will now be described in more detail
below.
B. DEFINITIONS
[0054] "Addiction antigens" are antigens associated with an
addiction or addictive substance. Such antigens include those that
can generate an antibody response against an addictive substance.
Such antigens can comprise an addictive substance or a portion
thereof.
[0055] "Adjuvant" means an agent that does not constitute a
specific antigen, but boosts the strength and longevity of immune
response to a concomitantly administered antigen. Such adjuvants
may include, but are not limited to stimulators of pattern
recognition receptors, such as Toll-like receptors, RIG-1 and
NOD-like receptors (NLR), mineral salts, such as alum, alum
combined with monphosphoryl lipid (MPL) A of Enterobacteria, such
as Escherihia coli, Salmonella minnesota, Salmonella typhimurium,
or Shigella flexneri or specifically with MPL.RTM. (AS04), MPL A of
above-mentioned bacteria separately, saponins, such as QS-21,
Quil-A, ISCOMs, ISCOMATRIX.TM., emulsions such as MF59.TM.,
Montanide.RTM. ISA 51 and ISA 720, AS02 (QS21+ squalene+MPL.RTM.),
liposomes and liposomal formulations such as AS01, synthesized or
specifically prepared microparticles and microcarriers such as
bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae,
Chlamydia trachomatis and others, or chitosan particles,
depot-forming agents, such as Pluronic.RTM. block co-polymers,
specifically modified or prepared peptides, such as muramyl
dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or
proteins, such as bacterial toxoids or toxin fragments.
[0056] In embodiments, adjuvants comprise agonists for pattern
recognition receptors (PRR), including, but not limited to
Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9
and/or combinations thereof. In other embodiments, adjuvants
comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like
Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably
the recited adjuvants comprise imidazoquinolines; such as R848;
adenine derivatives, such as those disclosed in U.S. Pat. No.
6,329,381 (Sumitomo Pharmaceutical Company), US Published Patent
Application 2010/0075995 to Biggadike et al., or WO 2010/018132 to
Campos et al.; immunostimulatory DNA; or immunostimulatory RNA. In
specific embodiments, synthetic nanocarriers incorporate as
adjuvants compounds that are agonists for toll-like receptors
(TLRs) 7 & 8 ("TLR 7/8 agonists"). Of utility are the TLR 7/8
agonist compounds disclosed in U.S. Pat. No. 6,696,076 to Tomai et
al., including but not limited to imidazoquinoline amines,
imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines,
and 1,2-bridged imidazoquinoline amines. Preferred adjuvants
comprise imiquimod and resiquimod (also known as R848). In specific
embodiments, an adjuvant may be an agonist for the DC surface
molecule CD40. In certain embodiments, to stimulate immunity rather
than tolerance, a synthetic nanocarrier incorporates an adjuvant
that promotes DC maturation (needed for priming of naive T cells)
and the production of cytokines, such as type I interferons, which
promote antibody immune responses. In embodiments, adjuvants also
may comprise immunostimulatory RNA molecules, such as but not
limited to dsRNA, poly I:C or poly I:poly C12U (available as
Ampligen.RTM., both poly I:C and poly I:polyC12U being known as
TLR3 stimulants), and/or those disclosed in F. Heil et al.,
"Species-Specific Recognition of Single-Stranded RNA via Toll-like
Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer
et al., "Immune modulation by chemically modified ribonucleosides
and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al.,
"Immunostimulatory oligoribonucleotides containing specific
sequence motif(s) and targeting the Toll-like receptor 8 pathway"
WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide
analogs with enhanced immunostimulatory activity" U.S. Pat. Appl.
Publ. 2006241076; G. Lipford et al., "Immunostimulatory viral RNA
oligonucleotides and use for treating cancer and infections" WO
2005097993 A2; G. Lipford et al, "Immunostimulatory G,U-containing
oligoribonucleotides, compositions, and screening methods" WO
2003086280 A2. In some embodiments, an adjuvant may be a TLR-4
agonist, such as bacterial lipopolysacccharide (LPS), VSV-G, and/or
HMGB-1. In some embodiments, adjuvants may comprise TLR-5 agonists,
such as flagellin, or portions or derivatives thereof, including
but not limited to those disclosed in U.S. Pat. Nos. 6,130,082,
6,585,980, and 7,192,725. In specific embodiments, synthetic
nanocarriers incorporate a ligand for Toll-like receptor (TLR)-9,
such as immunostimulatory DNA molecules comprising CpGs, which
induce type I interferon secretion, and stimulate T and B cell
activation leading to increased antibody production and cytotoxic T
cell responses (Krieg et al., CpG motifs in bacterial DNA trigger
direct B cell activation. Nature. 1995. 374:546-549; Chu et al. CpG
oligodeoxynucleotides act as adjuvants that switch on T helper 1
(Th1) immunity. J. Exp. Med. 1997. 186:1623-1631; Lipford et al.
CpG-containing synthetic oligonucleotides promote B and cytotoxic T
cell responses to protein antigen: a new class of vaccine
adjuvants. Eur. J. Immunol. 1997. 27:2340-2344; Roman et al
Immunostimulatory DNA sequences function as T helper-1-promoting
adjuvants. Nat. Med. 1997. 3:849-854; Davis et al. CpG DNA is a
potent enhancer of specific immunity in mice immunized with
recombinant hepatitis B surface antigen. J. Immunol. 1998.
160:870-876; Lipford et al., Bacterial DNA as immune cell
activator. Trends Microbiol. 1998. 6:496-500; U.S. Pat. No.
6,207,646 to Krieg et al.; U.S. Pat. No. 7,223,398 to Tuck et al.;
U.S. Pat. No. 7,250,403 to Van Nest et al.; or U.S. Pat. No.
7,566,703 to Krieg et al.
[0057] In some embodiments, adjuvants may be proinflammatory
stimuli released from necrotic cells (e.g., urate crystals). In
some embodiments, adjuvants may be activated components of the
complement cascade (e.g., CD21, CD35, etc.). In some embodiments,
adjuvants may be activated components of immune complexes. The
adjuvants also include complement receptor agonists, such as a
molecule that binds to CD21 or CD35. In some embodiments, the
complement receptor agonist induces endogenous complement
opsonization of the synthetic nanocarrier. In some embodiments,
adjuvants are cytokines, which are small proteins or biological
factors (in the range of 5 kD-20 kD) that are released by cells and
have specific effects on cell-cell interaction, communication and
behavior of other cells. In some embodiments, the cytokine receptor
agonist is a small molecule, antibody, fusion protein, or
aptamer.
[0058] In embodiments, at least a portion of the dose of adjuvant
may be coupled to synthetic nanocarriers, preferably, all of the
dose of adjuvant is coupled to synthetic nanocarriers. In other
embodiments, at least a portion of the dose of the adjuvant is not
coupled to the synthetic nanocarriers. In embodiments, the dose of
adjuvant comprises two or more types of adjuvants or multiple
adjuvants of the same type. For instance, and without limitation,
adjuvants that act on different TLR receptors may be combined. As
an example, in an embodiment a TLR 7/8 agonist may be combined with
a TLR9 agonist. In another embodiment, a TLR 7/8 agonist may be
combined with a TLR9 agonist. In yet another embodiment, a TLR9
agonist may be combined with a TLR9 agonist. In another embodiment,
two TLR9 agonists may be combined.
[0059] "Administering" or "administration" means providing a
material, such as a drug, to a subject in a manner that is
pharmacologically useful.
[0060] "Amount effective" is any amount of a composition provided
herein that produces one or more desired responses, such as one or
more desired immune responses. This amount can be for in vitro or
in vivo purposes. For in vivo purposes, the amount can be one that
a clinician would believe may have a clinical benefit for a subject
in need of a humoral immune response to a B cell antigen. Such
subjects include those that have or are at risk of having cancer,
an infection or infectious disease, a non-autoimmune or
degenerative disease or an addiction.
[0061] Amounts effective include those that involve the production
of an antibody response against a B cell antigen administered in
one of the inventive compositions provided herein. A subject's
antibody response can be monitored by routine methods. An amount
that is effective to produce one or more desired immune responses
can also be an amount of a composition provided herein that
produces a desired therapeutic endpoint or a desired therapeutic
result.
[0062] Amounts effective will depend, of course, on the particular
subject being treated; the severity of a condition, disease or
disorder; the individual patient parameters including age, physical
condition, size and weight; the duration of the treatment; the
nature of concurrent therapy (if any); the specific route of
administration and like factors within the knowledge and expertise
of the health practitioner. These factors are well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation. It is generally preferred that a maximum
dose be used, that is, the highest safe dose according to sound
medical judgment. It will be understood by those of ordinary skill
in the art, however, that a patient may insist upon a lower dose or
tolerable dose for medical reasons, psychological reasons or for
virtually any other reason.
[0063] In general, doses of the compositions of the invention can
range from about 10 .mu.g/kg to about 100,000 .mu.g/kg. In some
embodiments, the doses can range from about 0.1 mg/kg to about 100
mg/kg. In still other embodiments, the doses can range from about
0.1 mg/kg to about 25 mg/kg, about 25 mg/kg to about 50 mg/kg,
about 50 mg/kg to about 75 mg/kg or about 75 mg/kg to about 100
mg/kg. Alternatively, the dose can be administered based on the
number of synthetic nanocarriers. For example, useful doses include
greater than 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9 or 10.sup.10
synthetic nanocarriers per dose. Other examples of useful doses
include from about 1.times.10.sup.6 to about 1.times.10.sup.10,
about 1.times.10.sup.7 to about 1.times.10.sup.9 or about
1.times.10.sup.8 to about 1.times.10.sup.9 synthetic nanocarriers
per dose.
[0064] "Antibody response" refers to the generation of antibodies
specific for an antigen. An antibody response can target a desired
B cell antigen (i.e., a desired antibody response) or to an
off-target B cell antigen (i.e., an undesired antibody response).
Preferably, the desired antibody responses are specific to the
coupled B cell antigen of the compositions provided. Undesired
antibody responses can interfere with desired antibody responses
and include, for example, undesired antibody responses to the
synthetic nanocarrier or a component thereof (e.g., a polymer,
portion or unit thereof) of a synthetic nanocarrier. As a result,
the compositions provided herein have been devised to include
synthetic nanocarriers with an off-target response attenuating
polymeric coating that elicits a desired antibody response to a
coupled B cell antigen that is at least two-fold greater than an
undesired antibody response, such as to the synthetic nanocarrier
or component thereof. As provided elsewhere herein, the level of
antibody response can be measured as a titer with an ELISA.
[0065] Methods for measuring an antibody response are known to
those of ordinary skill in the art and are also exemplified below
in the EXAMPLES. In particular, the antibody response can be
quantitated, for example, as the number of antibodies,
concentration of antibodies or titer. The values can be absolute or
they can be relative. Assays for quantifying an antibody response
include antibody capture assays, enzyme-linked immunosorbent assays
(ELISAs), inhibition liquid phase absorption assays (ILPAAs),
rocket immunoelectrophoresis (RIE) assays and line
immunoelectrophoresis (LIE) assays. When a desired antibody
response is compared to an undesired antibody response the same
type of quantitative value (e.g., titer) and method of measurement
(e.g., ELISA) is used to make the comparison.
[0066] An ELISA method for measuring an antibody titer, for
example, may consist of the following steps (i) preparing an
ELISA-plate coating material such that the antibody target of
interest is coupled to a substrate polymer or other suitable
material (ii) preparing the coating material in an aqueous solution
(such as PBS) and delivering the coating material solution to the
wells of a multiwell plate for overnight deposition of the coating
onto the multiwell plate (iii) thoroughly washing the multiwell
plate with wash buffer (such as 0.05% Tween-20 in PBS) to remove
excess coating material (iv) blocking the plate for nonspecific
binding by applying a diluent solution (such as 10% fetal bovine
serum in PBS), (v) washing the blocking/diluent solution from the
plate with wash buffer (vi) diluting the serum sample(s) containing
antibodies and appropriate standards (positive controls) with
diluent as required to obtain a concentration that suitably
saturates the ELISA reponse (vii) serially diluting the plasma
samples on the multiwell plate such to cover a range of
concentrations suitable for generating an ELISA response curve
(viii) incubating the plate to provide for antibody-target binding
(ix) washing the plate with wash buffer to remove antibodies not
bound to antigen (x) adding an appropriate concentration of a
secondary detection antibody in same diluent such as a
biotin-coupled detection antibody capable of binding the primary
antibody (xi) incubating the plate with the applied detection
antibody, followed by washing with wash buffer (xii) adding an
enzyme such as streptavidin-HRP (horse radish peroxidase) that will
bind to biotin found on biotinylated antibodies and incubating
(xiii) washing the multiwell plate (xiv) adding substrate(s) (such
as TMB solution) to the plate (xv) applying a stop solution (such
as 2N sulfuric acid) when color development is complete (xvi)
reading optical density of the plate wells at a specific wavelength
for the substrate (450 nm with subtraction of readings at 570 nm)
(xvi) applying a suitable multiparameter curve fit to the data and
defining half-maximal effective concentration (EC50) as the
concentration on the curve at which half the maximum OD value for
the plate standards is achieved.
[0067] "Antigen" means a B cell antigen or T cell antigen. In
embodiments, antigens are coupled to the synthetic nanocarriers. In
other embodiments, antigens are not coupled to the synthetic
nanocarriers. "Type(s) of antigens" means molecules that share the
same, or substantially the same, antigenic characteristics.
[0068] An "at risk" subject is one in which a health practitioner
believes has a chance of having a disease or condition as provided
herein.
[0069] "Average", as used herein, refers to the arithmetic mean
unless otherwise noted.
[0070] "Average number of polymers" is an absolute or relative
value for the number of polymers averaged across a population of
synthetic nanocarriers. Methods for determining the average number
of polymers are known to those of ordinary skill in the art. For
example, the average number of polymers in a formulated population
may be obtained by determining the total weight of the polymer in
the population and dividing by the number-averaged molecular
weight. When the ratio of polymers as provided herein is calculated
for a particular synthetic nanocarrier population the same type of
value (absolute or relative) measured according to the same type of
assay is used.
[0071] "B cell antigen" means any antigen that is recognized by or
triggers an immune response in a B cell (e.g., an antigen that is
specifically recognized by a B cell or a receptor thereon). In some
embodiments, an antigen that is a T cell antigen is also a B cell
antigen. In other embodiments, the T cell antigen is not also a B
cell antigen. B cell antigens include, but are not limited to
proteins, peptides, small molecules, oligosaccharides, and
carbohydrates. In some embodiments, the B cell antigen comprises a
non-protein antigen (i.e., not a protein or peptide antigen). In
some embodiments, the B cell antigen comprises a carbohydrate
associated with an infectious agent. In some embodiments, the B
cell antigen comprises a glycoprotein or glycopeptide associated
with an infectious agent. The infectious agent can be a bacterium,
virus, fungus, protozoan, or parasite. In some embodiments, the B
cell antigen comprises a poorly immunogenic antigen. In some
embodiments, the B cell antigen comprises an abused substance or a
portion thereof. In some embodiments, the B cell antigen comprises
an addictive substance or a portion thereof. Addictive substances
include, but are not limited to, nicotine, a narcotic, a cough
suppressant, a tranquilizer, and a sedative. In some embodiments,
the B cell antigen comprises a toxin, such as a toxin from a
chemical weapon or natural sources. The B cell antigen may also
comprise a hazardous environmental agent. In some embodiments, the
B cell antigen comprises a self antigen. In other embodiments, the
B cell antigen comprises an alloantigen, an allergen, a contact
sensitizer, a degenerative disease antigen, a hapten, an infectious
disease antigen, a cancer antigen, an atopic disease antigen, an
autoimmune disease antigen, a non-autoimmune disease antigen, an
addictive substance, a xenoantigen, or a metabolic disease enzyme
or enzymatic product thereof.
[0072] Generally, as used herein and unless otherwise noted, "B
cell antigen" of the compositions provided refers to a B cell
antigen to which a target antibody response is desired and not to
an antigen to which an antibody response is not desired (e.g.,
against the carrier or synthetic component thereof (e.g., a polymer
of the synthetic nanocarrier)).
[0073] "Cancer antigens" are antigens associated with a cancer or
cancerous tumor. Such antigens can generate an antibody response
against a cancer or tumor cell. Such antigens can comprise an
antigen that is expressed in or on cancer or tumor cells but not in
or on normal or healthy cells. Such antigens can also comprise an
antigen that is expressed in or on cancer or tumor cells and on
normal or healthy cells but is expressed in or on cancer or tumor
cells at a greater level than on normal or healthy cells.
Preferably, the use of a cancer antigen in such an embodiment will
not lead to a substantial or detrimental immune response against
normal or healthy cells or will lead to a beneficial immune
response against the cancer or tumor cells that outweighs any
immune response against normal or healthy cells.
[0074] "Couple" or "Coupled" or "Couples" (and the like) means to
chemically associate one entity (for example a moiety) with
another. In some embodiments, the coupling is covalent, meaning
that the coupling occurs in the context of the presence of a
covalent bond between the two entities. In non-covalent
embodiments, the non-covalent coupling is mediated by non-covalent
interactions including but not limited to charge interactions,
affinity interactions, metal coordination, physical adsorption,
host-guest interactions, hydrophobic interactions, TT stacking
interactions, hydrogen bonding interactions, van der Waals
interactions, magnetic interactions, electrostatic interactions,
dipole-dipole interactions, and/or combinations thereof. In
embodiments, encapsulation is a form of coupling.
[0075] "Dosage form" means a pharmacologically and/or
immunologically active material in a medium, carrier, vehicle, or
device suitable for administration to a subject.
[0076] "Encapsulate" means to enclose at least a portion of a
substance within a synthetic nanocarrier. In some embodiments, a
substance is enclosed completely within a synthetic nanocarrier. In
other embodiments, most or all of a substance that is encapsulated
is not exposed to the local environment external to the synthetic
nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%,
10% or 5% (weight/weight) of the substance is exposed to the local
environment. Encapsulation is distinct from absorption, which
places most or all of a substance on a surface of a synthetic
nanocarrier, and leaves the substance exposed to the local
environment external to the synthetic nanocarrier. In some
embodiments, the polymeric coating provided herein encapsulates one
or more or all of the other substances of a synthetic nanocarrier
provided. In one embodiment, these other substances do not include
desired B cell antigen coupled to the polymeric coating at the
surface of the synthetic nanocarrier.
[0077] "Humoral response" means any immune response that results in
the production or stimulation of B cells and/or the production of
antibodies. Preferably, the humoral immune response is specific to
an antigen comprised within an inventive composition or
administered during the practice of an inventive method. Methods
for assessing whether a humoral response is induced are known to
those of ordinary skill in the art. Examples of such methods are
provided below in the Examples.
[0078] An "infection" or "infectious disease" is any condition or
disease caused by a microorganism, pathogen or other agent, such as
a bacterium, fungus, prion or virus. "An infection or infectious
disease antigen" is an antigen associated with an infection or
infectious disease. Such antigens include antigens that can be used
to generate an antibody response against a pathogen or other
infectious agent, or component thereof, or that can generate an
antibody response against infected cells.
[0079] "Maximum dimension of a synthetic nanocarrier" means the
largest dimension of a nanocarrier measured along any axis of the
synthetic nanocarrier. "Minimum dimension of a synthetic
nanocarrier" means the smallest dimension of a synthetic
nanocarrier measured along any axis of the synthetic nanocarrier.
For example, for a spheroidal synthetic nanocarrier, the maximum
and minimum dimension of a synthetic nanocarrier would be
substantially identical, and would be the size of its diameter.
Similarly, for a cuboidal synthetic nanocarrier, the minimum
dimension of a synthetic nanocarrier would be the smallest of its
height, width or length, while the maximum dimension of a synthetic
nanocarrier would be the largest of its height, width or length. In
an embodiment, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or greater than 100 nm. In
an embodiment, a maximum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or less than 5 .mu.m.
Preferably, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is greater than 110 nm, more preferably
greater than 120 nm, more preferably greater than 130 nm, and more
preferably still greater than 150 nm. Aspects ratios of the maximum
and minimum dimensions of inventive synthetic nanocarriers may vary
depending on the embodiment. For instance, aspect ratios of the
maximum to minimum dimensions of the synthetic nanocarriers may
vary from 1:1 to 1,000, 000:1, preferably from 1:1 to 100, 000:1,
more preferably from 1:1 to 10,000:1, more preferably from 1:1 to
1000:1, still more preferably from 1:1 to 100:1, and yet more
preferably from 1:1 to 10:1. Preferably, a maximum dimension of at
least 75%, preferably at least 80%, more preferably at least 90%,
of the synthetic nanocarriers in a sample, based on the total
number of synthetic nanocarriers in the sample is equal to or less
than 3 .mu.m, more preferably equal to or less than 2 .mu.m, more
preferably equal to or less than 1 .mu.m, more preferably equal to
or less than 800 nm, more preferably equal to or less than 600 nm,
and more preferably still equal to or less than 500 nm. In
preferred embodiments, a minimum dimension of at least 75%,
preferably at least 80%, more preferably at least 90%, of the
synthetic nanocarriers in a sample, based on the total number of
synthetic nanocarriers in the sample, is equal to or greater than
100 nm, more preferably equal to or greater than 120 nm, more
preferably equal to or greater than 130 nm, more preferably equal
to or greater than 140 nm, and more preferably still equal to or
greater than 150 nm. Measurement of synthetic nanocarrier
dimensions (e.g., diameter) is obtained by suspending the synthetic
nanocarriers in a liquid (usually aqueous) media and using dynamic
light scattering (DLS) (e.g. using a Brookhaven ZetaPALS
instrument). For example, a suspension of synthetic nanocarriers
can be diluted from an aqueous buffer into purified water to
achieve a final synthetic nanocarrier suspension concentration of
approximately 0.01 to 0.1 mg/mL. The diluted suspension may be
prepared directly inside, or transferred to, a suitable cuvette for
DLS analysis. The cuvette may then be placed in the DLS, allowed to
equilibrate to the controlled temperature, and then scanned for
sufficient time to acquire a stable and reproducible distribution
based on appropriate inputs for viscosity of the medium and
refractive indicies of the sample. The effective diameter, or mean
of the distribution, is then reported. "Dimension" or "size" or
"diameter" of synthetic nanocarriers means the mean of a particle
size distribution obtained using dynamic light scattering.
[0080] "Non-autoimmune or degenerative antigens" are antigens
associated with non-autoimmune or degenerative diseases or
conditions. Such antigens can result in an antibody response that
can be indicative of and/or present when the non-autoimmune or
degenerative disease or condition occurs or is present in a
subject. Such antigens can also be used to generate an antibody
response, the generation of which may be beneficial in the
treatment or prevention of the disease or condition or one or more
symptoms thereof.
[0081] "Off-target response attenuating polymeric coating" refers
to a composition comprising one or more polymers present at at
least a portion of the surface of a synthetic nanocarrier and that
when the synthetic nanocarrier is coupled to a B cell antigen, the
synthetic nanocarrier, or population thereof, generates an antibody
response against the B cell antigen that is at least two-fold
greater than an off-target (or undesired) antibody response, such
as against the synthetic nanocarrier or component thereof (such as
a polymer of the coating). In one embodiment, the antibody response
against the B cell antigen and the off-target antibody response are
of the same type, such as both an IgG or IgA antibody response. In
another embodiment, the synthetic nanocarrier, or population
thereof, generates an antibody response that is at least 3-, 4-,
5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 20-, 25-, 30-,
35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95- or
100-fold greater. In another embodiment, the response is measured
as an antibody titer (e.g., IgG or IgA EC50) with an ELISA. In
another embodiment, the coating may be present throughout the
surface of a synthetic nanocarrier.
[0082] The coating may comprise a number of polymers of the same
type or it may comprise a number of polymers of two or more
different types. The polymers of the coating may comprise PEG, a
polyethyloxazoline, a polyamino acid, polycarbonate, hydrophilic
polyacetal, hydrophilic polyketal, polysaccharide, polypropylene or
polyethyleneimine, or some combination thereof. The coating may
comprise a number of the same type of the aforementioned polymers
or may comprise a number of two or more types of the aforementioned
polymers. In one embodiment, the coating comprises a number of
polymers that comprise one or more of the aforementioned types of
polymers, and it is the antibody response to one or more of these
aforementioned types of polymers that is at least two-fold less
than the antibody response to the target B cell antigen. In one
embodiment, the antibody response is at least 3-, 4-, 5-, 6-, 7-,
8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 20-, 25-, 30-, 35-, 40-, 45-,
50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95- or 100-fold less.
In another embodiment, the coating comprises polymers comprising
PEG, and it is the antibody response to PEG that is at least
two-fold less than the antibody response to the target B cell
antigen. In a further embodiment, the antibody response to PEG is
at least 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-,
20-, 25-, 30-, 35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-,
85-, 90-, 95- or 100-fold less. Again, the response may be measured
as an antibody titer (e.g., IgG or IgA EC50) with an ELISA, and/or
both responses are of the same type (e.g., both IgG or IgA antibody
responses).
[0083] Preferably, in one embodiment, the coating comprises a
polymer (e.g., one of the aforementioned polymers) with a molecular
weight of greater than 2000 g/mole, 3000 g/mole, 4000 g/mole or
5000 g/mole. In another embodiment, the polymer has a molecular
weight of between 2000-5000 g/mole, between 2500-5000 g/mole,
between 3000-5000 g/mole, between 3500-5000 g/mole or between
4000-5000 g/mole. The B cell antigen may be coupled to this polymer
or to another polymer of the coating. The B cell antigen may also
be coupled to another portion of the synthetic nanocarriers such as
to the surface of the synthetic nanocarriers but not to the
coating. In another embodiment, wherein the B cell antigen is
coupled to another polymer of the coating, the other polymer also
has a molecular weight of greater than 2000 g/mole, 3000 g/mole,
4000 g/mole or 5000 g/mole. In another embodiment, the other
polymer has a molecular weight of between 2000-5000 g/mole, between
2500-5000 g/mole, between 3000-5000 g/mole, between 3500-5000
g/mole or between 4000-5000 g/mole. In a certain preferred
embodiment, the polymer and other polymer of the coating both have
a molecular weight of 5000 g/mole. The molecular weight may be a
weight average molecular weight or a number average molecular
weight.
[0084] In another preferred embodiment, the ratio of the average
number of polymers coupled to the B cell antigen of the coating
across the population of synthetic nanocarriers to the average
number of polymers not coupled to the B cell antigen of the coating
across the population of synthetic nanocarriers, the ratio of the
average number of polymers coupled to the B cell antigen of the
coating across the population of synthetic nanocarriers to the
average number of polymers coupled to the B cell antigen of the
coating across the population of synthetic nanocarriers plus the
average number of polymers not coupled to the B cell antigen of the
coating across the population of synthetic nanocarriers, or the
ratio of the average number of polymers not coupled to the B cell
antigen of the coating across the population of synthetic
nanocarriers to the average number of polymers coupled to the B
cell antigen of the coating across the population of synthetic
nanocarriers plus the average number of polymers not coupled to the
B cell antigen of the coating across the population of synthetic
nanocarriers is between 0.001 and 1, 0.01 and 1, 0.1 and 1, 0.25
and 1, 0.5 and 1 or 0.75 and 1 or as elsewhere provided. In yet
another preferred embodiment, the ratio by weight is 0.1, 0.25 or
0.5. In one embodiment, this ratio is calculated based on the
polymeric coatings of the synthetic nanocarriers. In another
embodiment, this ratio is calculated based on the synthetic
nanocarriers as a whole. The polymers coupled to the B cell antigen
and the polymers not coupled to the B cell antigen may be the same
type of polymer or may be different types of polymers. In one
embodiment, the polymers coupled to the B cell antigen and/or the
polymers not coupled to the B cell antigen have a molecular weight
of greater than 2000 g/mole, 3000 g/mole, 4000 g/mole or 5000
g/mole. In another embodiment, the polymers coupled to the B cell
antigen and/or the polymers not coupled to the B cell antigen have
a molecular weight of between 2000-5000 g/mole, between 2500-5000
g/mole, between 3000-5000 g/mole, between 3500-5000 g/mole or
between 4000-5000 g/mole. Again, in a certain preferred embodiment,
the polymer and other polymer both have a molecular weight of 5000
g/mole. The molecular weight may be the weight average molecular
weight or the number average molecular weight.
[0085] "Off-target antibody response" is any undesired antibody
response as provided herein. Generally, the off-target antibody
response is an antibody response not specific to the B cell antigen
coupled to the synthetic nanocarriers to which an antibody response
is desired. In some embodiments, if the intended antibody response
is IgG or IgA, it is desirable that this desired antibody response
be at least two-fold greater than the off-target response by that
same class. In some embodiments, an off-target IgM response that is
of similar or greater magnitude than a desired IgG or IgA response
may occur. Generally, IgM tends to be a transient low-affinity
response whereas IgG is a longer-lasting higher-affinity
response.
[0086] In one embodiment, the off-target antibody response is an
antibody response against the synthetic nanocarrier or component
thereof, such as a polymer (or portion thereof), such as of the
coating.
[0087] "Pharmaceutically acceptable excipient" means a
pharmacologically inactive material used together with the recited
synthetic nanocarriers to formulate the inventive compositions.
[0088] Pharmaceutically acceptable excipients comprise a variety of
materials known in the art, including but not limited to
saccharides (such as glucose, lactose, and the like), preservatives
such as antimicrobial agents, reconstitution aids, colorants,
saline (such as phosphate buffered saline), and buffers.
[0089] "Ratio by weight averaged across the population of synthetic
nanocarriers" refers to the ratio of absolute or relative values
for two weights averaged across a population of synthetic
nanocarriers. When the ratio of the weight of polymers is
calculated for a particular synthetic nanocarrier population the
same type of value (absolute or relative) measured according to the
same type of assay is used. Methods for determining the weight of a
certain type of polymer in synthetic nanocarriers are known to
those of ordinary skill in the art. Examples of methods are also
provided elsewhere herein. Alternatively, well-described polymers,
such as those with information provided by a manufacturer can be
formulated at a certain ratio.
[0090] "Same type of polymer" means polymers that share the same,
or substantially the same, chemical structure. Polymers that are
the same type of polymers may have the same or different molecular
weights. In a preferred embodiment, polymers that are the same type
of polymer also have the same molecular weight.
[0091] "Subject" means animals, including warm blooded mammals such
as humans and primates; avians; domestic household or farm animals
such as cats, dogs, sheep, goats, cattle, horses and pigs;
laboratory animals such as mice, rats and guinea pigs; fish;
reptiles; zoo and wild animals; and the like.
[0092] "Synthetic nanocarrier(s)" means a discrete object that is
not found in nature, and that possesses at least one dimension that
is less than or equal to 5 microns in size. Albumin nanoparticles
are generally included as synthetic nanocarriers, however in
certain embodiments the synthetic nanocarriers do not comprise
albumin nanoparticles. In embodiments, synthetic nanocarriers do
not comprise chitosan. In certain other embodiments, the synthetic
nanocarriers do not comprise chitosan. In other embodiments,
inventive synthetic nanocarriers are not lipid-based nanoparticles.
In further embodiments, inventive synthetic nanocarriers do not
comprise a phospholipid.
[0093] A synthetic nanocarrier can be, but is not limited to, one
or a plurality of lipid-based nanoparticles (also referred to
herein as lipid nanoparticles, i.e., nanoparticles where the
majority of the material that makes up their structure are lipids),
polymeric nanoparticles, metallic nanoparticles, surfactant-based
emulsions, dendrimers, buckyballs, nanowires, virus-like particles
(i.e., particles that are primarily made up of viral structural
proteins but that are not infectious or have low infectivity),
peptide or protein-based particles (also referred to herein as
protein particles, i.e., particles where the majority of the
material that makes up their structure are peptides or proteins)
(such as albumin nanoparticles) and/or nanoparticles that are
developed using a combination of nanomaterials such as
lipid-polymer nanoparticles. Synthetic nanocarriers may be a
variety of different shapes, including but not limited to
spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and
the like. Synthetic nanocarriers according to the invention
comprise one or more surfaces. Exemplary synthetic nanocarriers
that can be adapted for use in the practice of the present
invention comprise: (1) the biodegradable nanoparticles disclosed
in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymeric
nanoparticles of Published US Patent Application 20060002852 to
Saltzman et al., (3) the lithographically constructed nanoparticles
of Published US Patent Application 20090028910 to DeSimone et al.,
(4) the disclosure of WO 2009/051837 to von Andrian et al., (5) the
nanoparticles disclosed in Published US Patent Application
2008/0145441 to Penades et al., (6) the protein nanoparticles
disclosed in Published US Patent Application 20090226525 to de los
Rios et al., (7) the virus-like particles disclosed in published US
Patent Application 20060222652 to Sebbel et al., (8) the nucleic
acid coupled virus-like particles disclosed in published US Patent
Application 20060251677 to Bachmann et al., (9) the virus-like
particles disclosed in WO2010047839A1 or WO2009106999A2, (10) the
nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010) or (11) apoptotic cells, apoptotic bodies or
the synthetic or semisynthetic mimics disclosed in U.S. Publication
2002/0086049. In embodiments, synthetic nanocarriers may possess an
aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or
greater than 1:10.
[0094] Synthetic nanocarriers according to the invention that have
a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
with hydroxyl groups that activate complement or alternatively
comprise a surface that consists essentially of moieties that are
not hydroxyl groups that activate complement. In a preferred
embodiment, synthetic nanocarriers according to the invention that
have a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
that substantially activates complement or alternatively comprise a
surface that consists essentially of moieties that do not
substantially activate complement. In a more preferred embodiment,
synthetic nanocarriers according to the invention that have a
minimum dimension of equal to or less than about 100 nm, preferably
equal to or less than 100 nm, do not comprise a surface that
activates complement or alternatively comprise a surface that
consists essentially of moieties that do not activate complement.
In embodiments, synthetic nanocarriers exclude virus-like
particles. In embodiments, when synthetic nanocarriers comprise
virus-like particles, the virus-like particles comprise non-natural
adjuvant (meaning that the VLPs comprise an adjuvant other than
naturally occurring RNA generated during the production of the
VLPs). In embodiments, synthetic nanocarriers may possess an aspect
ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or
greater than 1:10.
[0095] "T cell antigen" means any antigen that is recognized by and
triggers an immune response in a T cell (e.g., an antigen that is
specifically recognized by a T cell receptor on a T cell or an NKT
cell via presentation of the antigen or portion thereof bound to a
Class I or Class II major histocompatability complex molecule
(MHC), or bound to a CD1 complex). In some embodiments, an antigen
that is a T cell antigen is also a B cell antigen. In other
embodiments, the T cell antigen is not also a B cell antigen. T
cell antigens generally are proteins or peptides. T cell antigens
may be an antigen that stimulates a CD8+ T cell response, a CD4+ T
cell response, or both. The nanocarriers, therefore, in some
embodiments can effectively stimulate both types of responses.
[0096] In some embodiments the T cell antigen is a T helper cell
antigen (i.e. one that can generate an enhanced response to a B
cell antigen, preferably an unrelated B cell antigen, through
stimulation of T cell help). In embodiments, a T helper cell
antigen may comprise one or more peptides obtained or derived from
tetanus toxoid, Epstein-Barr virus, influenza virus, respiratory
syncytial virus, measles virus, mumps virus, rubella virus,
cytomegalovirus, adenovirus, diphtheria toxoid, or a PADRE peptide
(known from the work of Sette et al. U.S. Pat. No. 7,202,351). In
other embodiments, a T helper cell antigen may comprise one or more
lipids, or glycolipids, including but not limited to:
.alpha.-galactosylceramide (.alpha.-GalCer), .alpha.-linked
glycosphingolipids (from Sphingomonas spp.), galactosyl
diacylglycerols (from Borrelia burgdorferi), lypophosphoglycan
(from Leishmania donovani), and phosphatidylinositol tetramannoside
(PIM4) (from Mycobacterium leprae). For additional lipids and/or
glycolipids useful as a T helper cell antigen, see V. Cerundolo et
al., "Harnessing invariant NKT cells in vaccination strategies."
Nature Rev Immun, 9:28-38 (2009). In embodiments, CD4+ T-cell
antigens may be derivatives of a CD4+ T-cell antigen that is
obtained from a source, such as a natural source. In such
embodiments, CD4+ T-cell antigen sequences, such as those peptides
that bind to MHC II, may have at least 70%, 80%, 90%, or 95%
identity to the antigen obtained from the source. In embodiments,
the T cell antigen, preferably a T helper cell antigen, may be
coupled to, or uncoupled from, a synthetic nanocarrier. In some
embodiments, the T cell antigen is encapsulated in the synthetic
nanocarriers of the compositions.
[0097] "Vaccine" means a composition of matter that improves the
immune response to a particular pathogen or disease. A vaccine
typically contains factors that stimulate a subject's immune system
to recognize a specific antigen as foreign and eliminate it from
the subject's body. A vaccine also establishes an immunologic
`memory` so the antigen will be quickly recognized and responded to
if a person is re-challenged. Vaccines can be prophylactic (for
example to prevent future infection by any pathogen), or
therapeutic (for example a vaccine against a tumor specific antigen
for the treatment of cancer). In embodiments, a vaccine may
comprise dosage forms according to the invention.
[0098] "Weight", as used herein, refers to mass unless otherwise
noted. When a molecular weight of a polymer is measured, it can be
measured as the weight average molecular weight or a number average
molecular weight. "Weight average molecular weight" for the
polymers of the compositions provided herein is calculated by the
following formula:
M _ w = i N i M i 2 i N i M i , Formula 1 ##EQU00001##
where Ni is the number of molecules of molecular weight Mi. The
weight average molecular weight can be determined by a variety of
methods including light scattering, small angle neutron scattering
(SANS), X-ray scattering, Nuclear Magnetic Resonance (NMR) and
sedimentation velocity. An example of an alternative for weight
average molecular weight is to perform gel permeation
chromatography using suitable traceable-weight standards to
establish a retention-time versus weight curve, and calculating the
mean weight-averaged molecular weight of a sample polymer from the
mean of the integrated sample peak as compared to the calibration
curve. The "number average molecular weight" can be determined by
NMR. For example, number average molecular weight can be determined
by proton NMR wherein the ratio of the polymer repeating units to
the end group is established and then multiplied by theoretical
repeating unit molecular weight. Alternatively, in the case of a
titratable (e.g., acid or base) end group polymer, a known weight
concentration may be established and then titrated in the presense
of an indicator dye with an appropriate neutralizing agent of known
molar concentration to provide moles of end group per mass of
polymer.
C. INVENTIVE COMPOSITIONS
[0099] Provided herein are compositions comprising synthetic
nanocarriers that provide optimized target antibody generation to a
B cell antigen relative to off-target antibody generation. These
synthetic nanocarriers comprise a B cell antigen and an off-target
response attenuating polymeric coating. It has been found that
optimized target antibody generation relative to off-target
antibody generation results when a polymeric coating comprises
certain B cell antigen content and/or polymer molecular weights and
compositions.
[0100] It has been found that coatings that provide optimized B
cell antigen response relative to off-target antibody response may
comprise polymers with certain molecular weights (as weight average
or number average) with polymers with greater molecular weights
having better effect. The coating may comprise one type of polymer
(with an aforementioned molecular weight) but may also comprise one
or more other types of polymers. The one or more other types of
polymers may also have the aforementioned molecular weights. The
one or more types of polymers of the coating may be in the form of
a polymeric matrix.
[0101] The target B cell antigen may be coupled to one of the types
of polymers of the coating or to more than one of the types of
polymers of the coating. In another embodiment, the target B cell
antigen is coupled to the polymer of the coating for which an
attenuated antibody response is desired. When a target B cell
antigen is coupled to one or more of the types of polymers of the
coating, the target B cell antigen is coupled to all or less than
all of the polymer molecules of the one or more types of polymers
of the coating. The target B cell antigen may also be coupled to
another component of the synthetic nanocarriers, such as the
surface of the synthetic nanocarrier, but not to the coating. The
target B cell antigen can be coupled, in some embodiments, by any
means known in the art. In one embodiment, the target B cell
antigen is coupled via a bond or linker.
[0102] It has also been found that the amount of antigen coupled to
the off-target response attenuating polymeric coating can also
provide optimized B cell antigen response relative to off-target
antibody response. It has been found that an increased amount of
antigen present in the coating of the synthetic nanocarrier
provides attenuated off-target antibody response relative to target
antibody response. In another preferred embodiment, the ratio of
the average number of polymers coupled to the B cell antigen across
the population of synthetic nanocarriers to the average number of
polymers not coupled to the B cell antigen across the population of
synthetic nanocarriers, the ratio of the average number of polymers
coupled to the B cell antigen across the population of synthetic
nanocarriers to the average number of polymers coupled to the B
cell antigen across the population of synthetic nanocarriers plus
the average number of polymers not coupled to the B cell antigen
across the population of synthetic nanocarriers, or the ratio of
the average number of polymers not coupled to the B cell antigen
across the population of synthetic nanocarriers to the average
number of polymers coupled to the B cell antigen across the
population of synthetic nanocarriers plus the average number of
polymers not coupled to the B cell antigen across the population of
synthetic nanocarriers may be between 0.001 and 1, 0.01 and 1, 0.1
and 1, 0.25 and 1, 0.5 and 1 or 0.75 and 1. The ratio can be
calculated based on an assessment of the polymeric coating across
the population of synthetic nanocarriers or of the synthetic
nanocarriers as a whole across the population of synthetic
nanocarriers. The polymers coupled to the B cell antigen and the
polymers not coupled to the B cell antigen of the coating may be
the same type of polymer or may be different types of polymers.
These polymers may also have the molecular weights provided above
in some embodiments.
[0103] As mentioned above, the polymers of the coating may comprise
a number of polymers of the same type or it may comprise a number
of polymers of two or more different types. The polymers of the
coating may comprise PEG, a polyethyloxazoline, a polyamino acid,
polycarbonate, hydrophilic polyacetal, hydrophilic polyketal,
polypropylene, polysaccharide or polyethyleneimine, or some
combination thereof. In preferred embodiments, the polymers of the
coating comprise PEG.
[0104] The off-target response attenuating polymeric coating may be
a coating on a number of different types of synthetic nanocarriers.
Accordingly, a wide variety of synthetic nanocarriers can be used
according to the invention. In some embodiments, synthetic
nanocarriers are spheres or spheroids. In some embodiments,
synthetic nanocarriers are flat or plate-shaped. In some
embodiments, synthetic nanocarriers are cubes or cubic. In some
embodiments, synthetic nanocarriers are ovals or ellipses. In some
embodiments, synthetic nanocarriers are cylinders, cones, or
pyramids.
[0105] In some embodiments, it is desirable to use a population of
synthetic nanocarriers that is relatively uniform in terms of size,
shape, and/or composition so that each synthetic nanocarrier has
similar properties. For example, at least 80%, at least 90%, or at
least 95% of the synthetic nanocarriers, based on the total number
of synthetic nanocarriers, may have a minimum dimension or maximum
dimension that falls within 5%, 10%, or 20% of the average diameter
or average dimension of the synthetic nanocarriers. In some
embodiments, a population of synthetic nanocarriers may be
heterogeneous with respect to size, shape, and/or composition.
[0106] Synthetic nanocarriers can be solid or hollow and can
comprise one or more layers. In some embodiments, each layer has a
unique composition and unique properties relative to the other
layer(s). To give but one example, synthetic nanocarriers may have
a core/shell structure, wherein the core is one layer (e.g. a
polymeric core) and the shell is a second layer (e.g. a lipid
bilayer or monolayer). Synthetic nanocarriers may comprise a
plurality of different layers.
[0107] In some embodiments, synthetic nanocarriers may optionally
comprise one or more lipids. In some embodiments, a synthetic
nanocarrier may comprise a liposome. In some embodiments, a
synthetic nanocarrier may comprise a lipid bilayer. In some
embodiments, a synthetic nanocarrier may comprise a lipid
monolayer. In some embodiments, a synthetic nanocarrier may
comprise a micelle. In some embodiments, a synthetic nanocarrier
may comprise a core comprising a polymeric matrix surrounded by a
lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In some
embodiments, a synthetic nanocarrier may comprise a non-polymeric
core (e.g., metal particle, quantum dot, ceramic particle, bone
particle, viral particle, proteins, nucleic acids, carbohydrates,
etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid
monolayer, etc.).
[0108] In some embodiments, synthetic nanocarriers can comprise one
or more other polymers. In some embodiments, various elements of
the synthetic nanocarriers can be coupled with such polymers. Such
other polymers may form a polymeric matrix, and the components of
the synthetic nanocarriers may be covalently associated with the
polymeric matrix. In some embodiments, covalent association is
mediated by a linker. In some embodiments, a component may be
noncovalently associated with the polymeric matrix. For example, in
some embodiments, a component may be encapsulated within,
surrounded by, and/or dispersed throughout a polymeric matrix.
Alternatively or additionally, a component can be associated with a
polymeric matrix by hydrophobic interactions, charge interactions,
van der Waals forces, etc. In some embodiments, where the polymers
of the coating form a polymeric matrix, components can also be
coupled thereto by these aforementioned methods.
[0109] A wide variety of polymers and methods for forming polymeric
matrices therefrom are known conventionally. In general, a
polymeric matrix comprises one or more polymers. Polymers may be
natural or unnatural (synthetic) polymers. Polymers may be
homopolymers or copolymers comprising two or more monomers. In
terms of sequence, copolymers may be random, block, or comprise a
combination of random and block sequences. Typically, polymers in
accordance with the present invention are organic polymers.
[0110] Examples of polymers suitable for use in the synthetic
nanocarriers, as part of the coating or other portion of the
synthetic nanocarriers, include, but are not limited to
polyethylenes, polycarbonates (e.g. poly(1,3-dioxan-2one)),
polyanhydrides (e.g. poly(sebacic anhydride)), polypropylfumerates,
polyamides (e.g. polycaprolactam), polyacetals, polyethers,
polyesters (e.g., polylactide, polyglycolide,
polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g.
poly(.beta.-hydroxyalkanoate))), poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates, polyureas,
polystyrenes, and polyamines, polylysine, polylysine-PEG
copolymers, and poly(ethyleneimine), poly(ethylene imine)-PEG
copolymers. In some embodiments, polymers in accordance with the
present invention include polymers which have been approved for use
in humans by the U.S. Food and Drug Administration (FDA) under 21
C.F.R. .sctn.177.2600, including but not limited to polyesters
(e.g., polylactic acid, poly(lactic-co-glycolic acid),
polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one));
polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g.,
polyethylene glycol); polyurethanes; polymethacrylates;
polyacrylates; and polycyanoacrylates.
[0111] In some embodiments, polymers can be hydrophilic. For
example, polymers may comprise anionic groups (e.g., phosphate
group, sulphate group, carboxylate group); cationic groups (e.g.,
quaternary amine group); or polar groups (e.g., hydroxyl group,
thiol group, amine group). In some embodiments, a synthetic
nanocarrier comprising a hydrophilic polymeric matrix generates a
hydrophilic environment within the synthetic nanocarrier. In some
embodiments, polymers can be hydrophobic. In some embodiments, a
synthetic nanocarrier comprising a hydrophobic polymeric matrix
generates a hydrophobic environment within the synthetic
nanocarrier. Selection of the hydrophilicity or hydrophobicity of
the polymer may have an impact on the nature of materials that are
incorporated (e.g. coupled) within the synthetic nanocarrier.
[0112] In some embodiments, polymers may be modified with one or
more moieties and/or functional groups. A variety of moieties or
functional groups can be used in accordance with the present
invention. In some embodiments, polymers may be modified with
polyethylene glycol (PEG), with a carbohydrate, and/or with acyclic
polyacetals derived from polysaccharides (Papisov, 2001, ACS
Symposium Series, 786:301). Certain embodiments may be made using
the general teachings of U.S. Pat. No. 5,543,158 to Gref et al., or
WO publication WO2009/051837 by Von Andrian et al.
[0113] In some embodiments, polymers may be modified with a lipid
or fatty acid group. In some embodiments, a fatty acid group may be
one or more of butyric, caproic, caprylic, capric, lauric,
myristic, palmitic, stearic, arachidic, behenic, or lignoceric
acid. In some embodiments, a fatty acid group may be one or more of
palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic,
gamma-linoleic, arachidonic, gadoleic, arachidonic,
eicosapentaenoic, docosahexaenoic, or erucic acid.
[0114] In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEG copolymers and copolymers of lactide and
glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG
copolymers, and derivatives thereof. In some embodiments,
polyesters include, for example, poly(caprolactone),
poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),
poly(serine ester), poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0115] In some embodiments, a polymer may be PLGA. PLGA is a
biocompatible and biodegradable co-polymer of lactic acid and
glycolic acid, and various forms of PLGA are characterized by the
ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic
acid, D-lactic acid, or D,L-lactic acid. The degradation rate of
PLGA can be adjusted by altering the lactic acid:glycolic acid
ratio. In some embodiments, PLGA to be used in accordance with the
present invention is characterized by a lactic acid:glycolic acid
ratio of approximately 85:15, approximately 75:25, approximately
60:40, approximately 50:50, approximately 40:60, approximately
25:75, or approximately 15:85.
[0116] In some embodiments, polymers may be one or more acrylic
polymers. In certain embodiments, acrylic polymers include, for
example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid
anhydride), methyl methacrylate, polymethacrylate, poly(methyl
methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate
copolymer, glycidyl methacrylate copolymers, polycyanoacrylates,
and combinations comprising one or more of the foregoing polymers.
The acrylic polymer may comprise fully-polymerized copolymers of
acrylic and methacrylic acid esters with a low content of
quaternary ammonium groups.
[0117] In some embodiments, polymers can be cationic polymers. In
general, cationic polymers are able to condense and/or protect
negatively charged strands of nucleic acids (e.g. DNA, or
derivatives thereof). Amine-containing polymers such as
poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and
Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethylene
imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA,
1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo
et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al.,
1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,
Bioconjugate Chem., 4:372) are positively-charged at physiological
pH, form ion pairs with nucleic acids, and mediate transfection in
a variety of cell lines. In embodiments, the inventive synthetic
nanocarriers may not comprise (or may exclude) cationic
polymers.
[0118] In some embodiments, polymers can be degradable polyesters
bearing cationic side chains (Putnam et al., 1999, Macromolecules,
32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon
et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am.
Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules,
23:3399). Examples of these polyesters include
poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem.
Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,
Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633).
[0119] The properties of these and other polymers and methods for
preparing them are well known in the art (see, for example, U.S.
Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404;
6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600;
5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and
4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et
al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem.
Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et
al., 1999, Chem. Rev., 99:3181). More generally, a variety of
methods for synthesizing certain suitable polymers are described in
Concise Encyclopedia of Polymer Science and Polymeric Amines and
Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles
of Polymerization by Odian, John Wiley & Sons, Fourth Edition,
2004; Contemporary Polymer Chemistry by Allcock et al.,
Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in
U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
[0120] In some embodiments, polymers can be linear or branched
polymers. In some embodiments, polymers can be dendrimers. In some
embodiments, polymers can be substantially cross-linked to one
another. In some embodiments, polymers can be substantially free of
cross-links. In some embodiments, polymers can be used in
accordance with the present invention without undergoing a
cross-linking step. It is further to be understood that inventive
synthetic nanocarriers may comprise block copolymers, graft
copolymers, blends, mixtures, and/or adducts of any of the
foregoing and other polymers. Those skilled in the art will
recognize that the polymers listed herein represent an exemplary,
not comprehensive, list of polymers that can be of use in
accordance with the present invention.
[0121] In some embodiments, synthetic nanocarriers may comprise
metal particles, quantum dots, ceramic particles, etc. In some
embodiments, a non-polymeric synthetic nanocarrier is an aggregate
of non-polymeric components, such as an aggregate of metal atoms
(e.g., gold atoms).
[0122] In some embodiments, synthetic nanocarriers may optionally
comprise one or more amphiphilic entities. In some embodiments, an
amphiphilic entity can promote the production of synthetic
nanocarriers with increased stability, improved uniformity, or
increased viscosity. In some embodiments, amphiphilic entities can
be associated with the interior surface of a lipid membrane (e.g.,
lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities
known in the art are suitable for use in making synthetic
nanocarriers in accordance with the present invention. Such
amphiphilic entities include, but are not limited to,
phosphoglycerides; phosphatidylcholines; dipalmitoyl
phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine
(DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester;
diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol
(DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol
(PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid,
such as palmitic acid or oleic acid; fatty acids; fatty acid
monoglycerides; fatty acid diglycerides; fatty acid amides;
sorbitan trioleate (Span.RTM.85) glycocholate; sorbitan monolaurate
(Span.RTM.20); polysorbate 20 (Tween.RTM.20); polysorbate 60
(Tween.RTM.60); polysorbate 65 (Tween.RTM.65); polysorbate 80
(Tween.RTM.80); polysorbate 85 (Tween.RTM.85); polyoxyethylene
monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester
such as sorbitan trioleate; lecithin; lysolecithin;
phosphatidylserine; phosphatidylinositol; sphingomyelin;
phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic
acid; cerebrosides; dicetylphosphate;
dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine;
hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl
sterate; isopropyl myristate; tyloxapol; poly(ethylene
glycol)5000-phosphatidylethanolamine; poly(ethylene
glycol)400-monostearate; phospholipids; synthetic and/or natural
detergents having high surfactant properties; deoxycholates;
cyclodextrins; chaotropic salts; ion pairing agents; and
combinations thereof. An amphiphilic entity component may be a
mixture of different amphiphilic entities. Those skilled in the art
will recognize that this is an exemplary, not comprehensive, list
of substances with surfactant activity. Any amphiphilic entity may
be used in the production of synthetic nanocarriers to be used in
accordance with the present invention.
[0123] In some embodiments, synthetic nanocarriers may optionally
comprise one or more carbohydrates. Carbohydrates may be natural or
synthetic. A carbohydrate may be a derivatized natural
carbohydrate. In certain embodiments, a carbohydrate comprises
monosaccharide or disaccharide, including but not limited to
glucose, fructose, galactose, ribose, lactose, sucrose, maltose,
trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid,
galactoronic acid, mannuronic acid, glucosamine, galatosamine, and
neuramic acid. In certain embodiments, a carbohydrate is a
polysaccharide, including but not limited to pullulan, cellulose,
microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),
hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran,
glycogen, hydroxyethylstarch, carageenan, glycon, amylose,
chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,
starch, chitin, inulin, konjac, glucommannan, pustulan, heparin,
hyaluronic acid, curdlan, and xanthan. In embodiments, the
inventive synthetic nanocarriers do not comprise (or specifically
exclude) carbohydrates, such as a polysaccharide. In certain
embodiments, the carbohydrate may comprise a carbohydrate
derivative such as a sugar alcohol, including but not limited to
mannitol, sorbitol, xylitol, erythritol, maltitol, and
lactitol.
[0124] Compositions according to the invention comprise inventive
synthetic nanocarriers in combination with pharmaceutically
acceptable excipients, such as preservatives, buffers, saline, or
phosphate buffered saline. The compositions may be made using
conventional pharmaceutical manufacturing and compounding
techniques to arrive at useful dosage forms. In an embodiment,
inventive synthetic nanocarriers are suspended in sterile saline
solution for injection together with a preservative.
[0125] In embodiments, when preparing synthetic nanocarriers as
carriers for antigens and/or adjuvants for use in vaccines, methods
for coupling the antigens and/or adjuvants to the synthetic
nanocarriers may be useful. If the antigen and/or adjuvant is a
small molecule it may be of advantage to attach the antigen and/or
adjuvant to a polymer prior to the assembly of the synthetic
nanocarriers. In embodiments, it may also be an advantage to
prepare the synthetic nanocarriers with surface groups that are
used to couple the antigen and/or adjuvant to the synthetic
nanocarrier through the use of these surface groups rather than
attaching the antigen and/or adjuvant to a polymer and then using
this polymer conjugate in the construction of synthetic
nanocarriers.
[0126] In certain embodiments, the coupling can be a covalent
linker. In embodiments, peptides according to the invention can be
covalently coupled to the external surface via a 1,2,3-triazole
linker formed by the 1,3-dipolar cycloaddition reaction of azido
groups on the surface of the nanocarrier with antigen or adjuvant
containing an alkyne group or by the 1,3-dipolar cycloaddition
reaction of alkynes on the surface of the nanocarrier with antigens
or adjuvants containing an azido group. Such cycloaddition
reactions are preferably performed in the presence of a Cu(I)
catalyst along with a suitable Cu(I)-ligand and a reducing agent to
reduce Cu(II) compound to catalytic active Cu(I) compound. This
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be
referred as the click reaction.
[0127] Additionally, the covalent coupling may comprise a covalent
linker that comprises an amide linker, a disulfide linker, a
thioether linker, a hydrazone linker, a hydrazide linker, an imine
or oxime linker, an urea or thiourea linker, an amidine linker, an
amine linker, and a sulfonamide linker.
[0128] An amide linker is formed via an amide bond between an amine
on one component such as the antigen or adjuvant with the
carboxylic acid group of a second component such as the
nanocarrier. The amide bond in the linker can be made using any of
the conventional amide bond forming reactions with suitably
protected amino acids or antigens or adjuvants and activated
carboxylic acid such N-hydroxysuccinimide-activated ester.
[0129] A disulfide linker is made via the formation of a disulfide
(S--S) bond between two sulfur atoms of the form, for instance, of
R1-S--S--R2. A disulfide bond can be formed by thiol exchange of an
antigen or adjuvant containing thiol/mercaptan group (--SH) with
another activated thiol group on a polymer or nanocarrier or a
nanocarrier containing thiol/mercaptan groups with an antigen or
adjuvant containing activated thiol group.
[0130] A triazole linker, specifically a 1,2,3-triazole of the
form
##STR00001##
wherein R1 and R2 may be any chemical entities, is made by the
1,3-dipolar cycloaddition reaction of an azide attached to a first
component such as the nanocarrier with a terminal alkyne attached
to a second component such as the peptide. The 1,3-dipolar
cycloaddition reaction is performed with or without a catalyst,
preferably with Cu(I)-catalyst, which links the two components
through a 1,2,3-triazole function. This chemistry is described in
detail by Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596,
(2002) and Meldal, et al, Chem. Rev., 2008, 108(8), 2952-3015 and
is often referred to as a "click" reaction or CuAAC.
[0131] In embodiments, a polymer containing an azide or alkyne
group, terminal to the polymer chain is prepared. This polymer is
then used to prepare a synthetic nanocarrier in such a manner that
a plurality of the alkyne or azide groups are positioned on the
surface of that nanocarrier. Alternatively, the synthetic
nanocarrier can be prepared by another route, and subsequently
functionalized with alkyne or azide groups. The antigen or adjuvant
is prepared with the presence of either an alkyne (if the polymer
contains an azide) or an azide (if the polymer contains an alkyne)
group. The antigen or adjuvant is then allowed to react with the
nanocarrier via the 1,3-dipolar cycloaddition reaction with or
without a catalyst which covalently couples the antigen to the
particle through the 1,4-disubstituted 1,2,3-triazole linker.
[0132] A thioether linker is made by the formation of a
sulfur-carbon (thioether) bond in the form, for instance, of
R1-S--R2. Thioether can be made by either alkylation of a
thiol/mercaptan (--SH) group on one component such as the antigen
or adjuvant with an alkylating group such as halide or epoxide on a
second component such as the nanocarrier. Thioether linkers can
also be formed by Michael addition of a thiol/mercaptan group on
one component such as an antigen or adjuvant to an
electron-deficient alkene group on a second component such as a
polymer containing a maleimide group or vinyl sulfone group as the
Michael acceptor. In another way, thioether linkers can be prepared
by the radical thiol-ene reaction of a thiol/mercaptan group on one
component such as an antigen or adjuvant with an alkene group on a
second component such as a polymer or nanocarrier.
[0133] A hydrazone linker is made by the reaction of a hydrazide
group on one component such as the antigen or adjuvant with an
aldehyde/ketone group on the second component such as the
nanocarrier.
[0134] A hydrazide linker is formed by the reaction of a hydrazine
group on one component such as the antigen or adjuvant with a
carboxylic acid group on the second component such as the
nanocarrier. Such reaction is generally performed using chemistry
similar to the formation of amide bond where the carboxylic acid is
activated with an activating reagent.
[0135] An imine or oxime linker is formed by the reaction of an
amine or N-alkoxyamine (or aminooxy) group on one component such as
the antigen or adjuvant with an aldehyde or ketone group on the
second component such as the nanocarrier.
[0136] An urea or thiourea linker is prepared by the reaction of an
amine group on one component such as the antigen or adjuvant with
an isocyanate or thioisocyanate group on the second component such
as the nanocarrier.
[0137] An amidine linker is prepared by the reaction of an amine
group on one component such as the antigen or adjuvant with an
imidoester group on the second component such as the
nanocarrier.
[0138] An amine linker is made by the alkylation reaction of an
amine group on one component such as the antigen or adjuvant with
an alkylating group such as halide, epoxide, or sulfonate ester
group on the second component such as the nanocarrier.
Alternatively, an amine linker can also be made by reductive
amination of an amine group on one component such as the antigen or
adjuvant with an aldehyde or ketone group on the second component
such as the nanocarrier with a suitable reducing reagent such as
sodium cyanoborohydride or sodium triacetoxyborohydride.
[0139] A sulfonamide linker is made by the reaction of an amine
group on one component such as the antigen or adjuvant with a
sulfonyl halide (such as sulfonyl chloride) group on the second
component such as the nanocarrier.
[0140] A sulfone linker is made by Michael addition of a
nucleophile to a vinyl sulfone. Either the vinyl sulfone or the
nucleophile may be on the surface of the nanocarrier or attached to
the antigen or adjuvant.
[0141] The antigen or adjuvant can also be conjugated to the
nanocarrier via non-covalent conjugation methods. For example, a
negative charged antigen or adjuvant can be conjugated to a
positive charged nanocarrier through electrostatic adsorption. An
antigen or adjuvant containing a metal ligand can also be
conjugated to a nanocarrier containing a metal complex via a
metal-ligand complex.
[0142] In embodiments, the antigen or adjuvant can be attached to a
polymer, for example polylactic acid-block-polyethylene glycol,
prior to the assembly of the synthetic nanocarrier or the synthetic
nanocarrier can be formed with reactive or activatible groups on
its surface. In the latter case, the antigen or adjuvant may be
prepared with a group which is compatible with the attachment
chemistry that is presented by the synthetic nanocarriers' surface.
In other embodiments, a peptide antigen can be attached to VLPs or
liposomes using a suitable linker. A linker is a compound or
reagent capable of coupling two molecules together. In an
embodiment, the linker can be a homobifuntional or
heterobifunctional reagent as described in Hermanson 2008. For
example, a VLP or liposome synthetic nanocarrier containing a
carboxylic group on the surface can be treated with a
homobifunctional linker, adipic dihydrazide (ADH), in the presence
of EDC to form the corresponding synthetic nanocarrier with the ADH
linker. The resulting ADH linked synthetic nanocarrier is then
conjugated with a peptide containing an acid group via the other
end of the ADH linker on NC to produce the corresponding VLP or
liposome peptide conjugate.
[0143] For detailed descriptions of available conjugation methods,
see Hermanson G T "Bioconjugate Techniques", 2nd Edition Published
by Academic Press, Inc., 2008. In addition to covalent attachment
the adjuvant can be coupled by adsorbtion to a pre-formed synthetic
nanocarrier or it can be coupled by encapsulation during the
formation of the synthetic nanocarrier.
[0144] In some embodiments, a component, such as an antigen or
adjuvant, may be isolated. Isolated refers to the element being
separated from its native environment and present in sufficient
quantities to permit its identification or use. This means, for
example, the element may be (i) selectively produced by expression
cloning or (ii) purified as by chromatography or electrophoresis.
Isolated elements may be, but need not be, substantially pure.
Because an isolated element may be admixed with a pharmaceutically
acceptable excipient in a pharmaceutical preparation, the element
may comprise only a small percentage by weight of the preparation.
The element is nonetheless isolated in that it has been separated
from the substances with which it may be associated in living
systems, i.e., isolated from other lipids or proteins. Any of the
elements provided herein may be isolated. Any of the antigens
provided herein can be included in the compositions in isolated
form.
D. METHODS OF MAKING AND USING THE INVENTIVE COMPOSITIONS AND
RELATED METHODS
[0145] Synthetic nanocarriers may be prepared using a wide variety
of methods known in the art. For example, synthetic nanocarriers
can be formed by methods as nanoprecipitation, flow focusing
fluidic channels, spray drying, single and double emulsion solvent
evaporation, solvent extraction, phase separation, milling,
microemulsion procedures, microfabrication, nanofabrication,
sacrificial layers, simple and complex coacervation, and other
methods well known to those of ordinary skill in the art.
Alternatively or additionally, aqueous and organic solvent
syntheses for monodisperse semiconductor, conductive, magnetic,
organic, and other nanomaterials have been described (Pellegrino et
al., 2005, Small, 1:48; Murray et al., 2000, Ann Rev. Mat. Sci.,
30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional
methods have been described in the literature (see, e.g., Doubrow,
Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy,"
CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control.
Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275;
and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; U.S.
Pat. Nos. 5,578,325 and 6,007,845; P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010)).
[0146] Various materials may be encapsulated into synthetic
nanocarriers as desirable using a variety of methods including but
not limited to C. Astete et al., "Synthesis and characterization of
PLGA nanoparticles" J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3,
pp. 247-289 (2006); K. Avgoustakis "Pegylated Poly(Lactide) and
Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties
and Possible Applications in Drug Delivery" Current Drug Delivery
1:321-333 (2004); C. Reis et al., "Nanoencapsulation I. Methods for
preparation of drug-loaded polymeric nanoparticles" Nanomedicine
2:8-21 (2006); P. Paolicelli et al., "Surface-modified PLGA-based
Nanoparticles that can Efficiently Associate and Deliver Virus-like
Particles" Nanomedicine. 5(6):843-853 (2010). Other methods
suitable for encapsulating materials into synthetic nanocarriers
may be used, including without limitation methods disclosed in U.S.
Pat. No. 6,632,671 to Unger Oct. 14, 2003.
[0147] In certain embodiments, synthetic nanocarriers are prepared
by a nanoprecipitation process or spray drying. Conditions used in
preparing synthetic nanocarriers may be altered to yield particles
of a desired size or property (e.g., hydrophobicity,
hydrophilicity, external morphology, "stickiness," shape, etc.).
The method of preparing the synthetic nanocarriers and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may depend on the materials to be coupled to the
synthetic nanocarriers and/or the composition of the polymer
matrix.
[0148] If particles prepared by any of the above methods have a
size range outside of the desired range, particles can be sized,
for example, using a sieve.
[0149] Elements of the inventive synthetic nanocarriers may be
coupled to the overall synthetic nanocarrier, e.g., by one or more
covalent bonds, or may be coupled by means of one or more linkers.
Additional methods of functionalizing synthetic nanocarriers may be
adapted from Published US Patent Application 2006/0002852 to
Saltzman et al., Published US Patent Application 2009/0028910 to
DeSimone et al., or Published International Patent Application
WO/2008/127532 A1 to Murthy et al.
[0150] Alternatively or additionally, synthetic nanocarriers can be
coupled to elements directly or indirectly via non-covalent
interactions. In non-covalent embodiments, the non-covalent
coupling is mediated by non-covalent interactions including but not
limited to charge interactions, affinity interactions, metal
coordination, physical adsorption, host-guest interactions,
hydrophobic interactions, TT stacking interactions, hydrogen
bonding interactions, van der Waals interactions, magnetic
interactions, electrostatic interactions, dipole-dipole
interactions, and/or combinations thereof. Such couplings may be
arranged to be on an external surface or an internal surface of an
inventive synthetic nanocarrier. In embodiments, encapsulation
and/or absorption is a form of coupling.
[0151] In embodiments, the inventive synthetic nanocarriers can be
combined with other adjuvants by admixing in the same vehicle or
delivery system. Such adjuvants may include, but are not limited to
mineral salts, such as alum, alum combined with monphosphoryl lipid
(MPL) A of Enterobacteria, such as Escherihia coli, Salmonella
minnesota, Salmonella typhimurium, or Shigella flexneri or
specifically with MPL.RTM. (AS04), MPL A of above-mentioned
bacteria separately, saponins, such as QS-21, Quil-A, ISCOMs,
ISCOMATRIX.TM., emulsions such as MF59.TM., Montanide.RTM. ISA 51
and ISA 720, AS02 (QS21+ squalene+MPL.RTM.), liposomes and
liposomal formulations such as AS01, synthesized or specifically
prepared microparticles and microcarriers such as bacteria-derived
outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia
trachomatis and others, or chitosan particles, depot-forming
agents, such as Pluronic.RTM. block co-polymers, specifically
modified or prepared peptides, such as muramyl dipeptide,
aminoalkyl glucosaminide 4-phosphates, such as RC529, or proteins,
such as bacterial toxoids or toxin fragments. The doses of such
other adjuvants can be determined using conventional dose ranging
studies.
[0152] In embodiments, the inventive synthetic nanocarriers can be
combined with an antigen different, similar or identical to those
coupled to a nanocarrier (with or without adjuvant, utilizing or
not utilizing another delivery vehicle) administered separately at
a different time-point and/or at a different body location and/or
by a different immunization route or with another antigen and/or
adjuvant-carrying synthetic nanocarrier administered separately at
a different time-point and/or at a different body location and/or
by a different immunization route.
[0153] Populations of synthetic nanocarriers may be combined to
form pharmaceutical dosage forms according to the present invention
using traditional pharmaceutical mixing methods. These include
liquid-liquid mixing in which two or more suspensions, each
containing one or more subset of nanocarriers, are directly
combined or are brought together via one or more vessels containing
diluent. As synthetic nanocarriers may also be produced or stored
in a powder form, dry powder-powder mixing could be performed as
could the re-suspension of two or more powders in a common media.
Depending on the properties of the nanocarriers and their
interaction potentials, there may be advantages conferred to one or
another route of mixing.
[0154] Typical compositions that comprise synthetic nanocarriers
may comprise inorganic or organic buffers (e.g., sodium or
potassium salts of phosphate, carbonate, acetate, or citrate) and
pH adjustment agents (e.g., hydrochloric acid, sodium or potassium
hydroxide, salts of citrate or acetate, amino acids and their
salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol),
surfactants (e.g., polysorbate 20, polysorbate 80,
polyoxyethylene9-10 nonyl phenol, sodium desoxycholate), solution
and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol,
trehalose), osmotic adjustment agents (e.g., salts or sugars),
antibacterial agents (e.g., benzoic acid, phenol, gentamicin),
antifoaming agents (e.g., polydimethylsilozone), preservatives
(e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers
and viscosity-adjustment agents (e.g., polyvinylpyrrolidone,
poloxamer 488, carboxymethylcellulose) and co-solvents (e.g.,
glycerol, polyethylene glycol, ethanol).
[0155] Compositions according to the invention comprise synthetic
nanocarriers in combination with pharmaceutically acceptable
excipients. The compositions may be made using conventional
pharmaceutical manufacturing and compounding techniques to arrive
at useful dosage forms. Techniques suitable for use in practicing
the present invention may be found in Handbook of Industrial
Mixing: Science and Practice, Edited by Edward L. Paul, Victor A.
Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons,
Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed.
Edited by M. E. Auten, 2001, Churchill Livingstone. In an
embodiment, inventive synthetic nanocarriers are suspended in
sterile saline solution for injection together with a
preservative.
[0156] It is to be understood that the compositions of the
invention can be made in any suitable manner, and the invention is
in no way limited to compositions that can be produced using the
methods described herein. Selection of an appropriate method may
require attention to the properties of the particular moieties
being associated.
[0157] In some embodiments, the synthetic nanocarriers are
manufactured under sterile conditions or are terminally sterilized.
This can ensure that resulting composition are sterile and
non-infectious, thus improving safety when compared to non-sterile
compositions. This provides a valuable safety measure, especially
when subjects receiving synthetic nanocarriers have immune defects,
are suffering from infection, and/or are susceptible to infection.
In some embodiments, inventive synthetic nanocarriers may be
lyophilized and stored in suspension or as lyophilized powder
depending on the formulation strategy for extended periods without
losing activity.
[0158] The compositions of the invention can be administered by a
variety of routes, including or not limited to subcutaneous,
intranasal, oral, intravenous, intraperitoneal, intramuscular,
transmucosal, transmucosal, sublingual, rectal, ophthalmic,
pulmonary, intradermal, transdermal, transcutaneous or intradermal
or by a combination of these routes. Routes of administration also
include administration by inhalation or pulmonary aerosol.
Techniques for preparing aerosol delivery systems are well known to
those of skill in the art (see, for example, Sciarra and Cutie,
"Aerosols," in Remington's Pharmaceutical Sciences, 18th edition,
1990, pp. 1694-1712; incorporated by reference).
[0159] Doses of dosage forms contain varying amounts of populations
of synthetic nanocarriers and/or varying amounts of antigens,
adjuvants, etc., according to the invention. The amount of
synthetic nanocarriers and/or other elements present in the
inventive dosage forms can be varied according to the nature of the
elements, the therapeutic benefit to be accomplished, and other
such parameters. In embodiments, dose ranging studies can be
conducted to establish optimal therapeutic amount of the population
of synthetic nanocarriers and the amount of antigens to be present
in the dosage form. In embodiments, the synthetic nanocarriers and
the antigens are present in the dosage form in an amount effective
to generate an immune response to the antigens upon administration
to a subject. It may be possible to determine amounts of the
antigens effective to generate an immune response using
conventional dose ranging studies and techniques in subjects.
Inventive dosage forms may be administered at a variety of
frequencies. In a preferred embodiment, at least one administration
of the dosage form is sufficient to generate a pharmacologically
relevant response. In more preferred embodiment, at least two
administrations, at least three administrations, or at least four
administrations, of the dosage form are utilized to ensure a
pharmacologically relevant response.
[0160] The compositions and methods described herein can be used to
induce, enhance, suppress, modulate, direct, or redirect an immune
response. The compositions and methods described herein can be used
in the diagnosis, prophylaxis and/or treatment of conditions such
as cancers, infectious diseases, metabolic diseases, degenerative
diseases, non-autoimmune diseases or other disorders and/or
conditions. The compositions and methods described herein can also
be used for the prophylaxis or treatment of an addiction, such as
an addiction to an illegal drug, an over-the-counter drug, a
prescription drug. In some embodiments, the addiction is to
cocaine, heroin, marijuana, methamphetamines, nicotine or a
narcotic. The compositions and methods described herein can also be
used for the prophylaxis and/or treatment of a condition resulting
from the exposure to a toxin, hazardous substance, environmental
toxin, or other harmful agent.
[0161] Examples of infectious disease include, but are not limited
to, viral infectious diseases, such as AIDS, Chickenpox
(Varicella), Common cold, Cytomegalovirus Infection, Colorado tick
fever, Dengue fever, Ebola hemorrhagic fever, Hand, foot and mouth
disease, Hepatitis, Herpes simplex, Herpes zoster, HPV, Influenza
(Flu), Lassa fever, Measles, Marburg hemorrhagic fever, Infectious
mononucleosis, Mumps, Norovirus, Poliomyelitis, Progressive
multifocal leukencephalopathy, Rabies, Rubella, SARS, Smallpox
(Variola), Viral encephalitis, Viral gastroenteritis, Viral
meningitis, Viral pneumonia, West Nile disease and Yellow fever;
bacterial infectious diseases, such as Anthrax, Bacterial
Meningitis, Botulism, Brucellosis, Campylobacteriosis, Cat Scratch
Disease, Cholera, Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo,
Legionellosis, Leprosy (Hansen's Disease), Leptospirosis,
Listeriosis, Lyme disease, Melioidosis, Rheumatic Fever, MRSA
infection, Nocardiosis, Pertussis (Whooping Cough), Plague,
Pneumococcal pneumonia, Psittacosis, Q fever, Rocky Mountain
Spotted Fever (RMSF), Salmonellosis, Scarlet Fever, Shigellosis,
Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia, Typhoid
Fever, Typhus and Urinary Tract Infections; parasitic infectious
diseases, such as African trypanosomiasis, Amebiasis, Ascariasis,
Babesiosis, Chagas Disease, Clonorchiasis, Cryptosporidiosis,
Cysticercosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis,
Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis,
Free-living amebic infection, Giardiasis, Gnathostomiasis,
Hymenolepiasis, Isosporiasis, Kala-azar, Leishmaniasis, Malaria,
Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Pinworm
Infection, Scabies, Schistosomiasis, Taeniasis, Toxocariasis,
Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis,
Trichomoniasis and Trypanosomiasis; fungal infectious disease, such
as Aspergillosis, Blastomycosis, Candidiasis, Coccidioidomycosis,
Cryptococcosis, Histoplasmosis, Tinea pedis (Athlete's Foot) and
Tinea cruris; prion infectious diseases, such as Alpers' disease,
Fatal Familial Insomnia, Gerstmann-Straussler-Scheinker syndrome,
Kuru and Variant Creutzfeldt-Jakob disease.
[0162] Examples of cancers include, but are not limited to breast
cancer; biliary tract cancer; bladder cancer; brain cancer
including glioblastomas and medulloblastomas; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; hematological neoplasms including acute
lymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell
acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic
myelogenous leukemia, multiple myeloma; AIDS-associated leukemias
and adult T-cell leukemia/lymphoma; intraepithelial neoplasms
including Bowen's disease and Paget's disease; liver cancer; lung
cancer; lymphomas including Hodgkin's disease and lymphocytic
lymphomas; neuroblastomas; oral cancer including squamous cell
carcinoma; ovarian cancer including those arising from epithelial
cells, stromal cells, germ cells and mesenchymal cells; pancreatic
cancer; prostate cancer; rectal cancer; sarcomas including
leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and
osteosarcoma; skin cancer including melanoma, Merkel cell
carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous
cell cancer; testicular cancer including germinal tumors such as
seminoma, non-seminoma (teratomas, choriocarcinomas), stromal
tumors, and germ cell tumors; thyroid cancer including thyroid
adenocarcinoma and medullar carcinoma; and renal cancer including
adenocarcinoma and Wilms tumor.
[0163] Examples of metabolic diseases include, but are not limited
to, disorders of carbohydrate metabolism, amino acid metabolism,
organic acid metabolism, fatty acid oxidation and mitochondrial
metabolism, prophyrin metabolism, purine or pyrimidine metabolism,
steroid metabolism, lysosomal mitochondrial function, peroxisomal
function, lysosomal storage, urea cycle disorders (e.g., N-acetyl
glutamate synthetase deficiency, carbamylphosphate synthase
deficiency, ornithine carbamyl transferase deficiency,
crginosuccinic aciduria, citrullinaemia, arginase deficiency),
amino acid disorders (e.g., Non-ketotic hyperglycinaemia,
tyrosinaemia (Type I), Maple syrup urine disease), organic
acidemias (e.g, isovaleric acidemia, methylmalonic acidemia,
propionic acidemia, glutaric aciduria type I, glutaric acidemia
type I & II), mitochondrial disorders (e.g., carboxylase
defects, mitochondrial myopathies, lactic acidosis (pyruvate
dehydrogenase complex defects), congenital lactic acidosis,
mitochondrial respiratory chain defects, cystinosis, Gaucher's
disease, Fabry's disease, Pompe's disease, mucopolysaccharoidosis
I, mucopolysaccharoidosis II, mucopolysaccharoidosis VI).
[0164] Examples of degenerative diseases include, but are not
limited to, mesenchyme/mesoderm degenerative disease, muscle
degenerative disease, endothelial degenerative disease,
neurodegenerative disease, degenerative joint disease (e.g.,
osteoarthritis), major types of degenerative heart disease (e.g.,
coronary heart disease, congenital heart disease, rheumatic heart
disease, angina pectoris), neurodegenerative disease (e.g.,
Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's
ataxia, Huntington's disease, Lewy body disease, Parkinson's
disease, spinal muscular atrophy), neuromuscular disorders (e.g.,
muscular dystrophy, duchenne muscular dystrophy,
facioscapulohumeral muscular dystrophy, myotonic muscular
dystrophy, congenital myopathy, familial cardiomyopathy, dilated
cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, or coronary artery disease).
EXAMPLES
Example 1
Formulations of Synthetic Nanocarriers
Materials for Lot #1
[0165] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA-R848 conjugate of 75/25
lactide/glycolide monomer composition and approximately 4100 Da
molecular weight having 5.2% w/w R848 content was synthesized by
conjugation of R848 to the terminal-acid of commercially-supplied
PLGA via an amide linkage. PLA-PEG-Nicotine with a
nicotine-terminated PEG block of 3,500 Da and DL-PLA block of
approximately 15,000 Da was synthesized. Polyvinyl alcohol
(Mw=9,000-10,000, 80% hydrolyzed) was purchased from SIGMA (Part
Number 360627).
Methods for Lot #1
[0166] Solutions were prepared as follows:
[0167] Solution 1: Ovalbumin peptide 323-339 amide acetate salt @
70 mg/mL was prepared by dissolution in 0.13N hydrochloric acid at
room temperature.
[0168] Solution 2: PLGA-R848 @ 75 mg/mL and PLA-PEG-Nicotine @ 25
mg/mL in dichloromethane was prepared by dissolving PLGA-R848 at
100 mg/mL in dichloromethane and PLA-PEG-Nicotine at 100 mg/mL in
dichloromethane, then combining 3 parts of the PLGA-R848 solution
to 1 part of the PLA-PEG-Nicotine solution.
[0169] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM phosphate
buffer, pH 8.
[0170] Solution 4: 70 mM phosphate buffer, pH 8.
[0171] A primary (W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2 mL) to the primary emulsion and sonicating at 30% amplitude for
40 seconds using the Branson Digital Sonifier 250. The secondary
emulsion was added to an open 50 mL beaker containing 30 mL of
stiffing 70 mM phosphate buffer solution and was stirred at room
temperature for not less than 2 hours to allow the dichloromethane
to evaporate and the nanocarriers to form in suspension. A portion
of the suspended nanocarriers was washed by transferring the
nanocarrier suspension to a centrifuge tube, spinning at 13800 rcf
for 60 minutes at 4.degree. C., removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. This washing
procedure was repeated and then the pellet was re-suspended in
phosphate buffered saline to achieve a nanocarrier suspension
having a nominal concentration of 10 mg/mL on a polymer basis. The
suspension which was stored frozen at -20C until use.
TABLE-US-00001 TABLE 1 Nanocarrier Effective TLR Agonist, % T-cell
helper peptide, ID Diameter (nm) w/w % w/w 1 197 1.5 0.8
Materials for Lot #2
[0172] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA-R848 conjugate of 75/25
lactide/glycolide monomer composition and approximately 4100 Da
molecular weight having 5.2% w/w R848 content was synthesized by
conjugation of R848 to the terminal-acid of commercially-supplied
PLGA via an amide linkage. PLA with an inherent viscosity of 0.19
dL/g was purchased from Boehringer Ingelheim (Ingelheim Germany.
Product Code R202H). PLA-PEG-Nicotine with a nicotine-terminated
PEG block of 3,500 Da and DL-PLA block of approximately 15,000 Da
was synthesized. Polyvinyl alcohol (Mw=9,000-10,000, 80%
hydrolyzed) was purchased from SIGMA (Part Number 360627).
Methods for Lot #2
[0173] Solutions were prepared as follows:
[0174] Solution 1: Ovalbumin peptide 323-339 amide acetate salt @
70 mg/mL was prepared by dissolution in 0.13N hydrochloric acid at
room temperature.
[0175] Solution 2: PLGA-R848 @ 75 mg/mL, PLA-PEG-Nicotine @ 6
mg/mL, and PLA at 19 mg/mL in dichloromethane was prepared by
dissolving PLGA-R848 at 100 mg/mL in dichloromethane,
PLA-PEG-Nicotine at 100 mg/mL in dichloromethane, and PLA at 100
mg/mL in dichloromethane and then combining 750 .mu.L of the
PLGA-R848 solution with 60 .mu.L of the PLA-PEG-Nicotine solution
and 190 .mu.L of the PLA solution.
[0176] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM phosphate
buffer, pH 8.
[0177] Solution 4: 70 mM phosphate buffer, pH 8.
[0178] A primary (W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2 mL) to the primary emulsion and sonicating at 10% amplitude for
40 seconds using the Branson Digital Sonifier 250. The secondary
emulsion was added to an open 50 mL beaker containing 30 mL of
stiffing 70 mM phosphate buffer solution and was stirred at room
temperature for not less than 2 hours to allow the dichloromethane
to evaporate and the nanocarriers to form in suspension. A portion
of the suspended nanocarriers was washed by transferring the
nanocarrier suspension to a centrifuge tube, spinning at 13800 rcf
for 60 minutes at 4.degree. C., removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. This washing
procedure was repeated and then the pellet was re-suspended in
phosphate buffered saline to achieve a nanocarrier suspension
having a nominal concentration of 10 mg/mL on a polymer basis. The
suspension which was stored frozen at -20C until use.
TABLE-US-00002 TABLE 2 Nanocarrier Effective TLR Agonist, % T-cell
helper peptide, ID Diameter (nm) w/w % w/w 2 212 1.4 1.8
Materials for Lot #3
[0179] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Part #4065609.) PLGA-R848 conjugate of 75/25
lactide/glycolide monomer composition and approximately 4100 Da
molecular weight having 5.2% w/w R848 content was synthesized by
conjugation of R848 to the terminal-acid of commercially-supplied
PLGA via an amide linkage. PLA-PEG-Nicotine with a
nicotine-terminated PEG block of 3,500 Da and DL-PLA block of
approximately 15,000 Da was synthesized. PLA-PEG-OMe block
co-polymer with a PEG-OMe (Methyl-ether capped PEG) block of 2,000
Da and DL-PLA block of approximately 19,000 Da was synthesized.
Polyvinyl alcohol (Mw=9,000-10,000, 80% hydrolyzed) was purchased
from SIGMA (Part Number 360627).
Methods for Lot #3
[0180] Solutions were prepared as follows:
[0181] Solution 1: Ovalbumin peptide 323-339 amide acetate salt @
70 mg/mL was prepared by dissolution in 0.13N hydrochloric acid at
room temperature.
[0182] Solution 2: PLGA-R848 @ 75 mg/mL, PLA-PEG-Nicotine @ 6
mg/mL, and PLA-PEG-OMe at 19 mg/mL in dichloromethane was prepared
by dissolving PLGA-R848 at 100 mg/mL in dichloromethane,
PLA-PEG-Nicotine at 100 mg/mL in dichloromethane, and PLA-PEG-OMe
at 100 mg/mL in dichloromethane and then combining 750 .mu.L of the
PLGA-R848 solution with 60 .mu.L of the PLA-PEG-Nicotine solution
and 190 .mu.L of the PLA-PEG-OMe solution.
[0183] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM phosphate
buffer, pH 8.
[0184] Solution 4: 70 mM phosphate buffer, pH 8.
[0185] A primary (W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2 mL) to the primary emulsion and sonicating at 10% amplitude for
40 seconds using the Branson Digital Sonifier 250. The secondary
emulsion was added to an open 50 mL beaker containing 30 mL of
stiffing 70 mM phosphate buffer solution and was stirred at room
temperature for not less than 2 hours to allow the dichloromethane
to evaporate and the nanocarriers to form in suspension. A portion
of the suspended nanocarriers was washed by transferring the
nanocarrier suspension to a centrifuge tube, spinning at 13800 rcf
for 60 minutes at 4.degree. C., removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. This washing
procedure was repeated and then the pellet was re-suspended in
phosphate buffered saline to achieve a nanocarrier suspension
having a nominal concentration of 10 mg/mL on a polymer basis. The
suspension which was stored frozen at -20C until use.
TABLE-US-00003 TABLE 3 Nanocarrier Effective TLR Agonist, % T-cell
helper peptide, ID Diameter (nm) w/w % w/w 3 197 1.8 0.9
Materials for Lots #4-#12
[0186] Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Product code 4065609.) PLGA-R848 of approximately 5,200 Da
made from PLGA of 3:1 lactide to glycolide ratio and having 12.7%
w/w conjugated R848 content was synthesized. PLA with an inherent
viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals
(756 Tom Martin Drive, Birmingham, Ala. 35211. Product Code 100 DL
2A.) PLA-PEG.sub.2k-OMe block co-polymer with a methyl ether
terminated PEG block of 2,000 Da and DL-PLA block of approximately
19,000 Da was synthesized. PLA-PEG.sub.5k-OMe block co-polymer with
a methyl ether terminated PEG block of 5,000 Da and DL-PLA block of
approximately 20,000 Da was synthesized. PLA-PEG-Nicotine block
copolymer having a nicotine-terminated PEG block of 5,000 Da and
DL-PLA block of approximately 21,000 Da was synthesized. Polyvinyl
alcohol (Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J.
T. Baker (Part Number U232-08).
Methods for Lots #4-#12
[0187] Solutions were prepared as follows:
[0188] Solution 1: Ovalbumin peptide 323-339 amide acetate salt @
20 mg/mL was prepared by dissolution in 0.13N hydrochloric acid at
room temperature.
[0189] Solution 2: Stock solutions, each containing one of the
individual polymers (PLGA-R848, PLA, PLA-PEG.sub.2k-OMe,
PLA-PEG.sub.5k-OMe, and PLA-PEG-Nicotine), were prepared in
dichloromethane at 100 mg/mL. These single-polymer stocks were
combined according to Table 4 to generate a unique "Solution 2" for
each of the nanocarrier lots.
[0190] Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
[0191] Solution 4: 70 mM phosphate buffer, pH 8.
[0192] A primary (W1/O) emulsion was first created using Solution 1
& Solution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion, vortexing to create a course
dispersion, and then sonicating at 30% amplitude for 40 seconds
using the Branson Digital Sonifier 250. The secondary emulsion was
added to an open 50 mL beaker containing 70 mM phosphate buffer
solution (30 mL) and stirred at room temperature for 2 to 3 hours
to allow the dichloromethane to evaporate and the nanocarriers to
form in suspension. A portion of the suspended nanocarriers was
washed by transferring the nanocarrier suspension to a centrifuge
tube, spinning at 21,000 rcf for 45 minutes, removing the
supernatant, and re-suspending the pellet in phosphate buffered
saline. This washing procedure was repeated and then the pellet was
re-suspended in phosphate buffered saline to achieve a nanocarrier
suspension having a nominal concentration of 10 mg/mL on a polymer
basis. The suspension was stored frozen at -20.degree. C. until
use.
TABLE-US-00004 TABLE 4 Composition of Solution 2 for Nanocarrier
Formulation Solution 2 Composition for Production of Example
Nanocarrier Lots 4 6 7 10 8 9 5 12 11 Polymer PLA-PEG- 0.25 mL 0.50
mL 0.25 mL 0.25 mL 0.375 mL 0.375 mL 0.375 mL -- -- Solution
Nicotine (100 mg/mL) PLA-PEG.sub.2k- -- -- 0.25 mL -- 0.125 mL --
-- -- -- OMe PLA-PEG.sub.5k- 0.25 mL -- 0.125 mL -- -- 0.50 mL OMe
PLA 0.25 mL -- -- -- -- -- 0.125 mL 0.50 mL -- PLGA-R848 0.50 mL
0.50 mL 0.50 mL 0.50 mL 0.50 mL 0.50 mL 0.50 mL 0.50 mL 0.50 mL
TABLE-US-00005 TABLE 5 PLA-PEG- PLA-PEG.sub.2k- PLA-PEG.sub.5k- Ova
Peptide R848 Nic OMe OMe PLA Load (% Load Gr. NC Lot # (% w/w) (%
w/w) (% w/w) (% w/w) w/w) (% w/w) 1 4 25 0 0 25 2.0 4.1 3 6 50 0 0
0 1.0 3.9 4 7 25 25 0 0 1.1 3.9 7 10 25 0 25 0 0.1 4.0 5 8 37.5
12.5 0 0 1.0 4.4 6 9 37.5 0 12.5 0 0.7 4.1 2 5 37.5 0 0 12.5 1.8
4.4 9 12 0 0 0 50 0.7 4.2 8 11 0 0 50 0 0 4.6
Example 2
Synthetic Nanocarriers with Increased Antigen Increases
Antigen-Specific Antibody Generation and Decreases Anti-Carrier
Antibody Generation
[0193] Mice were inoculated with nicotine-presenting
R848-adjuvanted nanocarrier formulations. Groups 2 through 4 were
evaluated for antigen-presentation and anti-carrier effect. The
nicotine-presenting conjugate in the nanocarrier is a
PLA-PEG3.5k-Nicotine construct of .about.15,350 Mw PLA and
.about.3500 Mw PEG. The study groups used formulations having
varied content of the PLA-PEG3.5k-Nicotine construct,
partially-substituting the construct with either a .about.20 k Mw
PLA polymer or with a PLA-PEG2k-OMe polymer of .about.18,700 Mw PLA
and 2000 Mw PEG. Mice were immunized at days 0, 14, and 28 and
serum was collected at days 26 and 40. The formulations are
described as tabulated below and the anti-nicotine and resultant
anti-PEG antibodies at day 40 are presented in FIG. 1.
TABLE-US-00006 TABLE 6 Synthetic Nanocarrier Formulations .mu.g
R848/mg NP released PLGA-R848 Polymer-Ag Replacement R848 Ova (24
hrs, Conjugate, % Description, % polymer, % of Load Peptide citrate
pH Gr. NC Lot # of NC mass of NC mass NC mass (%) Load (%) 4.5) 2 1
75% PLA-PEG-Nic, None 1.5 0.8 3.7 25% 3 2 75% PLA-PEG-Nic, R202H
PLA, 1.4 1.8 3.9 6% 19% 4 3 75% PLA-PEG-Nic, PLA-PEG, 19% 1.8 0.9
4.5 6%
[0194] Antibody titers to nicotine and PEG were determined by ELISA
using sera collected from immunized mice. Plates were coated with
100 .mu.L per well of either polylysine-nicotine (PLL-Nic),
PLA-PEG-OMe, or polylysine-PEG (PLL-PEG-OMe) and incubated
overnight at 4.degree. C. Plates were washed three times with wash
buffer (0.05% Tween-20 in PBS) and blocked at room temperature for
two hours using 200 .mu.L per well of 10% fetal bovine serum (FBS)
in PBS (diluent). Serum samples were added to the wells of the top
row of a 96-well plate and diluted 3-fold down the plate to obtain
an antibody titration curve. For a positive control, either a mouse
anti-nicotine monoclonal antibody or a biotinylated rabbit anti-PEG
monoclonal antibody (Epitomics, Catalog #2137-1) were used in two
columns of the plate. For negative controls, either serum from
unimmunized mice or isotype control antibodies were used. Plates
were incubated for two hours at room temperature and washed three
times with wash buffer. Secondary detection antibody (biotinylated
goat anti-mouse Ig, BD Biosciences, Catalog #553999) was diluted
1:1000 in diluent and 100 .mu.L was added to each well of the
plate. Plates were incubated for one hour at room temperature and
washed three times with wash buffer. Detection enzyme
(streptavidin-horseradish peroxidase, SA-HRP, BD Biosciences,
Catalog #554066) was diluted 1:1000 in diluent and 100 .mu.L was
added to each well of the plate. Plates were incubated for 30
minutes at room temperature in the dark and washed three times with
wash buffer (during each wash step, plates were incubated with wash
buffer for at least 30 seconds). TMB substrate (BD Biosciences,
Catalog #555214) was added to the plate (100 .mu.L per well) and
incubated for 15 minutes at room temperature in the dark. Stop
solution (2N sulfuric acid) was added to stop the enzymatic
reaction (50 .mu.L per well) and the optical density of the plates
was read using a plate reader at 450 nm wavelength with subtraction
of 570 nm. The half maximal effective concentration (EC50) of
antibodies was calculated based on the generated four-parameter
logistic curve-fit graph. The average OD value of two diluent-only
blanks (negative control) was subtracted from the rest of the wells
of the plate. The EC50 value of the average top OD value of the two
standards was used to determine the EC50 value for the rest of the
plate.
[0195] The data show a non-linear increase in anti-nicotine
(target) antibodies with higher nicotine content of the nanocarrier
(25% vs. 6% PLA-PEG3.5k-Nicotine); a 5-fold increase in
PLA-PEG3.5k-Nicotine yielded a 21 to 74-fold higher anti-nicotine
response while achieving a .about.31:1 ratio of anti-nicotine to
anti-PEG antibodies. Surprisingly, in the case where .about.75% of
the PLA-PEG3.5k-Nicotine was substituted with PLA-PEG2k-OMe, the
anti-PEG titer exceeded the nicotine titer to yield a 1:10 ratio of
anti-nicotine to anti-PEG antibodies. Anti-PEG antibody titers were
8-fold higher in formulations containing 6% PLA-PEG3.5k-Nicotine
than those containing 25% PLA-PEG3.5k-Nicotine. Additionally, in
the group that was inoculated with Lot 2 (contained 19% PLA polymer
instead of 19% PLA-PEG2k-OMe), anti-PEG antibody levels were nearly
absent.
Example 3
Synthetic Nanocarriers with Increased Antigen or Increased Polymer
Length Decreases Anti-Carrier Antibody Generation
[0196] Mice were inoculated with nicotine-presenting
R848-adjuvanted nanocarrier formulations. All formulations were
prepared on the same date using a consistent set of solutions and
materials. All tested nanocarriers were formulated with a 50%
PLGA-R848 polymer content, with the remaining 50% of the
composition made up of one or more of the following polymers:
PLA-PEG5k-Nicotine, PLA-PEG2k-OMe, PLA-PEG5k-OMe, or PLA.
TABLE-US-00007 TABLE 7 Synthetic Nanocarrier Formulations PLA-PEG-
PLA-PEG.sub.2k- PLA-PEG.sub.5k- Ova Peptide R848 Nic OMe OMe PLA
Load (% Load Gr. NC Lot # (% w/w) (% w/w) (% w/w) (% w/w) w/w) (%
w/w) 1 4 25 0 0 25 2.0 4.1 2 5 37.5 0 0 12.5 1.8 4.4 3 6 50 0 0 0
1.0 3.9 4 7 25 25 0 0 1.1 3.9 5 8 37.5 12.5 0 0 1.0 4.4 6 9 37.5 0
12.5 0 0.7 4.1 7 10 25 0 25 0 0.1 4.0 8 11 0 0 50 0 0 4.6 9 12 0 0
0 50 0.7 4.2
[0197] Following a prime and two-boost inoculation schedule, the
on-target (anti-nicotine) antibody titers and off-target (anti-PEG)
antibody titers were determined by ELISA as described above (except
PEG length in the ELISA coating materials was adjusted to match the
length used in the nanoparticles used for injections when
applicable) and are presented in FIG. 2.
[0198] The results revealed several surprising outcomes with
implications on nanocarrier vaccine formulations. For example, at
25% PLA-PEG5k-Nicotine content or higher, with no other sources of
PEG in the formulation, essentially no induction of anti-PEG
antibodies is observed. Additionally, the incorporation of
PLA-PEG5k-Nicotine above 25% of the particle content does not
further increase anti-nicotine antibody titers (plateau effect).
Incorporation of 100% more nicotine actually resulted in a decrease
in anti-nicotine antibody titers. Introduction of shorter-chain
filler PLA-PEG2k (PEG of 2000 Mw) leads to a significant anti-PEG
antibody titer, whereas a longer-PEG-chain filler PLA-PEG5k (PEG of
5000 Mw) results in limited anti-PEG titers. This result is evident
at two different content levels of the filler introduction whether
the anti-PEG response is considered as an absolute titer or as a
ratio to the intended anti-nicotine response.
* * * * *