U.S. patent application number 17/063122 was filed with the patent office on 2021-01-28 for optimized crosslinkers for trapping a target on a substrate.
The applicant listed for this patent is The University of North Carolina at Chapel Hill. Invention is credited to Alexander Chen, M. Gregory Forest, Christine Henry, Samuel Lai, Jay Newby, Jennifer Schiller, Timothy Wessler.
Application Number | 20210024618 17/063122 |
Document ID | / |
Family ID | 1000005137337 |
Filed Date | 2021-01-28 |
View All Diagrams
United States Patent
Application |
20210024618 |
Kind Code |
A1 |
Lai; Samuel ; et
al. |
January 28, 2021 |
OPTIMIZED CROSSLINKERS FOR TRAPPING A TARGET ON A SUBSTRATE
Abstract
The presently-disclosed subject matter relates to crosslinkers,
compositions, and methods for trapping a target of interest on a
substrate of interest. The methods may be used to inhibit and treat
pathogen infection and provide contraception. The methods may be
used to trap or separate particles and other substances. The
subject matter further relates to methods of identifying and
preparing optimal crosslinkers and methods for manipulating targets
of interest.
Inventors: |
Lai; Samuel; (Carrboro,
NC) ; Forest; M. Gregory; (Chapel Hill, NC) ;
Henry; Christine; (Chapel Hill, NC) ; Wessler;
Timothy; (Durham, NC) ; Chen; Alexander;
(Glenville, NY) ; Schiller; Jennifer; (Chapel
Hill, NC) ; Newby; Jay; (Carrboro, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill |
Chapel Hill |
NC |
US |
|
|
Family ID: |
1000005137337 |
Appl. No.: |
17/063122 |
Filed: |
October 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15977432 |
May 11, 2018 |
10793623 |
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17063122 |
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PCT/US2016/061574 |
Nov 11, 2016 |
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15977432 |
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62254856 |
Nov 13, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54346 20130101;
G01N 33/6854 20130101; C07K 16/1045 20130101; A61P 31/00 20180101;
A61K 38/00 20130101; C07K 16/087 20130101; G01N 33/557 20130101;
C07K 16/44 20130101; C07K 16/1235 20130101; G01N 2333/4725
20130101; G01N 33/56983 20130101; G01N 2333/16 20130101; C07K
2317/41 20130101 |
International
Class: |
C07K 16/12 20060101
C07K016/12; G01N 33/557 20060101 G01N033/557; G01N 33/68 20060101
G01N033/68; C07K 16/44 20060101 C07K016/44; G01N 33/569 20060101
G01N033/569; G01N 33/543 20060101 G01N033/543; A61P 31/00 20060101
A61P031/00; C07K 16/08 20060101 C07K016/08; C07K 16/10 20060101
C07K016/10 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with government support under Grant
Numbers R21EB017938 and U19AI096398 awarded by the National
Institutes of Health and Grant Number DMR-1151477 awarded by the
National Science Foundation. The government has certain rights in
the invention.
Claims
1-20. (canceled)
21. A method for preventing or treating a bacterial infection on a
mucosa in a subject, wherein the infection is caused by a bacteria,
said method comprising: administering to the subject in need
thereof an effective amount of a population of an antibody against
the bacteria, wherein the antibody in the population has been
selected so that the antibody associates with the mucins between
20% to less than 95% of the time, has a rate of binding to the
bacteria greater than about 1.times.10.sup.4 M.sup.-1s.sup.-1, and
has a diffusion coefficient between 20% to 99% less compared to the
diffusion coefficient of the antibody in water.
22. The method of claim 21, wherein the antibody has been selected
so that the antibody in the population associates with the mucins
about 75% of the time and has a diffusion coefficient about 75%
less compared to the diffusion coefficient of the antibody in
water.
23. The method of claim 21, wherein the mucosa is selected from an
oral mucosa, a nasal mucosa, a lung mucosa, a genital mucosa,
uterine mucosa, a vaginal mucosa, an ocular mucosa, and a
gastrointestinal mucosa.
24. The method of claim 21, wherein the administering comprises
topically administering the antibody to the mucosa of the
subject.
25. The method of claim 1, wherein the antibody is formulated into
a composition suitable for intranasal, oral, intravaginal, by
inhalation, or topical administration to a mucosal surface.
26. The method of claim 21, wherein the composition further
comprises a second antibody.
27. The method of claim 21, wherein the antibody in the population
has been selected so that the antibody has a trapping potency at a
sub-neutralization dose.
28. The method of claim 21, wherein the bacteria is selected from
the group including one or more of: Neisseria gonorrhoeae
(gonorrhea); Chlamydia trachomatis (chlamydia, lymphogranuloma
venereum); Treponema pallidum (syphilis); Haemophilus ducreyi
(chancroid); Klebsiella granulomatis or Calymmatobacterium
granulomatis (donovanosis), Mycoplasma genitalium, Ureaplasma
urealyticum (mycoplasmas), Salmonella, and Escherichia coli.
29. A method for preventing or treating an infection on a mucosa in
a subject, wherein the infection is caused by a bacteria, said
method comprising: administering to the subject in need thereof an
effective amount of a population of an IgM antibody against the
bacteria, wherein the IgM antibody in the population has been
selected so that the antibody is specific to the bacteria and
associates with the mucins between 30% to 85% of the time, has a
rate of binding to the bacteria of greater than about
1.times.10.sup.4 M.sup.-1s.sup.-1, and has a diffusion coefficient
between 30% to 85% less compared to the diffusion coefficient of
the antibody in water.
30. The method of claim 29, wherein the antibody has been selected
so that the antibody in the population associates with the mucins
about 75% of the time and has a diffusion coefficient about 75%
less compared to the diffusion coefficient of the antibody in
water.
31. The method of claim 29, wherein the mucosa is selected from an
oral mucosa, a nasal mucosa, a lung mucosa, a genital mucosa,
uterine mucosa, a vaginal mucosa, an ocular mucosa, and a
gastrointestinal mucosa.
32. The method of claim 29, wherein the administering comprises
topically administering the antibody to the mucosa of the
subject.
33. The method of claim 29, wherein the antibody is formulated into
a composition suitable for intranasal, oral, intravaginal, by
inhalation, or topical administration to a mucosal surface.
34. The method of claim 29, wherein the composition further
comprises a second antibody.
35. The method of claim 29, wherein the bacteria is selected from
the group including one or more of: Neisseria gonorrhoeae
(gonorrhea); Chlamydia trachomatis (chlamydia, lymphogranuloma
venereum); Treponema pallidum (syphilis); Haemophilus ducreyi
(chancroid); Klebsiella granulomatis or Calymmatobacterium
granulomatis (donovanosis), Mycoplasma genitalium, Ureaplasma
urealyticum (mycoplasmas), Salmonella, and Escherichia coli.
36. A method for preventing or treating a bacterial infection on a
mucosa in a subject, wherein the infection is caused by a bacteria
having a mobility of greater than 0.1 .mu.m.sup.2/s, said method
comprising: administering to the subject in need thereof an
effective amount of a population of an antibody that specifically
binds the bacteria, wherein the antibody has been selected so that
the antibody associates with the mucins between 20% to less than
95% of the time, has a rate of binding to the bacteria greater than
about 1.times.10.sup.4 M.sup.-1s.sup.-1, and has a diffusion
coefficient between 20% to 99% less compared to the diffusion
coefficient of the antibody in water.
37. The method of claim 36, wherein the bacteria comprises one or
more of: Neisseria gonorrhoeae (gonorrhea); Chlamydia trachomatis
(chlamydia, lymphogranuloma venereum); Treponema pallidum
(syphilis); Haemophilus ducreyi (chancroid); Klebsiella
granulomatis or Calymmatobacterium granulomatis (donovanosis),
Mycoplasma genitalium, Ureaplasma urealyticum (mycoplasmas),
Salmonella, and Escherichia coli.
Description
STATEMENT OF PRIORITY
[0001] This application is a continuation of and claims priority to
U.S. patent application Ser. No. 15/977,432, filed May 11, 2018,
now U.S. Pat. No. 10,793,623, which is a continuation-in-part of
PCT Application No. PCT/US2016/061574, filed Nov. 11, 2016, which
claims the benefit of U.S. Provisional Application Ser. No.
62/254,856, filed Nov. 13, 2015, the entire contents of each of
which are incorporated by reference herein.
FIELD OF THE INVENTION
[0003] The presently-disclosed subject matter relates to
crosslinkers, compositions, and methods for trapping a target of
interest on a substrate of interest. The methods may be used to
inhibit and treat pathogen infection and provide contraception. The
methods may be used to trap or separate particles and other
substances. The subject matter further relates to methods of
identifying and preparing optimal crosslinkers and methods for
manipulating targets of interest.
BACKGROUND OF THE INVENTION
[0004] Antibodies (Ab) produced by our immune system are found in
abundant quantities in both blood and mucosal secretions, and serve
as key messenger molecules that help regulate numerous complex
defense mechanisms against foreign pathogens (Casadevall et al.,
Nat. Immunol. 13:21 (2012); Corthesy, Future Microbiol. 5:817
(2010); Kozlowski et al., Curr. Mol. Med. 3:217 (2003)). For
example, Ab can directly block contact between viruses and target
cells, a process known as neutralization (Burton et al., Nat.
Immunol. 16:571 (2015); van Gils et al., Virology 435:46 (2013)).
Ab can also facilitate other protective functions, such as
ingestion and destruction of the pathogens (opsonization) or
infected cells (antibody-dependent cellular cytotoxicity, or ADCC)
by specialized immune cells, as well as activation of a cascade of
enzymes that lead to direct lysis of the pathogen membrane
(complement) (Dunkelberger et al., Cell Res. 20:34 (2010); Huber et
al., Olson W C, Trkola A (2008), Antibodies for HIV treatment and
prevention: window of opportunity? Curr. Top. Microbiol. Immunol.
317:39 (2008); Kilian et al., Function of mucosal immunoglobulins.
In: Ogra et al., editors. Handbook of Mucosal Immunology. San
Diego: Academic Press. pp. 127-137 (1994)). These various
protective mechanisms most certainly contribute in part to the
robust protection observed with topically delivered antibody
against mucosally transmitted infections in a multitude of animal
studies (Whaley et al., J Infect. Dis. 169:647 (1994); Zeitlin et
al., Virology 225:213 (1996); Veazey et al., Nat. Med. 9:343
(2003); Mascola et al, Nat. Med. 6:207 (2000)).
[0005] In the female reproductive tract, IgG is the predominant Ab
secreted into cervicovaginal mucus (CVM) coating the female
reproductive tract (Chipperfield et al., Infect. Immun. 11:215
(1975); Usala et al., J. Reprod. Med. 34:292 (1989); Wang et al.,
Mucosal Immunol. 7:1036 (2014)). We have recently shown that IgG
can facilitate an alternative mechanism of immune protection based
on trapping viruses in CVM (Wang et al., Mucosal Immunol. 7:1036
(2014)). Interestingly, since the diffusivity of IgG in mucus is
only slowed .about.10-20% compared to in buffer (Saltzman et al.,
Biophys. J 66:508 (1994); Olmsted et al., Biophys. J. 81:1930
(2001)), individual IgG appears to possess only weak and transient
affinity with mucins, and thus were thought incapable of
effectively crosslinking viruses to mucins. Nevertheless, as IgG
accumulates on the virus surface, the array of virion-bound IgG can
collectively impart to the individual virion multiple weak Ab-mucin
bonds, thereby generating sufficient avidity to slow or even
immobilize individual virions in mucus akin to a Velcro.RTM. patch.
Trapping viruses in mucus greatly reduces the flux of virus
reaching target cells in the vaginal epithelium, and trapped
viruses are eliminated along with natural mucus clearance
mechanisms, as evident by protection against vaginal Herpes
transmission using a non-neutralizing monoclonal IgG (Wang et al.,
Mucosal Immunol. 7:1036 (2014)).
[0006] Most viruses, including HIV, can quickly penetrate mucus
secretions, suggesting that there may be only a limited window of
opportunity for Ab to accumulate on the virus surface before they
can reach and infect the underlying vaginal epithelium (McKinley et
al., PLoS One 9:e100598 (2014)) [18]. The extent to which IgG can
hinder the diffusion of viruses in mucus, and consequently the
potency of protection based on IgG-mediated trapping of viruses, is
thus heavily influenced by the tandem effect of IgG-mucin affinity
as well as virion-binding kinetics of topically-delivered or
vaccine-elicited Ab. IT is desirable to develop more potent
`muco-trapping` IgG, as that can directly reduce the dose of IgG
needed for passive immunization of the vagina as well as enhance
protection against viruses with limited antigens on the virus
surface. Nevertheless, a conundrum quickly arises: although fewer
number of virus-bound IgG is needed to trap a virus if individual
IgG possesses increased affinity to mucins, the greater affinity to
mucins would also directly reduce the diffusional freedom of any
mucin-bound IgG and thus limits the rates with which the IgG can
bind to antigens on the virus surface. The exact IgG-mucin affinity
and IgG-antigen binding kinetics that maximizes viral trapping and
protection likely depend on specific characteristics of the virus,
such as its size and surface antigen density, and empirical
determination of both parameters experimentally is undoubtedly
challenging.
[0007] There is a need in the art for new compositions comprising
optimized Ab, and methods of using such compositions, to prevent
and treat infectious diseases and provide contraception, as well as
to manipulate targets by trapping on a substrate.
SUMMARY OF THE INVENTION
[0008] To better understand the subtle yet significant interplay
between the various kinetic and diffusive processes associated with
CVM laden with IgG with distinct mucin affinities, the introduction
of virus-laden semen and subsequent unfolding of events, a
mathematical model was developed whereby the kinetic and diffusion
constants of both IgG and viruses can be freely tuned. As a
proof-of-concept, a focus on HIV was chosen, given the sore need
for alternative strategies to prevent vaginal HIV transmission;
indeed, passive immunization has recently garnered attention as a
promising approach for HIV prophylaxis (Klein et al., Science
341:1199 (2013); Whaley et al., J Infect. Dis. 210 Suppl 3:S674
(2014)). The model described here allows one to simulate the
diffusion of HIV from seminal secretions through CVM containing
neutralizing IgG against HTV immediately after ejaculation, with
IgG concentration, IgG-antigen binding kinetics and IgG-mucin
affinity as tunable model parameters (FIG. 1). This allows one to
explore quantitatively whether tuning IgG-mucin affinity can
facilitate improved protection against vaginal HIV infection.
[0009] Development and validation of the mathematical model has
allowed optimization of antibodies and other crosslinkers that can
be used for preventing and treating infection, monitoring the
effectiveness of vaccines, providing contraception, as well as the
purification and manipulation of target molecules.
[0010] Thus, one aspect of the invention relates to a method of
selecting a crosslinker for trapping a target of interest on a
substrate of interest, comprising:
[0011] (a) determining an optimal target binding affinity and
substrate binding affinity for the target of interest and the
substrate of interest using a mathematical model;
[0012] (b) measuring the target binding affinity and substrate
binding affinity of one or more crosslinkers; and
[0013] (c) selecting a crosslinker that substantially matches the
optimal target binding affinity and substrate binding affinity
determined in step (a).
[0014] A further aspect of the invention relates to a method of
selecting a crosslinker for trapping a target of interest on a
substrate of interest, comprising:
[0015] (a) determining an optimal target binding affinity and
substrate binding affinity for the target of interest and the
substrate of interest using a mathematical model;
[0016] (b) altering the target binding affinity and/or substrate
binding affinity of one or more crosslinkers;
[0017] (c) measuring the target binding affinity and substrate
binding affinity of the one or more altered crosslinkers; and
[0018] (d) selecting an altered crosslinker that substantially
matches the optimal target binding affinity and substrate binding
affinity determined in step (a).
[0019] Another aspect of the invention relates to a method of
selecting a crosslinker for trapping a target of interest on a
substrate of interest, comprising:
[0020] (a) measuring the target binding affinity and substrate
binding affinity of one or more crosslinkers; and
[0021] (b) selecting a crosslinker that substantially matches a
predetermined optimal target binding affinity and substrate binding
affinity.
[0022] An additional aspect of the invention relates to a method of
selecting a crosslinker suitable for trapping a target of interest
on a substrate of interest, comprising:
[0023] (a) altering the target binding affinity and/or substrate
binding affinity of one or more crosslinkers;
[0024] (b) measuring the target binding affinity and substrate
binding affinity of the one or more crosslinkers; and
[0025] (c) selecting an altered crosslinker that substantially
matches a predetermined optimal target binding affinity and
substrate binding affinity.
[0026] In some embodiments, the target is a pathogen, a sperm cell,
or a particle. In some embodiments, the substrate is a biopolymer.
In some embodiments, the crosslinker is an antibody or an antibody
fragment or derivative.
[0027] A further aspect of the invention relates to a crosslinker
identified by the methods of the invention.
[0028] Another aspect of the invention relates to a crosslinker for
trapping a target of interest on a substrate of interest, wherein
the crosslinker associates with the substrate of interest at least
about 10% of the time but less than 99.9% of the time, and has a
rate of binding to the target of interest greater than about
10.sup.4 M.sup.-1s.sup.-1.
[0029] For example, described herein are methods for trapping a
foreign substance (e.g., a pathogen) in mucus containing mucins,
said method comprising contacting the foreign substance (e.g., the
pathogen) with an antibody in an amount effective to trap the
foreign substance (e.g., the pathogen) in mucus, wherein the
antibody associates with the mucins about 20% to less than 99% of
the time (e.g., about 25% to 95%, about 25% to 90%, about 25% to
85%, about 30% to 85%, etc.), has a rate of binding to the foreign
substance such as pathogen greater than about 1.times.10.sup.4
M.sup.-1s.sup.-1, and has a diffusion coefficient about 20% to 99%
(e.g., about 25% to 95%, about 25% to 90%, about 25% to 85%, about
30% to 85%, etc.), less compared to the diffusion coefficient of
the antibody in water.
[0030] For example, the antibody may associate with the mucins
about 75% of the time (e.g., between 30% and 85% of the time). As
described in greater detail herein, antibodies having a biding
affinity of less than 1.times.10.sup.4 M.sup.-1s.sup.-1, and that
associate with the mucins outside of this range (e.g., greater than
85-95% of the time, less than 25-30% of the time) may not be
effective.
[0031] As described in greater detail herein, the crosslinker may
be an antibody or portion of an antibody. Although any appropriate
antibody (e.g., IgG, IgM) may be used, in some variations, the
antibody is an IgG antibody or a fragment or derivative thereof. In
some variations, the antibody is an IgM antibody or a fragment or
derivative thereof. The antibody may bind to a non-neutralizing
epitope on the pathogen; alternatively, in some variations, the
antibody binds to a neutralizing epitope on the pathogen.
[0032] The antibody may have a trapping potency at a
sub-neutralization dose.
[0033] The antibody may be formulated into a composition suitable
for delivering to a mucosal surface. For example, the antibody may
be formulated as a topical composition (e.g., an aerosol or the
like). The composition may be formulated as a composition suitable
for oral administration. The composition may further comprise a
second antibody. The second antibody may be directed to a second
epitope of the same or a different epitope.
[0034] A method for trapping a foreign substance (e.g., a pathogen)
in mucus containing mucins may include: administering to a subject
an effective amount of an antibody to trap the foreign substance
such as the pathogen in mucus, wherein the antibody associates with
the mucins about 20% to less than 99% of the time (e.g., between
30%-85% of the time, between about 25% to 95%, between about 25% to
90%, between about 25% to 85%, between about 30% to 85%, etc.,
about 75% of the time, etc.), has a rate of binding to the foreign
substance such as the pathogen greater than about 1.times.10.sup.4
M.sup.-1s.sup.-1, and has a diffusion coefficient about 20% to 99%
(e.g., about 25% to 95%, about 25% to 90%, about 25% to 85%, about
30% to 85%, etc.), less compared to the diffusion coefficient of
the antibody in water.
[0035] Also described herein are methods for preventing or treating
an infection at a mucosa in a subject (e.g., at one or more of: an
oral mucosa, a nasal mucosa, a lung mucosa, a genital mucosa,
uterine mucosa, a vaginal mucosa an ocular mucosa and a
gastrointestinal mucosa), wherein the infection is caused by a
foreign substance (e.g., a pathogen), said method comprising:
administering to the subject in need thereof an effective amount of
an antibody, wherein the antibody associates with the mucins about
20% to less than 99% of the time (e.g., between about 25% to 95%,
between about 25% to 90%, between about 25% to 85%, between about
30% to 85%, etc., of the time, about 75% of the time, etc.), has a
rate of binding to the foreign substance such as the pathogen
greater than about 1.times.10.sup.4 M.sup.-1s.sup.-1, and has a
diffusion coefficient about 20% to 99% (e.g., about 25% to 95%,
about 25% to 90%, about 25% to 85%, about 30% to 85%, etc.) less
compared to the diffusion coefficient of the antibody in water.
[0036] Administering may include topically administering the
antibody to the mucosa of the subject. The antibody may be
formulated into a composition suitable for intranasal, oral,
intravaginal, by inhalation, or topical administration to a mucosal
surface.
[0037] An additional aspect of the invention relates to a
composition, pharmaceutical composition, or kit comprising one or
more crosslinkers of the invention.
[0038] A further aspect of the invention relates to a method of
trapping a target of interest on a substrate of interest, the
method comprising contacting the target of interest with the
crosslinker or composition of the invention in an amount effective
to trap the target of interest on the substrate of interest.
[0039] Another aspect of the invention relates to a method of
inhibiting an infection by a pathogen or a disease or disorder
caused by an infection by a pathogen in a subject in need thereof,
comprising administering to a mucosa of the subject the crosslinker
or composition of the invention in an amount effective to inhibit
the infection or the disease or disorder caused by the infection;
wherein the crosslinker specifically binds the pathogen.
[0040] An additional aspect of the invention relates to a method of
treating an infection by a pathogen or a disease or disorder caused
by an infection by a pathogen in a subject in need thereof,
comprising administering to a mucosa of the subject the crosslinker
or composition of the invention in an amount effective to treat the
infection or the disease or disorder caused by the infection;
wherein the crosslinker specifically binds the pathogen.
[0041] A further aspect of the invention relates to a method of
providing contraception in a female subject, comprising
administering to a mucosa of a reproductive tract of the subject
the crosslinker or composition of the invention in an amount
effective to provide contraception, wherein the crosslinker
specifically binds a sperm cell.
[0042] Another aspect of the invention relates to a computer
program product comprising: a computer readable storage medium
having computer readable code embodied in the medium, the computer
code comprising: computer readable code to perform operations to
determine an optimal target binding affinity and substrate binding
affinity for a target of interest and an substrate of interest
using a mathematical model.
[0043] An additional aspect of the invention relates to a computer
system, comprising: a processor; and a memory coupled to the
processor, the memory comprising computer readable program code
embodied therein that, when executed by the processor, causes the
processor to perform operations to determine an optimal target
binding affinity and substrate binding affinity for a target of
interest and an substrate of interest using a mathematical
model.
[0044] In general, the method and compositions described herein are
directed to trapping one or more target pathogen on a surface or in
a material to inhibit infection by the pathogen. The surface or
material may be any one in which pathogens are found, including
both natural surfaces/materials (e.g., mucus, extracellular matrix,
basement membranes, etc.) and man-made or applied
surfaces/materials (e.g., hydrogels). For example, any of the
methods described herein may include improving or enhancing
trapping of a pathogen in a hydrogel that is applied to a subject
as part of a film, bandage, gannent, implant, tool, or the like.
The hydrogel may be matched to the crosslinker (e.g., antibody)
with an affinity for the pathogen(s).
[0045] For example, described herein are methods for improving or
enhancing the barrier property of a hydrogel against a foreign
substance (e.g., a pathogen), said method comprising contacting the
foreign substance (e.g., the pathogen) with an IgG or IgM antibody,
or a fragment or derivative thereof in an amount effective to
immobilize the foreign substance (e.g., the pathogen), wherein the
antibody has a binding affinity less than about 10.sup.-2 M (e.g.,
between about 10.sup.-9 M and about 10.sup.-7 M) with a constituent
of the hydrogel. The hydrogel may be a biological hydrogel. The
antibody or fragment thereof may therefore bind to both the foreign
substance such as the pathogen and to the hydrogel; the binding to
the hydrogel may be within a predetermined range so that when a
composition (e.g., solution, aerosol, etc.) of the antibody is
exposed to the hydrogel it will bind to it with a binding affinity
within the ranges described herein as effective.
[0046] In general the antibody may bind to the pathogen with a
relatively high affinity. For example, the antibody (or fragment
thereof) may have a rate of binding to pathogen greater than
10.sup.4 M.sup.-1s.sup.-1.
[0047] As described above, any appropriate antibody or region of
antibody may be used; for example, the Fe region of the antibody
binds to the constituent of the biological hydrogel.
[0048] Any of these methods may include adding a hydrogel to the
patient (e.g., directly to a subject's skin, tissue, wound, etc.
and/or indirectly, via an implant, bandage, etc.). The hydrogel may
include a constituent of a biological hydrogels such as collagen,
laminin, actin, fibronectin, entactin and a combination
thereof.
[0049] In some embodiments, the antibody (or fragment thereof) may
be an IgG antibody with a concentration less than about 100
.mu.g/mL. In some variations, the antibody (or fragment thereof) is
an IgM antibody. As mentioned, the antibody binds to a
non-neutralizing epitope on the pathogen.
[0050] For example, described herein are methods for improving or
enhancing the barrier property of a hydrogel against a foreign
substance (e.g., a pathogen) in a subject, said method comprising
administering to the subject an effective amount of an IgG or IgM
antibody to immobilize the foreign substance (e.g., the pathogen),
wherein the antibody has a binding affinity of less than about
10.sup.-2 M (e.g., from 10.sup.-9 M to about 10.sup.-7 M) with a
constituent of the hydrogel. Any of these methods may also applying
the hydrogel to the subject. The hydrogel may be applied externally
or internally. The hydrogel may be applied via a device (e.g., a
bandage, implant or garment, including as a layer or coating on the
device.
[0051] A method for trapping a foreign substance (e.g., a pathogen)
in a hydrogel as described herein may be a method of treating a
pathogen and/or reducing the likelihood or infection by a foreign
substance such as a pathogen. For example, any of these methods may
include contacting the foreign substance (e.g., the pathogen) with
an IgG or IgM antibody in an amount effective to trap the pathogen,
wherein the antibody has a binding affinity less than about
10.sup.-2 M (e.g., between about 10.sup.-9 M and about 10.sup.-7 M)
with a constituent of the hydrogel.
[0052] The antibody may be directed to one or more pathogens,
including, but not limited to, classes of pathogens (e.g.,
bacteria, gram positive bacterial, grain negative bacteria,
etc.
[0053] For example, a method for preventing or treating an
infection in a subject (wherein the infection is caused by a
foreign substance such as a pathogen) may include: applying a
hydrogel to the subject; and administering to the subject an
effective amount of an IgG or IgM antibody, wherein the antibody
has a binding affinity of less than about 10.sup.-2 M (e.g., from
10.sup.-9 M to about 10.sup.-7 M) with a constituent of the
hydrogel.
[0054] As mentioned above, in general, the antibody may be
formulated in to a composition suitable for intranasal, oral, by
inhalation, or topical administration to a mucosal surface.
[0055] Also described herein are matrix complexes (e.g.,
extracellular matrix complexes) comprising a hydrogel, a plurality
of IgG antibodies or IgM antibodies, a plurality of immobilized
pathogens, wherein the Fe region of the plurality of IgG antibodies
or IgM antibodies binds to a constituent of the biological hydrogel
and the Fab region of the plurality of IgG antibodies or IgM
antibodies binds to the surface of the plurality of pathogens to
immobilize the plurality of the pathogens, and wherein the
plurality of IgG antibodies or IgM antibodies has a rate of binding
to the plurality of pathogen greater than 10.sup.4 M.sup.-1s.sup.-1
and a binding affinity of about 10.sup.-9 M to about 10.sup.-7 M
with the constituent of the biological hydrogel.
[0056] These and other aspects of the invention are set forth in
more detail in the description of the invention below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 shows a schematic of the model that captures the
dynamics of HIV from seminal secretions diffusing across
cervicovaginal mucus (CVM) layer containing HIV-binding IgG to
reach the underlying vaginal epithelium. To reduce infection, IgG
must bind to HIV in sufficient quantities to neutralize or to trap
the virions in mucus before HIV virions successfully penetrate CVM
and reach the vaginal epitheliuin. The model captures the tandem
effects of IgG-antigen binding kinetics (k.sub.on, k.sub.off) as
well as IgG-mucin interactions (m.sub.on, m.sub.off).
[0058] FIG. 2 shows the distribution of time viruses spend freely
diffusing or associated with mucins in CVM containing 10 .mu.g/mL
of NIH45-46 with different affinity to mucins, ranging from no
affinity at .alpha.=1 to very strong affinity at .alpha.=0.001. In
this simulation, Ab are allowed to accumulate on HIV for 30 mins
first prior to measuring the time of free diffusion or association
with mucins for the subsequent 90 mins.
[0059] FIGS. 3A-3D show the predicted trapping potency and
protection by 5 .mu.g/mL and 10 .mu.g/mL of NIH45-46 with varying
affinity to mucins as characterized by .alpha., which reflects the
ratio of the diffusion coefficients of the monoclonal IgG in mucus
vs. water. (A) Predicted fraction of HI load initially in semen
that can diffuse across CVM containing NIH45-46 over the first two
hours post-deposition. (B) Average number of NIH45-46 bound to HIV
arriving at the vaginal epitheliumn. Values below I represent HIV
that arrive at the vaginal epithelium without any bound NIH45-46.
(C-D) Extent of NIH45-46-mediated protection, as quantified by
infectivity relative to (C) no NIH45-46 present in CVM, or (D) the
same amount of NIH45-46 present but without any affinity to
mucins.
[0060] FIGS. 4A-4D show phase diagram mapping of the predicted
trapping potency and protection as a function of NIH45-46
concentration in CVM and IgG affinity to mucins as characterized by
.alpha., which reflects the ratio of the diffusion coefficients of
the monoclonal IgG in mucus vs. water. (A) Fraction of HIV load
initially in semen that can diffuse across CVM containing NIH45-46
over the first two hours post-deposition. (B) Average number of
NIH45-46 bound to HIV arriving the vaginal epithelium. (C-D) Extent
of NIH45-46-mediated protection, as quantified by infectivity
relative to (C) no NIH45-46 present in CVM, or (D) the same amount
of NIH45-46 present but without any affinity to mucins.
[0061] FIGS. 5A-5D show phase diagram mapping of the predicted
trapping potency and protection as a function of NIH45-46 unbinding
kinetics from HIV virions (k.sub.off) as well as accumulation
kinetics on HIV virions, which is influenced by both the local
NIH45-46 concentrations and binding rate (k.sub.on). (A) Fraction
of HIV load initially in semen that can diffuse across CVM
containing NIH45-46 over the first two hours post-deposition. (B)
Average number of NIH45-46 bound to HIV arriving the vaginal
epithelium. (C-D) Extent of NI-145-46-mediated protection, as
quantified by infectivity relative to (C) no NIH45-46 present in
CVM, or (D) the same amount of NIH45-46 present but without any
affinity to mucins.
[0062] FIG. 6 shows IgG profiles in genital secretions overlaying
the vaginal epithelium over time for IgG with distinct affinity to
mucins.
[0063] FIG. 7 shows simulations of crosslinker binding to
substrate.
[0064] FIGS. 8A-8C show diffusion rates of PEG-coated nanoparticles
in mCVM treated with different IgG antibodies. (A) Representative
trajectories for particles exhibiting effective diffusivities
within one SEM of the ensemble average at a time scale of 0.2667 s.
(B) Ensemble-averaged geometric mean square displacements
(<MSD>) as a function of time scale. * indicates a
statistically significant difference (p<0.05). (C) Distributions
of the logarithms of individual particle effective diffusivities
(D.sub.eff) at a time scale of 0.2667 s. Log D.sub.eff values to
the left of the dashed line correspond to particles with
displacements of less than 100 nm (i.e., less than the particle
diameter) within 0.2667 s. These small motions are consistent with
particles permanently stuck to the mucus gel, and most likely
reflect thermal motions of the gel itself. Data represent the
ensemble average of four independent experiments, with n.gtoreq.40
particles per frame (n.gtoreq.130 particle traces per experiment)
on average for each experiment.
[0065] FIGS. 9A-9D show diffusion rates of PEG-coated nanoparticles
in mCVM treated with different IgM antibodies. (A) Representative
trajectories for particles exhibiting effective diffusivities
within one SEM of the ensemble average at a time scale of 0.2667 s.
(B) Ensemble-averaged geometric mean square displacements
(<MSD>) as a function of time scale. (C) Distributions of the
logarithms of individual particle effective diffusivities
(D.sub.eff) at a time scale of 0.2667 s. Log D.sub.eff values to
the left of the dashed line correspond to particles that are
effectively trapped, with displacements of less than 100 nm (i.e.,
less than the particle diameter) within 0.2667 s. (D) Ensemble
averaged geometric effective diffusion coefficients at a timescale
of 0.2667 s for mucus treated with different IgG and IgM
antibodies. Distinct samples are indicated with different circles;
averages are indicated by solid lines. * indicates a statistically
significant difference (p<0.05). Data represent the ensemble
average of four independent experiments, with n.gtoreq.40 particles
per frame (n.gtoreq.120 particle traces per experiment) on average
for each experiment.
[0066] FIG. 10 shows representative transverse 50 .mu.m thick
frozen tissue sections showing distribution of PEG-coated
nanoparticles in mouse vagina treated with control or anti-PEG Ab.
Light gray corresponds to PEG-coated nanoparticles, and dark gray
corresponds to DAPI-stained cell nuclei.
[0067] FIG. 11 shows a schematic illustrating the effects of
anti-PEG antibodies in mucus on PEG-coated nanoparticles
administered to the vaginal mucosal surface. In the absence of
specific antibodies, PEG-coated nanoparticles can diffuse quickly
through the mucus layer and reach the vaginal epithelium as well as
enter into the rugae (folds in the vaginal epithelium), thereby
achieving more uniform coverage of the entire epithelial surface.
In contrast, when anti-PEG antibodies are present in mucus,
PEG-coated nanoparticles become immobilized in mucus, and are
largely localized within the mucus layer rather than in close
proximity to the vaginal epithelium.
[0068] FIGS. 12A-12D show a dot blot assay demonstrating binding of
anti-PEG antibodies to PS-PEG vs. control PS beads. PS and PS-PEG
beads were blotted onto nitrocellulose membrane and incubated with
(a) anti-PEG IgG, (b) anti-PEG IgM, (c) control IgG or (d) control
IgM.
[0069] FIGS. 13A-13C show nanoparticle agglutination in mCVM with
anti-PEG IgM was only observed when nanoparticles were pre-mixed
with anti-PEG IgM prior to addition to mucus (a); no agglutination
was observed when anti-PEG IgM was added to mucus first (b) or with
control IgM (c). Two representative images are shown for each
condition.
[0070] FIGS. 14A-14D show diffusion of nanoparticles that are
modified with polyethylene glycol and are muco-inert (PS-PEG) in
biotinylated basement membrane with neutravidin. (A) Representative
trajectories for PS-PEG particles with anti-PEG antibody or
biotinylated anti-PEG antibody or without antibody exhibiting
effective diffusivities within one SEM of the ensemble average at a
time scale of 1 s. (B) Distributions of the logarithms of
individual particle effective diffusivities (D.sub.eff) at a time
scale of 0.2667 s. Log D.sub.eff values to the left of the dashed
line correspond to particles with displacements of less than 100 nm
(i.e., roughly the particle diameter) within 0.2667 s. (C)
Ensemble-averaged geometric mean square displacements (<MSD>)
as a function of time scale. (D) Estimated time for 10% and 50% of
viruses and particles to diffuse through a 50 .mu.m thick mucus
layer. Data represent the ensemble average of 14 independent AM
specimens, with n.gtoreq.ZZ particles per frame on average
(n.gtoreq.ZZ particle traces per experiment) for each experiment.
Error bars represent standard error of the mean (SEM). * indicates
a statistically significant difference (p<0.05).
[0071] FIGS. 15A-15B show diffusion of PEG-modified nanoparticles
in biotinylated basement membrane with anti-PEG IgG, with or
without neutravidin. (A) Ensemble-averaged geometric mean square
displacements (<MSD>) as a function of time scale. (B)
Distributions of the logarithms of individual particle effective
diffusivities (D.sub.f) at a time scale of 0.2667 s. Log D.sub.eff
values to the left of the dashed line correspond to particles with
displacements of less than 100 nm (i.e., roughly the particle
diameter) within 0.2667 s. Data represent the ensemble average of 4
independent experiments per condition, with n.gtoreq.77 particles
per frame on average (n.gtoreq.92 particle traces per experiment)
for each experiment. Error bars represent standard error of the
mean (SEM).
[0072] FIGS. 16A-16B show diffusion of PEG-modified nanoparticles
in biotinylated basement membrane with biotinylated anti-PEG IgG
together with neutravidin, after 30 min and after 24 hr incubation.
(A) Ensemble-averaged geometric mean square displacements
(<MSD>) as a function of time scale. (B) Distributions of the
logarithms of individual particle effective diffusivities (De) at a
time scale of 0.2667 s. Log D.sub.eff values to the left of the
dashed line correspond to particles with displacements of less than
100 nm (i.e., roughly the particle diameter) within 0.2667 s. Data
represent the ensemble average of 4-5 independent experiments per
condition, with n.gtoreq.50 particles per frame on average
(n.gtoreq.61 particle traces per experiment) for each experiment.
Error bars represent standard error of the mean (SEM).
[0073] FIGS. 17A-17C show model prediction of the effect of the
timescale separation, .tau..sub.AP/.tau..sub.AM, on trapping.
Symbols show averages from 10 Monte Carlo simulations. Solid lines
show the .tau..sub.P/.tau..sub.M.fwdarw..infin. approximation. (A)
The effective diffusivity obtains a minimum for 0<.phi.<1
when .tau..sub.P/.tau..sub.M increases above 0.01. (B) Using the
effective diffusivity shown in (A), the probability of penetration
across a layer of thickness L=50 .mu.m within two hours. (C) A heat
map of the effective diffusivity vs. the anchor concentration (for
170 KD anchors) and timescale separation. Parameter values used
were D.sub.A/D.sub.P=20, N=15.
[0074] FIGS. 18A-18B show effective diffusivity vs. free fraction
of anchors. The approximation [eq:Deff] is shown as solid curves
and symbols show the Monte-Carlo simulation estimator [eq:4] with
106 time steps. Different curves are shown for different values of
(A) N, the maximum number of binding sites on the nanoparticle, and
(B) D.sub.A/D.sub.P, the ratio of the anchor diffusivity to the
nanoparticle diffusivity. Parameter values used were
.tau..sub.AP/.tau..sub.AM=20, N=15, and D.sub.A/D.sub.P=20.
[0075] FIGS. 19A-19C show heat maps of the (A) effective
diffusivity and (B-C) Probability of penetration of a gel layer
within two hours. The y axis is the anchor concentration for 170 KD
anchors. The x axis is (A-B) the fraction of free anchors and (C)
the thickness of matrix layer. Parameter values used were: (A)
D.sub.A/D.sub.P=20 and N=20; (B) D.sub.A/D.sub.P=20, N=20, and L=50
.mu.m; and (C) D.sub.A/D.sub.P=10, N=20, and .phi.=0.72.
[0076] FIG. 20 shows the effective diffusivity for the case of
saturation of binding sites by anchor species. Each pane shows a
heat map for a different values of the timescale separation
.tau..sub.AP/.tau..sub.AM. Parameter values used were
D.sub.A/D.sub.P=20, N=20, and [M]=10.sup.5/.mu.m.sup.3 (this
concentration is relevant for a 2% gel with 10 anchor binding sites
per matrix).
[0077] FIG. 21 shows sensitivity analysis for
.tau..sub.P/.tau..sub.M, N, and D.sub.A/D.sub.P. The sensitivity
was defined as
.differential. .differential. p log ( D eff D P ) .apprxeq. .DELTA.
D eff D eff .DELTA. p , ##EQU00001##
where p can be .tau..sub.P/.tau..sub.M, N, or D.sub.A/D.sub.P.
Fixed parameters were D.sub.A/D.sub.P=20, N=20,
.tau..sub.P/.tau..sub.M=20, and [A]k.sub.on/k.sub.off=2.
[0078] FIGS. 22A-22B show antibody deglycosylation. (A) Heavy chain
of reduced, denatured glycosylated IgG (left) and deglycosylated
IgG (right) and (B) heavy chain of reduced, denatured glycosylated
IgM (left) and deglycosylated IgM (right). Top, silver stain, and
bottom, lectin blot for both.
[0079] FIGS. 23A-23E show PS-COOH, PS-PEG in Matrigel. (A)
Representative particle trajectories of 200 nm PEG-modified
polystyrene beads in Matrigel or PS-COOH. (B) Log Deff. (C) MSD
trajectories. (D) Avg Deff at t=1 s. (E) % Mobile.
[0080] FIGS. 24A-24E show initial IgG anti-PEG in Matrigel (A)
Representative particle trajectories of 200 nm PEG-modified
polystyrene beads in Matrigel with non-specific antibody or
anti-PEG IgG (5 or 10 .mu.g/mL). (B) Log Deff. (C) MSD
trajectories. (D) Avg Deff at t=1 s. (E) % Mobile.
[0081] FIGS. 25A-25E show initial IgM anti-PEG in Matrigel. (A)
Representative particle trajectories of 200 nm PEG-modified
polystyrene beads in Matrigel with non-specific antibody or
anti-PEG IgM(1, 3, or 5 .mu.g/mL). (B) Log Deff. (C) MSD
trajectories. (D) Avg Deff at t=1 s. (E) % Mobile.
[0082] FIGS. 26A-26E show deglycosylated IgG, IgM anti-PEG in
Matrigel. (A) Representative particle trajectories of 200 nm
PEG-modified polystyrene beads in Matrigel with non-specific
antibody or deglycosylated anti-PEG IgG or IgM. (B) Log Deff. (C)
MSD trajectories. (D) Avg Deff at t=1 s. (E) % Mobile.
[0083] FIGS. 27A-27E show IgG, IgM anti-PEG in LAM. (A)
Representative particle trajectories of 200 nm PEG-modified
polystyrene beads in LAM with non-specific antibody or anti-PEG IgG
or IgM. (B) Log Deff. (C) MSD trajectories. (D) Avg Deff at t=1 s.
(E) % Mobile
[0084] FIG. 28 shows IgG anti-Salmonella typhimurium in BM and LAM
impairs the flux of Salmonella in a transwell experiment. OD600 of
Salmonella in bottom well was normalized to OD600 of LB alone and
of Salmonella through matrix without antibody. *p<0.05,
****p<0.0001.
[0085] FIGS. 29A-29B show anti-biotin IgG mediates trapping of
biotinylated PS-PEG in BM: (A) Average ensemble effective
diffusivities (<D.sub.eff>) at a time scale of 1 s. (B)
Fraction of mobile nanoparticles.
[0086] FIGS. 30A-30B show anti-PEG IgG effectively traps .about.100
nm PS-PEG in the presence of high concentrations of anti-biotin
IgG: (A) Average ensemble effective diffusivities
(<D.sub.eff>) at a time scale of 1 s. (B) Fraction of mobile
nanoparticles.
[0087] FIG. 31 shows deglycosylated IgG, IgM anti-PEG in Matrigel.
Fraction of mobile nanoparticles. * indicates a statistically
significant difference (p<0.001) compared to native antibody by
one-way ANOVA with post hoc idak test in FIG. 24E.
DETAILED DESCRIPTION OF THE INVENTION
[0088] The present invention is based, in part, on the development
of mathematical models of interactions between antibodies, targets,
and substrates. The models permit the identification and/or
development of crosslinkers that can bind the target to the
substrates and the use of such crosslinkers in methods for
preventing and treating infection, providing contraception,
monitoring the effectiveness of vaccines, and manipulating (e.g.,
separating, purifying, washing) the target.
[0089] The present invention is explained in greater detail below.
This description is not intended to be a detailed catalog of all
the different ways in which the invention may be implemented, or
all the features that may be added to the instant invention. For
example, features illustrated with respect to one embodiment may be
incorporated into other embodiments, and features illustrated with
respect to a particular embodiment may be deleted from that
embodiment. In addition, numerous variations and additions to the
various embodiments suggested herein will be apparent to those
skilled in the art in light of the instant disclosure which do not
depart from the instant invention. Hence, the following
specification is intended to illustrate some particular embodiments
of the invention, and not to exhaustively specify all permutations,
combinations and variations thereof.
[0090] Unless the context indicates otherwise, it is specifically
intended that the various features of the invention described
herein can be used in any combination. Moreover, the present
invention also contemplates that in some embodiments of the
invention, any feature or combination of features set forth herein
can be excluded or omitted. To illustrate, if the specification
states that a complex comprises components A, B and C, it is
specifically intended that any of A, B or C, or a combination
thereof, can be omitted and disclaimed singularly or in any
combination.
[0091] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting of the invention.
[0092] Except as otherwise indicated, standard methods known to
those skilled in the art may be used for production of recombinant
and synthetic polypeptides, antibodies or antigen-binding fragments
thereof, manipulation of nucleic acid sequences, and production of
transformed cells. Such techniques are known to those skilled in
the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A
LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M.
AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green
Publishing Associates, Inc. and John Wiley & Sons, Inc., New
York).
[0093] All publications, patent applications, patents, nucleotide
sequences, amino acid sequences and other references mentioned
herein are incorporated by reference in their entirety.
I. Definitions
[0094] As used in the description of the invention and the appended
claims, the singular forms "a," "an" and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise.
[0095] As used herein, "and/or" refers to and encompasses any and
all possible combinations of one or more of the associated listed
items, as well as the lack of combinations when interpreted in the
alternative ("or").
[0096] Moreover, the present invention also contemplates that in
some embodiments of the invention, any feature or combination of
features set forth herein can be excluded or omitted.
[0097] Furthermore, the term "about," as used herein when referring
to a measurable value such as an amount of a compound or agent of
this invention, dose, time, temperature, and the like, is meant to
encompass variations of .+-.20%, .+-.10%, .+-.5%, .+-.1%, .+-.0.5%,
or even .+-.0.1% of the specified amount.
[0098] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as reaction conditions,
and so forth used in the specification and claims are to be
understood as being modified in all instances by the term "about".
Accordingly, unless indicated to the contrary, the numerical
parameters set forth in this specification and claims are
approximations that can vary depending upon the desired properties
sought to be obtained by the presently-disclosed subject
matter.
[0099] As used herein, ranges can be expressed as from "about" one
particular value, and/or to "about" another particular value. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that each unit between two particular units is
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0100] The transitional phrase "consisting essentially of" means
that the scope of a claim is to be interpreted to encompass the
specified materials or steps recited in the claim, and those that
do not materially affect the basic and novel characteristic(s) of
the claimed invention.
[0101] As used herein, the term "polypeptide" encompasses both
peptides and proteins, unless indicated otherwise.
[0102] A "nucleic acid" or "nucleotide sequence" is a sequence of
nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences
(including both naturally occurring and non-naturally occurring
nucleotide), but is preferably either single or double stranded DNA
sequences.
[0103] As used herein, an "isolated" antibody means an antibody
separated or substantially free from at least some of the other
components of the naturally occurring organism or virus, for
example, the cell structural components or other polypeptides or
nucleic acids commonly found associated with the antibody. The term
also encompasses antibodies that have been prepared
synthetically.
[0104] By the terms "treat," "treating," or "treatment of" (or
grammatically equivalent terms) it is meant that the severity of
the subject's condition is reduced or at least partially improved
or ameliorated and/or that some alleviation, mitigation or decrease
in at least one clinical symptom is achieved and/or there is a
delay in the progression of the condition.
[0105] As used herein, the terms "prevent," "prevents," or
"prevention" and "inhibit," "inhibits," or "inhibition" (and
grammatical equivalents thereof) are not meant to imply complete
abolition of disease and encompasses any type of prophylactic
treatment that reduces the incidence of the condition, delays the
onset of the condition, and/or reduces the symptoms associated with
the condition after onset.
[0106] An "effective," "prophylactically effective," or
"therapeutically effective" amount as used herein is an amount that
is sufficient to provide some improvement or benefit to the
subject. Alternatively stated, an "effective," "prophylactically
effective," or "therapeutically effective" amount is an amount that
will provide some delay, alleviation, mitigation, or decrease in at
least one clinical symptom in the subject. Those skilled in the art
will appreciate that the effects need not be complete or curative,
as long as some benefit is provided to the subject.
[0107] As used herein, the term "trapping potency" refers to the
ability of a crosslinker that specially binds to a target to
inhibit movement of the target on a substrate. Trapping potency can
be measured by methods known in the art and as disclosed herein.
Trapping potency can be quantitated, e.g., as the amount of
crosslinker (e.g., concentration of crosslinker in mucus) needed to
reduce the mobility of at least 50% of the target in the presence
of the substrate to at least one-tenth of its mobility in solution
(e.g., saline). Mobility in the presence of the substrate can be
measured using techniques well known in the art and described
herein. Alternatively, trapping potency can be quantitated as the
reduction in percentage of target (e.g., pathogens or sperm) that
penetrate mucus.
[0108] As used herein, the term "bind specifically" or
"specifically binds" in reference to a crosslinker of the
presently-disclosed subject matter means that the crosslinker of
the invention will bind with a target, but does not substantially
bind to other unrelated molecules. In certain embodiments, the term
refers to a crosslinker that exhibits at least about 60% binding,
e.g., at least about 70%, 80%, 90%, or 95% binding, to the target
relative to binding to other unrelated molecules.
[0109] The term "crosslinker" refers to a molecule that can
non-covalently bind a target of interest to a substrate of
interest.
II. Methods of Identifying Crosslinkers
[0110] The present invention is based on the development of
mathematical models that may be used to identify a crosslinker
suitable for trapping a target of interest on a substrate of
interest. The models may be used to identify the optimal binding
affinity between a crosslinker and any given target of interest and
substrate of interest for trapping the target on the substrate. The
mathematical model takes into account several factors that are
important for determining optimal trapping, including two or more
factors selected from the fraction of time the crosslinker spends
associated with the substrate, the rate of binding of the
crosslinker to the target, the crosslinker concentration, the size
of the target, the diffusivity of the target, the diffusivity of
the crosslinker, and the number of target binding sites. In some
embodiments, the model includes at least three, four, or five or
more of these factors.
[0111] As would be understood by one of skill in the art, the
optimal binding affinity for the crosslinker may vary for each
target of interest and substrate of interest. In some embodiments,
the methods of the present invention may involve determining
optimal binding affinities for a given target of interest and
substrate of interest using the mathematical models of the
invention and then identifying crosslinkers that match the optimal
affinities. In other embodiments, crosslinkers are identified that
match optimal affinities that have been predetermined for a given
target of interest and substrate of interest, e.g., the
mathematical models have been used previously (prior to
identification of the crosslinkers) to determine optimal affinities
for the given target of interest and substrate of interest.
[0112] In some embodiments, of the invention, the methods can be
used to screen for crosslinkers that match the optimal target
binding affinity and substrate binding affinity. In other
embodiments, one or more preexisting crosslinkers may be modified
to alter the target binding affinity and/or substrate binding
affinity of the one or more crosslinkers, which are then screened
to identify optimal crosslinkers. The phrase "altering binding
affinity" refers to the physical and/or chemical modification of a
crosslinker to increase or decrease the binding affinity. For
example, one or more crosslinkers may be modified, e.g., by random
mutagenesis, and the resulting molecules screened for the desired
binding affinity, e.g., using phage display or other techniques
known in the art. As another example, a library of molecules, e.g.,
a combinatorial library, can be prepared and screened for the
desired binding affinity.
[0113] Thus, one aspect of the invention relates to a method of
selecting a crosslinker for trapping a target of interest on a
substrate of interest, comprising:
[0114] (a) determining an optimal target binding affinity and
substrate binding affinity for the target of interest and the
substrate of interest using a mathematical model;
[0115] (b) measuring the target binding affinity and substrate
binding affinity of one or more crosslinkers; and
[0116] (c) selecting a crosslinker that substantially matches the
optimal target binding affinity and substrate binding affinity
determined in step (a).
[0117] A further aspect of the invention relates to a method of
selecting a crosslinker for trapping a target of interest on a
substrate of interest, comprising:
[0118] (a) determining an optimal target binding affinity and
substrate binding affinity for the target of interest and the
substrate of interest using a mathematical model;
[0119] (b) altering the target binding affinity and/or substrate
binding affinity of one or more crosslinkers;
[0120] (c) measuring the target binding affinity and substrate
binding affinity of the one or more altered crosslinkers; and
[0121] (d) selecting an altered crosslinker that substantially
matches the optimal target binding affinity and substrate binding
affinity determined in step (a).
[0122] Another aspect of the invention relates to a method of
selecting a crosslinker for trapping a target of interest on a
substrate of interest, comprising:
[0123] (a) measuring the target binding affinity and substrate
binding affinity of one or more crosslinkers; and
[0124] (b) selecting a crosslinker that substantially matches a
predetermined optimal target binding affinity and substrate binding
affinity.
[0125] An additional aspect of the invention relates to a method of
selecting a crosslinker for trapping a target of interest on a
substrate of interest, comprising:
[0126] (a) altering the target binding affinity and/or substrate
binding affinity of one or more crosslinkers;
[0127] (b) measuring the target binding affinity and substrate
binding affinity of the one or more crosslinkers; and
[0128] (c) selecting an altered crosslinker that substantially
matches a predetermined optimal target binding affinity and
substrate binding affinity.
[0129] The terms "for trapping" and "suitable for trapping" as used
herein refer to a crosslinker that provides a binding interaction
between a target and a substrate that results in the desired level
of immobilization (trapping) of the target on the substrate.
[0130] The term "optimal" as used herein with respect to binding
affinity refers to a binding affinity that results in the desired
level of immobilization of the target on the substrate.
[0131] The term "substantially matches the optimal binding
affinity" as used herein refers to a crosslinker that has a binding
affinity that differs from the calculated affinity by no more than
about 20%, e.g., no more than about 15%. 10%, 5%, 4%, 3%, 2%, or
1%. The crosslinker may substantially match the calculated binding
affinity of the target binding affinity, the substrate binding
affinity, or both.
[0132] In some embodiments, the target of interest is a pathogen.
The crosslinker is useful for binding target pathogens to trap the
pathogen on a surface or in a material to inhibit infection by the
pathogen. The surface or material may be any one in which pathogens
are found. Examples include, without limitation, mucus,
extracellular matrix, basement membranes, hydrogels, and any other
gel/matrix systems. Target pathogens of the crosslinker can include
any pathogen that can infect a subject through a mucus membrane or
other surface as discussed below. Pathogens can be in the
categories of algae, bacteria, fungi, parasites (helminths,
protozoa), viruses, and subviral agents. Target pathogens further
include synthetic systems comprising an antigen having an epitope,
for example particles or particulates (e.g., polystyrene beads)
comprising attached proteins, e.g., as might be used for
bioterrorism.
[0133] In some embodiments, the target of interest is a particle or
other particulate matter. The particle may be a microparticle
(diameter less than 1 mm) or a nanoparticle (diameter less than 1
.mu.m). The particulate matter may be, for example, proteins,
nucleic acids, polymers, toxins, and/or small molecules. In some
embodiments, the crosslinker binds directly to the particle or
particulate matter. In other embodiments, the particle comprises
attached proteins, nucleic acids, polymers, and/or small molecules
and the crosslinker binds to the attached moieties.
[0134] In some embodiments, the substrate of interest is a polymer,
e.g., a biopolymer, and the crosslinker traps the target on the
polymer. As used herein, the term "biopolymer" refers to a polymer
composed of naturally-occurring units. In certain embodiments, the
biopolymer is, without limitation, mucin, extracellular matrix,
laminin, collagen, actin, or fibronectin. In certain embodiments,
the substrate of interest is mucin and the crosslinker traps the
target in mucus.
[0135] In certain embodiments, the crosslinker is an antibody or an
antibody fragment or derivative as described below. In some
embodiments, the crosslinker is not a naturally occurring molecule.
In some embodiments, the crosslinker is derived from a naturally
occurring molecule (e.g., an antibody), such as by chemical
modification of the molecule.
[0136] The methods of the present invention, including steps of
determining optimal binding affinities, can be readily incorporated
into kit or system formats that are well known in the art. The
terms "kit" and "system," as used herein refer, e.g., to
combinations of reagents, or one or more reagents in combination
with one or more other types of elements or components (e.g., other
types of reagents, containers, packages such as packaging intended
for commercial sale, electronic hardware components, etc.).
III. Crosslinkers and Compositions
[0137] The presently-disclosed subject matter includes crosslinkers
for modulating the movement of a target of interest on a substrate
of interest. In particular, the presently-disclosed subject matter
relates to crosslinkers and compositions capable of trapping a
target of interest on a substrate of interest. As used herein, the
term "trapping" refers to the noncovalent binding of a target to a
substrate such that the target is at least temporarily restrained
in its movement. The crosslinkers can be used advantageously to
trap pathogens and sperm in mucus, thereby inhibiting transport of
pathogens or sperm across or through mucus secretions, or to trap a
target on a substrate for purposes of purification, washing,
filtration, etc.
[0138] A crosslinker of the invention is a molecule that can
noncovalently bind a target of interest to a substrate of interest.
In certain embodiments, the crosslinker is a non-naturally
occurring molecule. In some embodiments, the crosslinker is an
antibody or an antibody fragment or derivative. In other
embodiments, the crosslinker is a target binding moiety covalently
linked to a substrate binding moiety. The binding moieties may be
naturally occurring moieties (e.g., polypeptide sequences) that are
synthetically linked together. The crosslinker may be a naturally
occurring molecule that has been modified to be non-naturally
occurring, e.g., to increase stability or to modify the binding
capacity.
[0139] One aspect of the invention relates to crosslinkers having
optimal binding affinities for both the target of interest and the
substrate of interest to provide trapping of the target on the
substrate. One important factor for optimization is the affinity of
the crosslinker for the substrate, i.e., the amount of time the
crosslinker spends associated with the substrate. In certain
embodiments, the crosslinker associates with the substrate of
interest with an affinity that is sufficient to trap the target of
interest on the substrate of interest but not so high that the
crosslinker is overly limited in its ability to move in the
presence of the substrate. Many naturally occurring antibodies bind
the substrate weakly, e.g., an a value of about 0.8-0.9, where
.alpha.=0 is permanent affinity and .alpha.=1 is no affinity. The
optimized crosslinkers of the invention will generally have a
higher affinity for the substrate of interest than many naturally
occurring antibodies. In some embodiments, the crosslinker
associates with the substrate of interest at least about 1% of the
time but less than 100% of the time, e.g., about 1% of the time to
about 95% of the time, e.g., about 1% of the time to about 90% of
the time, e.g., about 50% of the time to about 90% of the time,
e.g., about 65% of the time to about 85% of the time, e.g., about
75% of the time e.g., about 1% of the time to about 50% of the
time, e.g., about 10% of the time to about 45% of the time, e.g.,
about 20% of the time to about 40% of the time, e.g., about 25% of
the time to about 30% of the time. In some embodiments, the
crosslinker associates with the substrate of interest about 1%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 99% of the time or any range therein.
In some embodiments, the crosslinkers of the invention will have an
a value of about 0.01 to about 0.9, e.g., about 0.05 to about 0.7,
e.g., about 0.1 to about 0.5, e.g., about 0.2 to about 0.4, e.g.,
about 0.25 to about 0.3. In some embodiments, the crosslinkers of
the invention will have an a value of about 0.01, 0.05, 0.1, 0.15,
0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75,
0.8, 0.85, or 0.9 or any range therein. In some embodiments, the
crosslinker associates with the substrate of interest, and has a
reduced diffusion coefficient compared to the diffusion coefficient
of the crosslinker in water. In some embodiments, the crosslinker
is a modified non-native antibody when associates with the
substrate of interest such as mucin, has a diffusion coefficient
reduced about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 95%, or about 99% or any
range therein compared to the diffusion coefficient of the antibody
in water. In some embodiments, the antibody has a diffusion
coefficient about 20% to 99% less or about 25% to 95% less compared
to the diffusion coefficient of the antibody in water. In some
embodiments, the antibody associates with the mucins about 20% to
99% of the time, about 25% to 95% of the time or about 30% to about
85% of the time. In one embodiment, the antibody associates with
the mucins about 75% of the time and has a diffusion coefficient
about 75% less compared to the diffusion coefficient of the
antibody in water. Surprisingly, in mucus, native antibody, such as
native IgG only associates with mucin about 5 to 10% of the time
and has a diffusion coefficient reduced about 5 to 10% compared to
the diffusion coefficient of the native antibody in water.
[0140] Another important factor for optimization is the rate at
which the crosslinker binds to the target of interest. A rapid rate
of binding to the target of interest has been found to be an
important characteristic, even if the rate of release form the
target of interest is also rapid. This is in contrast to most
characterizations of antibodies which are based on the affinity of
the antibody to the target, not the rate of binding. In certain
embodiments, the crosslinker has a rapid rate of binding to the
target of interest. In some embodiments, the crosslinker has a rate
of binding to the target of interest greater than about 10.sup.4
M.sup.-1s.sup.-1, e.g., greater than about 2.5.times.10.sup.4
M.sup.-1s.sup.-1, 5.times.10.sup.4 M.sup.-1s.sup.-1,
1.times.10.sup.5 M.sup.-1s.sup.-1, 2.5.times.10.sup.5
M.sup.-1s.sup.-1, 5.times.10.sup.5 M.sup.-1s.sup.-1,
1.times.10.sup.6 M.sup.-1s.sup.-1, 2.5.times.10.sup.6
M.sup.-1s.sup.-1, 5.times.10.sup.6 M.sup.-1s.sup.-1, or
1.times.10.sup.7 M.sup.-1s.sup.-1.
[0141] In certain embodiments of the invention, the crosslinker is
an antibody, an antibody fragment or derivative, or a molecule that
has binding affinities similar to antibodies. The prevailing view
of how antibodies protect a subject at mucosal surfaces assumes
that neutralization of the pathogen is the primary mechanism of
protection. Surprisingly and unexpectedly, in light of this
widespread view, the present inventors disclose herein that
neutralization is not necessary to protect against infection at
mucosal surfaces in a subject. Indeed, it is demonstrated herein
that sub-neutralization doses of antibodies to neutralizing
epitopes of pathogens can be quite effective at inhibiting
infection. Furthermore, it is demonstrated herein that use of
antibodies to non-neutralizing epitopes of pathogens can also be
quite effective at inhibiting infection.
[0142] Antibodies are naturally found in mucus. The current
thoughts on antibody-mediated mucosal protection are that secretory
IgA (sIgA) antibodies are important for protection because very
large amounts of this isotype are found in the gastrointestinal
tract. It is further thought that IgG does not play a role in
mucosal protection. However, IgG is the dominant isotype in genital
secretions and there is approximately a 50:50 ratio of IgG:IgA in
respiratory mucus secretions. In contrast to the prevailing thought
in the scientific community, it is shown herein that certain
antibodies, e.g., IgG, found in CVM can diffuse rapidly through the
CVM, slowed only slightly by weak, transient adhesive interactions
with mucins within the mucus. This rapid diffusion allows
antibodies to accumulate rapidly on pathogen or sperm surfaces.
When a plurality of antibodies have accumulated on the surface of a
pathogen, the adhesive interactions between the plurality of
antibodies and the mucus become sufficient to trap the bound
pathogen or sperm in the mucus, thereby preventing
infectionlproviding contraception. Pathogens or sperm trapped in
CVM cannot reach their target cells in the mucosal surface, and
will instead be shed with post-coital discharge and/or inactivated
by spontaneous thermal degradation as well as additional protective
factors in mucus, such as defensins (Cole, Curr. Top. Microbiol.
Immunol. 306:199 (2006); Doss et al., J. Leukoc. Biol. 87:79
(2010). As disclosed herein, this pathogen trapping activity
provides for protection without neutralization, and can effectively
inhibit infection at sub-neutralization doses and/or using
antibodies to non-neutralizing epitopes of a pathogen.
[0143] In addition to trapping pathogens or sperm in mucus, the
crosslinkers (e.g., antibodies) may be used to bind a target of
interest to a substrate of interest in any location in vitro or in
vivo. In certain embodiments, the crosslinkers may be used to
manipulate a target by binding it to a substrate, e.g., to
separate, purify, wash, or filter the target. For example, the
substrate may be part of a chromatography, dialysis, or filtration
medium.
[0144] In some embodiments, the crosslinker of the invention is a
mixture of crosslinkers that bind to different targets or bind to
different portions of the same target.
[0145] The crosslinker is useful for binding target pathogens to
trap the pathogen in mucus to inhibit infection by the pathogen.
Target pathogens of the crosslinker can include any pathogen that
can infect a subject through a mucus membrane. Pathogens can be in
the categories of algae, bacteria, fungi, parasites (helminths,
protozoa), viruses, and subviral agents. Target pathogens further
include synthetic systems comprising an antigen having an epitope,
for example particles or particulates (e.g., polystyrene beads)
comprising attached proteins, e.g., as might be used for
bioterrorism.
[0146] Pathogens include those that cause sexually-transmitted
diseases (listed with the diseases caused by such pathogens),
including, without limitation, Neisseria gonorrhoeae (gonorrhea);
Chlamydia trachomatis (chlamydia, lymphogranuloma venereum);
Treponema pallidum (syphilis); Haemophilus ducreyi (chancroid);
Kilebsiella granulomatis or Calymmatobacterium granulomatis
(donovanosis), Mycoplasma genitalium, Ureaplasma urealyticum
(mycoplasmas); human immunodeficiency virus HIV-I and HIV-2 (HIV,
AIDS); HTLV-1 (T-lymphotrophic virus type 1); herpes simplex virus
type 1 and type 2 (HSV-1 and HSV-2); Epstein-Barr virus;
cytomegalovirus; human herpesvirus 6; varicella-zoster virus; human
papillomaviruses (genital warts); hepatitis A virus, hepatitis B
virus, hepatitis C virus (viral hepatitis); molluscum contagiosum
virus (MCV); Trichomona vaginalis (trichomoniasis); and yeasts,
such as Candida albicans (vulvovaginal candidiasis). The antibodies
and compositions may also be active against other diseases that are
transmitted by contact with bodily fluids that may also be
transmissible by sexual contact and are capable of being prevented
by administration of the compositions according to this invention.
Accordingly, the phrase "sexually transmitted diseases (STDs)" is
to be interpreted herein as including any disease that is capable
of being transmitted in the course of sexual contact, whether or
not the genital organs are the site of the resulting pathology.
[0147] Pathogens also include those that cause respiratory
diseases, including, without limitation, influenza (including
influenza A, B, and C); severe acute respiratory syndrome (SARS);
respiratory syncytial virus (RSV); parainfluenza; adenovirus; human
rhinovirus; coronavirus; and norovirus.
[0148] Other pathogens include, without limitation, Salmonella and
Escherichia coli.
[0149] Pathogens include those that affect non-human animals, such
as livestock, e.g., swine (e.g., porcine epidemic diarrhea virus
(PEDV), transmissible gastroenteritis virus (TGEV), rotavirus,
classical swine fever virus (CSFV), porcine circovirus type 2
(PCV2), encephalomyocarditis virus (EMCV), porcine reproductive and
respiratory syndrome virus (PRRSV), porcine parvovirus (PPV),
pseudorabies virus (PRV), Japanese encephalitis virus (JEV),
Brucella, Leptospira, Salmonella, and Lawsonia intracellularis,
Pasteurella multocida, Brachyspira hyodysenteriae, Mycoplasma
hyopneumoniae), ruminants (e.g., bovine virus diarrhoea virus
(BVDV), border disease virus (BDV), bovine papular stomatitis virus
(BPSV), pseudocowpox virus (PCPV), Pasteurella haemolytica,
Pasteurella multocida, Haemophilus somnus, Haemophilus agnii,
Moraxella bovis, Mycoplasma mycoides, The/eria annulata,
Myvcobacterium avium paratuberculosis), ungulates (e.g., Brucella
abortus, Mycobacterium bovis, Theileria parva, Rift Valley fever
virus, foot-and-mouth disease virus, lumpy skin disease virus),
horses (e.g., Rhodococcus equi, Salmonella choleraesuis,
Pasteurella multocida, equine herpesvirus-1, equine herpesvirus-4,
equine influenza virus, Streptococcus equi), poultry (e.g., fowl
pox virus, Newcastle disease virus, Marek's disease virus, avian
influenza virus, infectious bursal disease virus (IBDV), avian
infectious bronchitis virus (IBV)), and the like.
[0150] The terms virus and viral pathogen are used interchangeably
herein, and further refer to various strains of virus, e.g.,
influenza is inclusive of new strains of influenza, which would be
readily identifiable to one of ordinary skill in the art. The terms
bacterium, bacteria, and bacterial pathogen are used
interchangeably herein, and further refer to antibiotic-resistant
or multidrug resistant strains of bacterial pathogens. As used
herein when referring to a bacterial pathogen, the term
"antibiotic-resistant strain" or "multidrug resistant strain"
refers to a bacterial pathogen that is capable of withstanding an
effect of an antibiotic or drug used in the art to treat the
bacterial pathogen (i.e., a non-resistant strain of the bacterial
pathogen).
[0151] In some embodiments, it is contemplated that a crosslinker
according to the presently-disclosed subject matter is capable of
broadly binding to viruses containing lipid envelopes, which are
not necessarily specific to one virus.
[0152] As noted above, it was surprisingly discovered that
sub-neutralization doses of a crosslinker can be used to
effectively trap a target pathogen or sperm in mucus. As such, in
some embodiments, wherein the crosslinker specifically binds a
neutralizing epitope of the target pathogen, a sub-neutralization
dose can be used. A sub-neutralization doses is a dose below that
which would be needed to achieve effective neutralization.
[0153] As will be recognized by one of skill in the art, doses
appropriate for trapping bacterial pathogens can be higher in some
embodiments than the doses appropriate for trapping viral
pathogens. It will further be recognized that appropriate doses may
differ between pathogens, between mucosal surfaces, and also
between individuals.
[0154] It is further proposed herein that crosslinkers that
selectively bind non-neutralizing epitopes of a target pathogen can
be used to effectively trap the target pathogen in mucus. As such,
in some embodiments, the crosslinker specifically binds a
non-neutralizing epitope, e.g., one or more non-neutralizing
epitopes.
[0155] The presently-disclosed subject matter further includes a
crosslinker that selectively binds a conserved epitope of a target
pathogen. A benefit of targeting a conserved epitope would be to
preserve efficacy of the crosslinker as against new strains of the
pathogen. Targeting such epitopes has been avoided at times in the
past because they were viewed as being ineffective targets;
however, in view of the disclosure herein that non-neutralizing
epitopes can serve as effective targets and/or that
sub-neutralization doses can be effective for inhibiting infection,
previously dismissed conserved epitopes of target pathogens can be
seen as effective targets.
[0156] Crosslinkers of the invention are useful for binding sperm
to trap the sperm in mucus to inhibit fertilization of an egg by
the sperm. Sperm specific antigens that can be used as antibody
targets are well known in the art. See, e.g., U.S. Pat. Nos.
8,211,666, 8,137,918, 8,110,668, 8,012,932, 7,339,029, 7,230,073,
and 7,125,550, each incorporated by reference in its entirety.
[0157] As noted above, it was determined that the low-affinity
binding interactions that an antibody forms with mucins are
Fc-dependent. As such, the presently-disclosed subject matter
includes antibodies having a preserved and/or engineered Fc region.
Such antibodies can be, for example, one or more of IgG, IgA, IgM,
IgD, or IgE. In certain embodiments, the antibodies are IgG. In
some embodiments, the antibodies are one or more subclasses of IgG,
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, or any
combination thereof.
[0158] In some embodiments, the crosslinker has a sufficient
binding rate and/or binding affinity to the target pathogen or
sperm to accumulate on the surface of the pathogen or sperm at
sufficient levels to trap the pathogen or sperm within one hour
after administration of the crosslinker at a crosslinker
concentration of less than about 5 .mu.g/ml. The term "trap" in
this instance refers to reduction of further movement through the
mucus. In some embodiments, the target pathogen or sperm is trapped
within about 30 minutes, e.g., about 25, 20, 15, or 10 minutes
after administration of the crosslinker. In some embodiments, the
crosslinker traps the target pathogen or sperm at a crosslinker
concentration of less than about 4, 3, 2, or 1 .mu.g/ml.
[0159] In some embodiments, the crosslinker binds to a target of
interest that is a particle. The particle may be a microparticle
(diameter less than 1 mm) or a nanoparticle (diameter less than 1
.mu.m). In some embodiments, the crosslinker binds directly to the
particle. In other embodiments, the particle comprises attached
proteins, nucleic acids, polymers, and/or small molecules and the
crosslinker binds to the attached moieties.
[0160] In some embodiments, the crosslinker binds to a substrate of
interest that is a polymer, e.g., a biopolymer, and the crosslinker
traps the target on the polymer. In certain embodiments, the
biopolymer is, without limitation, mucin, extracellular matrix,
laminin, collagen, actin or fibronectin. In certain embodiments,
the substrate of interest is mucin and the crosslinker traps the
target in mucus.
[0161] The following discussion is presented as a general overview
of the techniques available for the production of antibodies;
however, one of skill in the art will recognize that many
variations upon the following methods are known.
[0162] The term "antibody" or "antibodies" as used herein refers to
all types of immunoglobulins, including IgG, IgM, IgA, IgD, and
IgE. The antibody can be monoclonal or polyclonal and can be of any
species of origin, including (for example) mouse, rat, rabbit,
horse, goat, sheep, camel, or human, or can be a chimeric or
humanized antibody. See, e.g., Walker et al., Molec. Immunol.
26:403 (1989). The antibodies can be recombinant monoclonal
antibodies produced according to the methods disclosed in U.S. Pat.
No. 4,474,893 or 4,816,567. The antibodies can also be chemically
constructed according to the method disclosed in U.S. Pat. No.
4,676,980. The antibody can be a single monoclonal antibody or a
combination of different monoclonal antibodies, e.g., that bind to
the same target of interest or different targets of interest.
[0163] Antibody fragments and derivatives included within the scope
of the present invention include, for example, Fab, Fab',
F(ab').sub.2, and Fv fragments; domain antibodies, diabodies;
vaccibodies, linear antibodies; single-chain antibody molecules;
and multispecific antibodies formed from antibody fragments. Such
fragments and derivatives can be produced by known techniques. For
example, F(ab').sub.2 fragments can be produced by pepsin digestion
of the antibody molecule, and Fab fragments can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragments.
Alternatively, Fab expression libraries can be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity (Huse et al., Science 254:1275 (1989)). In some
embodiments, the term "antibody fragment or derivative" as used
herein may also include any protein construct that is capable of
binding a target of interest and associate with a substrate of
interest to trap the target on the substrate.
[0164] Antibodies of the invention may be altered or mutated for
compatibility with species other than the species in which the
antibody was produced. For example, antibodies may be humanized or
camelized. Humanized forms of non-human (e.g., murine) antibodies
are chimeric immunoglobulins, immunoglobulin chains or fragments or
derivatives thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementarity determining region (CDR) of
the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the framework (FR)
regions (i.e., the sequences between the CDR regions) are those of
a human immunoglobulin consensus sequence. The humanized antibody
optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunmoglobulin (Jones et al., Nature 321:522 (1986); Riechmann et
al., Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol.
2:593 (1992)).
[0165] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can essentially be performed
following the method of Winter and co-workers (Jones et al., Nature
321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen
et al., Science 239:1534 (1988)), by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues (e.g., all of the CDRs or a portion thereof) and
possibly some FR residues are substituted by residues from
analogous sites in rodent antibodies.
[0166] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
(Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al.,
J. Mol. Biol. 222:581 (1991)). The techniques of Cole et al. and
Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol. 147:86 (1991)). Similarly, human antibodies can be made by
introducing human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, for example, in
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,016, and in the following scientific publications:
Marks et al., Bio/Technology 10:779 (1992); Lonberg et al., Nature
368:856 (1994); Morrison, vature 368:812 (1994); Fishwild et al.,
Nature iotechnol. 14:845 (1996); Neuberger, Nature Biotechnol.
14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65
(1995).
[0167] Immunogens (antigens) are used to produce antibodies
specifically reactive with target polypeptides. Recombinant or
synthetic polypeptides and peptides, e.g., of at least 5 (e.g., at
least 7 or 10) amino acids in length, or greater, are the preferred
immunogens for the production of monoclonal or polyclonal
antibodies. In one embodiment, an immunogenic polypeptide conjugate
is also included as an immunogen. The peptides are used either in
pure, partially pure or impure form. Suitable polypeptides and
epitopes for target pathogens and sperm are well known in the art.
Polynucleotide and polypeptide sequences are available in public
sequence databases such as GENBANK.RTM./GENPEPT.RTM.. Large numbers
of neutralizing and non-neutralizing antibodies that specifically
bind to target pathogens and sperm have been described in the art
and can be used as starting material to prepare the antibodies of
the present invention. Alternatively, new antibodies can be raised
against target pathogens and sperm using the techniques described
herein and well known in the art.
[0168] Recombinant polypeptides are expressed in eukaryotic or
prokaryotic cells and purified using standard techniques. The
polypeptide, or a synthetic version thereof, is then injected into
an animal capable of producing antibodies. Either monoclonal or
polyclonal antibodies can be generated for subsequent use in
immunoassays to measure the presence and quantity of the
polypeptide.
[0169] Methods of producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, e.g., a purified
or synthetic peptide, a peptide coupled to an appropriate carrier
(e.g., glutathione-S-transferase, keyhole limpet hemocyanin, etc.),
or a peptide incorporated into an immunization vector such as a
recombinant vaccinia virus is optionally mixed with an adjuvant and
animals are immunized with the mixture. The animal's immune
response to the immunogen preparation is monitored by taking test
bleeds and determining the titer of reactivity to the peptide of
interest. When appropriately high titers of antibody to the
immunogen are obtained, blood is collected from the animal and
antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the peptide is performed where
desired. Antibodies, including binding fragments and single chain
recombinant versions thereof, against the polypeptides are raised
by immunizing animals, e.g., using immunogenic conjugates
comprising a polypeptide covalently attached (conjugated) to a
carrier protein as described above. Typically, the immunogen of
interest is a polypeptide of at least about 10 amino acids, in
another embodiment the polypeptide is at least about 20 amino acids
in length, and in another embodiment, the fragment is at least
about 30 amino acids in length. For example, the polypeptide can
comprise amino acids acid residues 1 through 200 from the
N-terminal of the papillomavirus L2 protein. The immunogenic
conjugates are typically prepared by coupling the polypeptide to a
carrier protein (e.g., as a fusion protein) or, alternatively, they
are recombinantly expressed in an immunization vector.
[0170] Monoclonal antibodies are prepared from cells secreting the
desired antibody. These antibodies are screened for binding to
normal or modified peptides, or screened for agonistic or
antagonistic activity. Specific monoclonal and polyclonal
antibodies will usually bind with a KD of at least about 50 mM,
e.g., at least about 1 mM, e.g., at least about 0.1 mM or better.
In some instances, it is desirable to prepare monoclonal antibodies
from various mammalian hosts, such as mice, rodents, primates,
humans, etc. Description of techniques for preparing such
monoclonal antibodies are found in Kohler and Milstein 1975 Nature
256:495-497. Summarized briefly, this method proceeds by injecting
an animal with an immunogen, e.g., an immunogenic peptide either
alone or optionally linked to a carrier protein. The animal is then
sacrificed and cells taken from its spleen, which are fused with
myeloma cells. The result is a hybrid cell or "hybridoma" that is
capable of reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which secrete a
single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0171] Alternative methods of immortalization include
transformation with Epstein Barr Virus, oncogenes, or retroviruses,
or other methods known in the art. Colonies arising from single
immortalized cells are screened for production of antibodies of the
desired specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells is enhanced by various
techniques, including injection into the peritoneal cavity of a
vertebrate (preferably mammalian) host. The polypeptides and
antibodies of the present invention are used with or without
modification, and include chimeric antibodies such as humanized
murine antibodies. Other suitable techniques involve selection of
libraries of recombinant antibodies in phage or similar vectors.
See, Huse et al. 1989 Science 246:1275-1281; and Ward et al 1989
Nature 341:544-546.
[0172] Antibodies specific to the target polypeptide can also be
obtained by phage display techniques known in the art.
[0173] Antibodies can sometimes be labeled by joining, either
covalently or noncovalently, a substance which provides a
detectable signal. A wide variety of labels and conjugation
techniques are known and are reported extensively in both the
scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Such antibodies are useful for detecting
or diagnosing the presence of a microbe on which an antigen is
found.
[0174] Crosslinkers with altered binding affinity to a target of
interest and/or a substrate of interest may be created by methods
known in the art. Methods include starting with a known crosslinker
or crosslinkers (e.g., antibodies) and modifying the structure of
the crosslinker to produce a crosslinker with a desired binding
affinity. Methods of modification include, without limitation,
random mutagenesis or targeted mutagenesis, e.g., to increase the
number of moieties on crosslinkers that can interact with the
scaffold, such as N-glycans and Fc domains on antibodies, and
chemical modification, e.g., whereby a moiety which some affinity
to the scaffold is covalently linked to the crosslinker of
interest. Screening of modified crosslinkers for altered binding
affinity may be carried out using techniques routinely used in the
art.
[0175] As would be recognized by one skilled in the art, the
crosslinkers of the presently-disclosed subject matter can also be
formed into suitable compositions, e.g., pharmaceutical
compositions for administration to a subject in order to treat or
prevent an infection caused by a target pathogen or a disease or
disorder caused by infection by a target pathogen or to provide
contraception. In one embodiment, the compositions comprise,
consist essentially of, or consist of a crosslinker of the
invention in a prophylactically or therapeutically effective amount
and a pharmaceutically-acceptable carrier. In other embodiments,
the compositions comprise, consist essentially of, or consist of a
crosslinker of the invention in an amount suitable for in vitro
uses, such as manipulation of a target of interest. Such
compositions may further comprise water, buffer, or another carrier
for the crosslinker.
[0176] Pharmaceutical compositions containing the compositions
comprise, consist essentially of, or consist of a crosslinker of
the invention as disclosed herein can be formulated in combination
with any suitable pharmaceutical vehicle, excipient or carrier that
would commonly be used in this art, including such conventional
materials for this purpose, e.g., saline, dextrose, water,
glycerol, ethanol, and combinations thereof. As one skilled in this
art would recognize, the particular vehicle, excipient or carrier
used will vary depending on the subject and the subject's
condition, and a variety of modes of administration would be
suitable for the compositions of the invention. Suitable methods of
administration of any pharmaceutical composition disclosed in this
application include, but are not limited to, topical, oral,
intranasal, buccal, inhalation, anal, and vaginal administration,
wherein such administration achieves delivery of the crosslinker to
a mucus membrane of interest.
[0177] The composition can be any type of composition suitable for
delivering crosslinker to a mucosal surface and can be in various
forms known in the art, including solid, semisolid, or liquid form
or in lotion form, either oil-in-water or water-in-oil emulsions,
in aqueous gel compositions. Compositions include, without
limitation, gel, paste, suppository, douche, ovule, foam, film,
spray, ointment, pessary, capsule, tablet, jelly, cream, milk,
dispersion, liposomes, powder/talc or other solid, suspension,
solution, emulsion, microemulsion, nanoemulsion, liquid, aerosol,
microcapsules, time-release capsules, controlled release
formulation, sustained release formulation or bioadhesive gel
(e.g., a mucoadhesive thermogelling composition) or in other forms
embedded in a matrix for the slow or controlled release of the
crosslinker to the surface onto which it has been applied or in
contact.
[0178] If topical administration is desired, the composition may be
formulated as needed in a suitable form, e.g., an ointment, cream,
gel, lotion, drops (such as eye drops and ear drops), or solution
(such as mouthwash). The composition may contain conventional
additives, such as preservatives, solvents to promote penetration,
and emollients. Topical formulations may also contain conventional
carriers such as cream or ointment bases, ethanol, or oleyl
alcohol. Other formulations for administration, including
intranasal administration, etc., are contemplated for use in
connection with the presently-disclosed subject matter. All
formulations, devices, and methods known to one of skill in the art
which are appropriate for delivering the crosslinker or composition
containing the crosslinker to one or more mucus membranes of a
subject can be used in connection with the presently-disclosed
subject matter.
[0179] The compositions used in the methods described herein may
include other agents that do not negatively impact or otherwise
affect the inhibitory and/or contraceptive effectiveness of the
components of the composition, including antibodies, antimicrobial
agents, and/or sperm-function inhibitors. For example, solid,
liquid or a mixture of solid and liquid pharmaceutically acceptable
carriers, diluents, vehicles, or excipients may be employed in the
pharmaceutical compositions. Suitable physiologically acceptable,
substantially inert carriers include water, a polyethylene glycol,
mineral oil or petrolatum, propylene glycol, hydroxyethylcellulose,
carboxymethyl cellulose, cellulosic derivatives, polycarboxylic
acids, linked polyacrylic acids, such as carbopols; and other
polymers such as poly(lysine), poly(glutamic acid), poly(maleic
acid), poly(lactic acid), thermal polyaspartate, and
aliphatic-aromatic resin; glycerin, starch, lactose, calcium
sulphate dihydrate, terra alba, sucrose, talc, gelatin, pectin,
acacia, magnesium stearate, stearic acid, syrup, peanut oil, olive
oil, saline solution, and the like.
[0180] The pharmaceutical compositions described herein useful in
the methods of the present invention may further include diluents,
fillers, binding agents, colorants, stabilizers, perfumes, gelling
agents, antioxidants, moisturizing agents, preservatives, acids,
and other elements known to those skilled in the art. For example,
suitable preservatives are well known in the art, and include, for
example, methyl paraben, propyl paraben, butyl paraben, benzoic
acid and benzyl alcohol.
[0181] For injection, the carrier will typically be a liquid, such
as sterile pyrogen-free water, pyrogen-free phosphate-buffered
saline solution, bacteriostatic water, or Cremophor EL[R] (BASF,
Parsippany, N.J.). For other methods of administration, the carrier
can be either solid or liquid.
[0182] For oral administration, the crosslinker can be administered
in solid dosage forms, such as capsules, tablets, and powders, or
in liquid dosage forms, such as elixirs, syrups, and suspensions.
Compositions can be encapsulated in gelatin capsules together with
inactive ingredients and powdered carriers, such as glucose,
lactose, sucrose, mannitol, starch, cellulose or cellulose
derivatives, magnesium stearate, stearic acid, sodium saccharin,
talcum, magnesium carbonate and the like. Examples of additional
inactive ingredients that can be added to provide desirable color,
taste, stability, buffering capacity, dispersion or other known
desirable features are red iron oxide, silica gel, sodium lauryl
sulfate, titanium dioxide, edible white ink and the like. Similar
diluents can be used to make compressed tablets. Both tablets and
capsules can be manufactured as sustained release products to
provide for continuous release of medication over a period of
hours. Compressed tablets can be sugar coated or film coated to
mask any unpleasant taste and protect the tablet from the
atmosphere, or enteric-coated for selective disintegration in the
gastrointestinal tract. Liquid dosage forms for oral administration
can contain coloring and flavoring to increase patient
acceptance.
[0183] Compositions suitable for buccal (sub-lingual)
administration include tablets or lozenges comprising the
crosslinker in a flavored base, usually sucrose and acacia or
tragacanth; and pastilles comprising the antibody in an inert base
such as gelatin and glycerin or sucrose and acacia. The composition
can comprise an orally dissolvable or degradable composition.
Alternately, the composition can comprise a powder or an
aerosolized or atomized solution or suspension comprising the
crosslinker. Such powdered, aerosolized, or atomized compositions,
when dispersed, preferably have an average particle or droplet size
in the range from about 0.1 to about 200 nanometers.
[0184] Compositions of the present invention suitable for
parenteral administration comprise sterile aqueous and non-aqueous
injection solutions of the crosslinker, which preparations are
preferably isotonic with the blood of the intended recipient. These
preparations can contain anti-oxidants, buffers, bacteriostats and
solutes which render the composition isotonic with the blood of the
intended recipient. Aqueous and non-aqueous sterile suspensions can
include suspending agents and thickening agents. The compositions
can be presented in unit/dose or multi-dose containers, for example
sealed ampoules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, saline or water-for-injection
immediately prior to use.
[0185] Extemporaneous injection solutions and suspensions can be
prepared from sterile powders, granules and tablets of the kind
previously described. For example, in one aspect of the present
invention, there is provided an injectable, stable, sterile
composition comprising a crosslinker, in a unit dosage form in a
sealed container. The crosslinker is provided in the form of a
lyophilizate which is capable of being reconstituted with a
suitable pharmaceutically acceptable carrier to form a liquid
composition suitable for injection thereof into a subject.
[0186] Compositions suitable for rectal administration are
preferably presented as unit dose suppositories. These can be
prepared by admixing the crosslinker with one or more conventional
solid carriers, for example, cocoa butter, and then shaping the
resulting mixture.
[0187] The crosslinker can alternatively be formulated for nasal
administration or otherwise administered to the lungs of a subject
by any suitable means, e.g., administered by an aerosol suspension
of respirable particles comprising the crosslinker, which the
subject inhales. The respirable particles can be liquid or solid.
The term "aerosol" includes any gas-borne suspended phase, which is
capable of being inhaled into the bronchioles or nasal passages.
Specifically, aerosol includes a gas-borne suspension of droplets,
as can be produced in a metered dose inhaler or nebulizer, or in a
mist sprayer. Aerosol also includes a dry powder composition
suspended in air or other carrier gas, which can be delivered by
insufflation from an inhaler device, for example. See Ganderton
& Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood
(1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier
Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth.
27:143 (1992). Aerosols of liquid particles comprising the
crosslinker can be produced by any suitable means, such as with a
pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is
known to those of skill in the art. See, e.g., U.S. Pat. No.
4,501,729. Aerosols of solid particles comprising the crosslinker
can likewise be produced with any solid particulate medicament
aerosol generator, by techniques known in the pharmaceutical
art.
[0188] Alternatively, one can administer the crosslinker in a local
rather than systemic manner, for example, in a depot or
sustained-release formulation.
[0189] One aspect of the invention relates to devices coated or
impregnated with the crosslinkers and compositions of the
invention. The device can be for delivery of the crosslinkers and
compositions of the invention to a mucus membrane, e.g., to the
vagina or uterus. In one embodiment, a device includes a solid
support adapted to be inserted into the vagina. The support can be
impregnated with or coated with a composition. The release of
crosslinkers from the devices may be controlled by the material
composing these devices, such as silicone elastomers, ethylene
vinyl acetate and polyurethane polymers. Devices, such as
cervicovaginal and rectal devices, include, without limitation, a
ring, rod, applicator, sponge, cervical cap, tampon, diaphragm, or
intrauterine device. Applicators can be those currently used
commercially to deliver spermicidal gels or anti-yeast compounds
and include, without limitation, plunger-type applicators,
pessaries, sprays, squeezable tubes, vaginal rings, cervical rings,
sponges, and the like. All such means for delivery are intended to
be encompassed by the present invention.
[0190] As noted herein, crosslinkers of the presently-disclosed
subject matter are capable of diffusing through mucus when they are
unbound, to allow the crosslinker to bind a target pathogen or
sperm at a desirable rate. It is also desirable that, when
crosslinkers are bound to the pathogen or sperm, the cumulative
effect of the crosslinker-mucin interactions effectively traps the
pathogen or sperm in the mucus. To facilitate this goal, in some
embodiments, it can be desirable to provide a composition that
includes more than one crosslinker, wherein each crosslinker
specifically binds a different portion (e.g., epitope) of the
target pathogen or sperm. Such a composition provides the ability
for an increased number of crosslinkers to become bound to the
pathogen or sperm, thereby strengthening the crosslinker-mucin
interactions that serve to trap the pathogen or sperm in the
mucus.
[0191] In some embodiments of the presently-disclosed subject
matter, a composition includes a first crosslinker and a second
crosslinker, as disclosed herein, wherein the first crosslinker
specifically binds a first portion (e.g., epitope) of the target
pathogen or sperm and the second crosslinker specifically binds a
second portion (e.g., epitope) of the target pathogen or sperm,
wherein said first portion is distinct from the second portion. In
certain embodiments, the composition includes three or more
different crosslinkers, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more
different crosslinkers, wherein each crosslinker specifically binds
a different portion or epitope of the target pathogen or sperm.
[0192] In some embodiments of the presently-disclosed subject
matter, a composition includes a first crosslinker that
specifically binds a gG surface glycoprotein of HSV, a second
crosslinker that specifically binds a gD surface glycoprotein of
HSV, and/or a third crosslinker that specifically binds a gB
surface glycoprotein of HSV.
[0193] It is also desirable to provide a composition that can
provide treatment or prevention of infection due to more than one
target pathogen. In some embodiments of the presently-disclosed
subject matter, a composition includes a first crosslinker and a
second crosslinker, as disclosed herein, wherein the first
crosslinker specifically binds a portion (e.g., epitope) of a first
target pathogen and the second crosslinker specifically binds a
portion (e.g., epitope) of second target pathogen. In certain
embodiments, the composition includes three or more different
crosslinkers, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different
crosslinkers, wherein each crosslinker specifically binds a portion
or epitope of a different target pathogen.
[0194] In other embodiments, a composition provides both
contraception and treatment or prevention of infection by one or
more target pathogens. In some embodiments of the
presently-disclosed subject matter, a composition includes a first
crosslinker and a second crosslinker, as disclosed herein, wherein
the first crosslinker specifically binds sperm and the second
crosslinker specifically binds a target pathogen. In certain
embodiments, the composition includes three or more different
crosslinkers, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different
crosslinkers, wherein one or more crosslinkers bind different
portions (e.g., epitopes) of sperm and one or more crosslinkers
specifically binds a portion (e.g., epitope) of a target pathogen
or multiple target pathogens.
[0195] In some embodiments, the pharmaceutical composition can
further include an additional active agent, e.g., a prophylactic or
therapeutic agent. For example, the additional active agent can be
an antimicrobial agent, as would be known to one of skill in the
art. The antimicrobial agent may be active against algae, bacteria,
fungi, parasites (helminuths, protozoa), viruses, and subviral
agents. Accordingly, the antimicrobial agent may be an
antibacterial, antifungal, antiviral, antiparasitic, or
antiprotozoal agent. The antimicrobial agent is preferably active
against infectious diseases.
[0196] Suitable antiviral agents include, for example,
virus-inactivating agents such as nonionic, anionic and cationic
surfactants, and C31 G (amine oxide and alkyl betaine),
polybiguanides, docosanol, acylcamitine analogs, octyl glycerol,
and antimicrobial peptides such as magainins, gramicidins,
protegrins, and retrocyclins. Mild surfactants, e.g., sorbitan
monolaurate, may advantageously be used as antiviral agents in the
compositions described herein. Other antiviral agents that may
advantageously be utilized in the compositions described herein
include nucleotide or nucleoside analogs, such as tenofovir,
acyclovir, amantadine, didanosine, foscamet, ganciclovir,
ribavirin, vidarabine, zalcitabine, and zidovudine. Further
antiviral agents that may be used include non-nucleoside reverse
transcriptase inhibitors, such as UC-781 (thiocarboxanilide),
pyridinones, TIBO, nevaripine, delavirdine, calanolide A,
capravirine and efavirenz. From these reverse transcriptase
inhibitors, agents and their analogs that have shown poor oral
bioavailability are especially suitable for administration to
mucosal tissue, in combination with antibodies and compositions of
the invention, to prevent sexual transmission of HIV. Other
antiviral agents that may be used are those in the category of HIV
entry blockers, such as cyanovirin-N, cyclodextrins, carregeenans,
sulfated or sulfonated polymers, mandelic acid condensation
polymers, monoclonal antibodies, chemokine receptor antagonists
such as TAK-779, SCH-C/D, and AMD-3100, and fusion inhibitors such
as T-20 and 1249.
[0197] Suitable antibacterial agents include antibiotics, such as
aminoglycosides, cephalosporins, including first, second and third
generation cephalosporins; macrolides, including erythromycins,
penicillins, including natural penicillins, penicillinase-resistant
penicillins, aminopenicillins, extended spectrum penicillins;
sulfonamides, tetracyclines, fluoroquinolones, metronidazole and
urinary tract antiseptics.
[0198] Suitable antifungal agents include amphotericin B, nystatin,
griseofulvin, flucytosine, fluconazole, potassium iodide,
intraconazole, clortrimazole, miconazole, ketoconazole, and
tolnaftate.
[0199] Suitable antiprotozoal agents include antimalarial agents,
such as chloroquine, primaquine, pyrimethamine, quinine, fansidar,
and mefloquine; amebicides, such as dioloxamide, emetine,
iodoquinol, metronidazole, paromomycine and quinacrine; pentamidine
isethionate, atovaquone, and eflornithine.
[0200] In certain embodiments, the additional active agent can be a
sperm-function inhibitor, e.g., an agent that has the ability to
inhibit the function of sperm, to otherwise inhibit fertilization
of an egg by sperm and/or to otherwise prevent pregnancy, such as
by killing and/or functionally inactivating sperm or by other
effects on the activity of the sperm. In some embodiments, the
active agent may have at least dual functions, such as acting as a
sperm-function inhibitor and as an antimicrobial agent.
[0201] Sperm-function inhibitors include, without limitation,
surfactants, including nonionic surfactants, cationic surfactants,
and anionic surfactants; spermicides, such as nonoxynol-9
(.alpha.-(4-Nonylphenyl)-.omega.-hydroxynona(oxyethylene); other
sperm-inactivators such as sulfated or sulfonated polymers such as
polystyrene sulfonate, mandelic acid condensation polymers,
cyclodextrins; antimicrobial peptides such as gramicidins,
magainins, indolicidin, and melittin; and acid-buffering
compositions, such as BufferGel and AcidForm. Nonionic surfactants
include, for example, sorbitan monolaurate, nonylphenoxypolyethoxy
ethanol, p-diisobutyphenoxypolyethoxy ethanol, polyoxyethylene (10)
oleyl ether and onyx-ol. Suitable anionic surfactants include,
without limitation, sodium alkyl sulfonates and the sodium
alkylbenzene sulfonates. Cationic surfactants include, for example,
the quaternary ammonium surfactants, such as cetyl pyrimidinium
chloride and benzalkonium chlorides. Zwitterionic surfactants such
as acylcarnitine analogs and C31G are especially suitable for their
mild skin and mucosal irritation properties.
[0202] The presently-disclosed subject matter further includes a
kit, including the crosslinker or composition comprising the
crosslinker as described herein; and optionally a device for
administering the crosslinker or composition. In some embodiments,
the kit can include multiple crosslinkers and/or compositions
containing such crosslinkers. In some embodiments, each of multiple
crosslinkers provided in such a kit can specifically bind to a
different portion (e.g., epitope) of the target pathogen or sperm.
In other embodiments, each of multiple crosslinkers provided in
such a kit can specifically bind to a portion (e.g., epitope) of a
different target pathogen or to and epitope of sperm. In some
embodiments, the kit can further include an additional active
agent, e.g., antimicrobial, such as an antibiotic, an antiviral, or
other antimicrobial, or a sperm-function inhibitor as would be
known to one of skill in the art. For in vitro uses, the kit may
further comprise buffers, reagents, and/or other components for
manipulating the target of interest.
IV. Prevention and Treatment of Infection
[0203] The presently-disclosed subject matter further includes
methods of inhibiting or treating an infection by a target pathogen
in a subject, including administering to a subject, e.g., to a
mucosa of the subject, a crosslinker and/or composition as
disclosed herein. The mucosa can be selected from, for example, a
respiratory tract mucosa (e.g., a nasal mucosa, a lung mucosa), a
reproductive tract mucosa (e.g., a genital mucosa, an uterine
mucosa, a vaginal mucosa), an ocular mucosa, and a gastrointestinal
mucosa (e.g., an oral mucosa, an anal mucosa), and any combination
thereof. The surface may be a non-mucosal surface, e.g., skin,
blood vessels, central nervous system, extracellular matrix, etc.
In certain embodiments, the methods comprise additional steps such
as one or more of isolating/preparing the crosslinkers, preparing a
composition of the crosslinkers, determining the level of
antibodies in the mucus of the subject before administering the
crosslinkers, and determining the level of crosslinkers in the
mucus of the subject after administering the crosslinkers.
[0204] The crosslinkers and compositions of the present invention
according to the methods described herein are administered or
otherwise applied by delivering the composition, typically to a
site of infection. The site of infection may be one where an
infection is already present (an actual site of infection) or where
an infection is likely to occur (a potential site of site of
infection in or on an uninfected individual). In some embodiments,
the crosslinkers and compositions may be topically delivered. In
other embodiments, the crosslinkers and compositions may be
systemically delivered such that the crosslinkers are secreted into
the mucus of the subject. Accordingly, the compositions as
described above may be delivered to a mucosal surface, e.g., to the
reproductive tract, e.g., to the vulva, including the vaginal
cavity, and/or to the gastrointestinal tract, e.g., the ano-rectal
and buccal cavities and/or to the respiratory tract, e.g., the
nasal cavity and the lungs. In the vaginal cavity, the compositions
may be applied to any portion of the uterus, including inside the
uterus and on the cervix, including the mucosa and/or lining of the
endo- and ecto-cervix. The ano-rectal cavity includes the perianal
surface and the lining of the anus. Topical delivery to the
gastrointestinal tract includes oral administration such that the
crosslinkers reach, e.g., the small and/or large intestines. For
example, oral administration of an enteric-coated solid oral dosage
form (e.g., tablet or capsule) can effectively carry the
crosslinkers through the stomach unharmed with release occurring in
the intestines.
[0205] An effective amount of the crosslinker can be administered.
As used herein, an "effective amount" of the crosslinker for
inhibition of infection refers to a dosage sufficient to inhibit
infection by the target pathogen. As used herein, an "effective
amount" of the crosslinker for treatment of infection refers to a
dosage sufficient to inhibit spread of the target pathogen from
infected cells to non-infected cells in the subject and/or to
inhibit spread of the target pathogen from the infected subject to
another subject, e.g., an infected or non-infected subject. The
effective amount can be an amount sufficient to trap an amount of
the target pathogen in mucus or another substrate. As will be
recognized by one of skill in the art, the amount can vary
depending on the patient and the target pathogen. The exact amount
that is required will vary from subject to subject, depending on
the species, age, and general condition of the subject, the
particular carrier or adjuvant being used, mode of administration,
and the like. As such, the effective amount will vary based on the
particular circumstances, and an appropriate effective amount can
be determined in a particular case by one of skill in the art using
only routine experimentation. In some instances, an effective
amount of the crosslinker that specifically binds the target
pathogen or sperm can be an amount that achieves a concentration of
the crosslinker in the mucus of about 0.1 .mu.g/mL to about 1000
.mu.g/mL, e.g., about 0.5 .mu.g/mL to about 100 .mu.g/mL, e.g.,
about 1 .mu.g/mL to about 50 .mu.g/mL or any range therein. In some
embodiments, the crosslinker may be administered in two or more
stages with different doses in each stage. For example, higher
doses can be administered initially in order to clear target
pathogens that are present in the mucus of exposed or infected
subjects and ensure that sufficient amounts of crosslinker remain
in the mucus to provide protection, e.g., for about 24 hours. In
later stages, lower doses can be administered to maintain
protective levels of the crosslinker. In other embodiments,
protective doses can be administered to subjects that are likely to
be exposed to a pathogen and higher doses can be administered if
infection occurs.
[0206] As will be recognized by one of skill in the art, the term
"inhibiting" or "inhibition" does not refer to the ability to
completely eliminate the possibility of infection in all cases.
Rather, the skilled artisan will understand that the term
"inhibiting" refers to reducing the chances of pathogens moving
through mucus beyond the mucus membrane such that infection of a
subject can occur, such as reducing chances of infection by a
pathogen when such pathogen is bound to trapping crosslinkers in
mucus. Such decrease in infection potential can be determined
relative to a control that has not been administered the
crosslinkers of the invention. In some embodiments, the decrease of
inhibition relative to a control can be about a 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease.
[0207] In some embodiments inhibiting or treating an infection in a
subject can comprise trapping a pathogen in mucus or another
substrate. As such, in some embodiments a method of trapping a
target pathogen in mucus is provided, which method includes
administering to a mucosa of the subject a crosslinker or
composition as described herein.
[0208] In some embodiments, a method of inhibiting or treating an
infection in a subject, and/or trapping a pathogen in the mucus of
a subject, involves administering to a mucosa of the subject a
composition comprising an isolated antibody that specifically binds
a non-neutralizing epitope of a target pathogen. The antibody can
be a non-neutralizing antibody. In some embodiments, the
non-neutralizing antibody is provided at a concentration above a
predetermined amount.
[0209] In some embodiments, a method of inhibiting or treating an
infection in a subject, and/or trapping a pathogen in the mucus of
a subject, involves administering to a mucosa of the subject a
composition comprising an isolated antibody that specifically binds
a neutralizing epitope of a target pathogen, wherein the antibody
is provided at a sub-neutralization dose.
[0210] As used herein, the term "subject" refers to humans and
other animals. Thus, veterinary treatment is provided in accordance
with the presently-disclosed subject matter. As such, the
presently-disclosed subject matter provides for the treatment of
mammals such as humans, as well as those mammals of importance due
to being endangered, such as Siberian tigers; of economic
importance, such as animals raised on farms; and/or animals of
social importance to humans, such as animals kept as pets or in
zoos. Examples of such animals include but are not limited to:
carnivores such as cats and dogs; swine, including pigs, hogs, and
wild boars; ruminants and/or ungulates such as cattle, oxen, sheep,
giraffes, deer, goats, bison, and camels; and horses. Thus, also
provided is the treatment of livestock, including, but not limited
to, domesticated swine, ruminants, ungulates, horses (including
race horses), poultry, and the like.
[0211] A subject in need of inhibiting an infection or a disease or
disorder caused by an infection is a subject that has been
identified as being at risk of infection. In some embodiments, the
subject is identified as having been exposed to the target
pathogen. In other embodiments, the subject is in contact with
other subjects that are infected or are likely to become infected,
e.g., a subject that is living with, working with, and/or attending
school with infected subjects.
[0212] A subject in need of treating an infection or a disease or
disorder caused by an infection is a subject that has been
diagnosed as infected with a pathogen or is suspected of being
infected with a pathogen, e.g., exhibiting symptoms of
infection.
[0213] In some embodiments, the subject is one that does not have
antibodies targeted to the pathogen or has antibodies targeted to
the pathogen that are not effective to inhibit or treat infection
by the pathogen.
[0214] In particular embodiments of the invention, more than one
administration (e.g., two, three, four, or more administrations) of
the crosslinker, composition, or device comprising the composition
can be employed over a variety of time intervals (e.g., hourly,
daily, weekly, monthly, etc.) to achieve prophylactic and/or
therapeutic effects.
[0215] In some embodiments, the method include administering a
crosslinker, and further administering an additional active agent,
e.g., prophylactic or therapeutic agent, e.g., an antimicrobial,
such as an antibiotic, an antiviral, or other antimicrobial as
would be known to one of skill in the art. The additional active
agents can be delivered concurrently with the crosslinkers and
compositions of the invention. The additional active agents can be
delivered in the same composition as the crosslinker or in separate
compositions. As used herein, the word "concurrently" means
sufficiently close in time to produce a combined effect (that is,
concurrently can be simultaneously, or it can be two or more events
occurring within a short time period before or after each
other).
[0216] As used herein, the term foreign substance means any
substance that can cause an infection. The foreign substance
includes, but is not limited to, a pathogen, a bacterium, a virus,
a foreign particle or particulate, a cell, e.g., a sperm cell, a
microorganism, a germ, a protozoa, an infective agent or an
infectious agent.
[0217] In some embodiments, the disclosure provides a method for
improving or enhancing the barrier property of hydrogels such as
biological hydrogels against a foreign substance (e.g., a
pathogen). The method includes/comprises contacting the foreign
substance (e.g., the pathogen) with an antibody in an amount
effective to immobilize the foreign substance (e.g., the pathogen),
wherein the antibody has a binding affinity K.sub.D less than about
10.sup.-2 M with a constituent of the hydrogels. For example, the
antibody's binding affinity with a constituent of the hydrogels can
be less than about 5.times.10.sup.-3 M, less than about 10.sup.-3
M, less than about 5.times.10.sup.-4 M, less than about 10.sup.-4
M, less than about 5.times.10.sup.-5 M, less than about 10.sup.-5
M, less than about 5.times.10.sup.-6 M, less than about 10.sup.-6
M, less than about 5.times.10.sup.-7 M, less than about 10.sup.-7
M, less than about 10.sup.-8 M, less than about 5.times.10.sup.-9
M, or less than about 10.sup.-9 M, or any range therein. In some
embodiments, the antibody's binding affinity with a constituent of
the hydrogels can be between about 10.sup.-9 [M] and about
10.sup.-7 [M], between about 10.sup.-9 [M] and about 10.sup.-8 [M],
between about 10.sup.-8 [M] and about 10.sup.-7 [M], between about
7.times.10.sup.-9 [M] and about 2.times.10.sup.-7 [M], between
about 3.times.10.sup.-9 [M] and about 2.times.10.sup.-8 [M],
between about 2.times.10.sup.-8 [M] and about 2.times.10.sup.-7
[M], between about 3.times.10.sup.-9 [M] and about
7.times.10.sup.-9 [M], or between about 10.sup.-9 M and 10.sup.-2
M. The antibody can be a monoclonal or polyclonal antibody.
Examples of the antibodies can be used include, but are not limited
to, IgG, IgA, IgM, or a fragment or derivative thereof. In some
instances, antibody can be IgG or IgM. When an IgG or IgM antibody
is used, it can have a binding affinity K.sub.D of less than about
2.times.10.sup.-7 [M], e.g., less than about 10.sup.-7 [M],
9.times.10.sup.-8 [M], 8.times.10.sup.-8 [M], 7.times.10.sup.-8,
6.times.10.sup.-8 [M], 5.times.10.sup.8 [M], 4.times.10.sup.-8 [M],
3.times.10.sup.-8 [M], 2.times.10.sup.-8 [M], 10.sup.-8 [M],
9.times.10.sup.-9 [M], 8.times.10.sup.-9 [M], 7.times.10.sup.-9
[M], 6.times.10.sup.-9 [M], 5.times.10.sup.-9 [M],
4.times.10.sup.-9 [M], 3.times.10.sup.-9 [M], 2.times.10.sup.-9
[M], or 10.sup.-9 [M], or any range therein.
[0218] In some embodiments, the antibody has a rate of binding to
pathogen greater than about 10.sup.4 M.sup.-1s.sup.-1, e.g.,
greater than about 2.5.times.10.sup.4 M.sup.-1s.sup.-1,
5.times.10.sup.4 M.sup.-1s.sup.-1, 1.times.10.sup.5
M.sup.-1s.sup.-1, 2.5.times.10.sup.5M.sup.-1s.sup.-1,
5.times.10.sup.5 M.sup.-1s.sup.-1, 1.times.10.sup.6
M.sup.-1s.sup.-1, 2.5.times.10.sup.6M.sup.-1s.sup.-1,
5.times.10.sup.6 M.sup.-1s.sup.-1, or 1.times.10.sup.7
M.sup.-1s.sup.-1. The pathogen can be a virus, a bacterium or a
microorganism.
[0219] The constituents of the biological hydrogels can include
collagen, laminin, actin, fibronectin, entactin and a combination
thereof.
[0220] In some embodiments, the antibody binds to a
non-neutralizing epitope on the pathogen. In other embodiments, the
antibody binds to a neutralizing epitope on the pathogen.
[0221] In some embodiments, the disclosure provides a method for
improving or enhancing the barrier property of extracellular
matrices, such as biological hydrogels against a foreign substance
(e.g., a pathogen) in a subject. The method includes/comprises
administering to the subject an effective amount of an IgG or IgM
antibody to immobilize the foreign substance such as the pathogen,
wherein the antibody has a binding affinity less than about
10.sup.-2 M (e.g., from about 10.sup.-9 M to about 10.sup.-7 M)
with a constituent of the biological hydrogels.
[0222] In some embodiments, the disclosure provides a method for
tuning the barrier property of biological hydrogels against a
foreign substance (e.g., a pathogen) in a subject. The method
includes/comprises administering to the subject an effective amount
of an IgG or IgM antibody to immobilize the foreign substance such
as the pathogen, wherein the antibody has a binding affinity less
than about 10.sup.-2 M (e.g., from about 10.sup.-9 M to about
10.sup.-7 M) with a constituent of the biological hydrogels.
[0223] In some embodiments, the disclosure provides a method for
trapping a foreign substance (e.g., a pathogen) in biological
hydrogels. The method includes contacting the pathogen with an IgG
or IgM antibody in an amount effective to trap the pathogen,
wherein the antibody has a binding affinity less than about
10.sup.-2 M (e.g., between about 10-9 N and about 10.sup.-7 M) with
a constituent of the biological hydrogels.
[0224] In some embodiments, the disclosure provides a method for
preventing or treating an infection in a subject, wherein the
infection is caused by a foreign substance (e.g., a pathogen) in an
extracellular matrix of a basement membrane. The method
includes/comprises administering to the subject in need thereof an
effective amount of an IgG or IgM antibody, wherein the antibody
has a binding affinity less than about 10.sup.-2 M (e.g., from
about 10.sup.-9 M to about 10.sup.-7 M) with a constituent of the
extracellular matrix. The extracellular matrix can be a biological
hydrogel. The basement membrane can be an epithelium, such as
respiratory tract or gastrointestinal tract.
[0225] In some embodiments, the Fc region of the antibodies
described herein can bind to one or more constituents of the
biological hydrogel and the Fab region of the antibodies described
herein can bind to the surface of the pathogens.
[0226] In some embodiments, the disclosure provides an
extracellular matrix complex or mixture, which includes/comprises a
biological hydrogel, a plurality of IgG antibodies or IgM
antibodies, a plurality of immobilized pathogens, wherein the Fc
region of the plurality of IgG antibodies or IgM antibodies binds
to a constituent of the biological hydrogel and the Fab region of
the plurality of IgG antibodies or IgM antibodies binds to the
surface of the plurality of pathogens to immobilize the plurality
of the pathogens, and wherein the plurality of IgG antibodies or
IgM antibodies has a rate of binding to the plurality of pathogen
greater than 10.sup.4 M.sup.-1s.sup.-1 and a binding affinity less
than about 10.sup.-2 M (e.g., from about 10.sup.-9 M to about
10.sup.-7 M) with the constituent of the biological hydrogel.
[0227] In some embodiments, the antibody can be formulated into a
pharmaceutical composition suitable for intranasal, oral,
intravaginal, by inhalation, or topical administration. The
composition can be administered or delivered to a mucosal surface,
a respiratory tract, a gastrointestinal tract or a skin
surface.
[0228] In some embodiments, the disclosure provides a method for
preventing or treating an infection in a subject, wherein the
infection is caused by a foreign substance. The method includes
applying a hydrogel to the subject and administering to the subject
an effective amount of an IgG or IgM antibody, wherein the antibody
has a binding affinity of less than about 10.sup.-2 M with a
constituent of the hydrogel.
V. Contraception
[0229] One aspect of the invention relates to methods of
contraception. One embodiment relates to a method of providing
contraception, e.g., inhibiting fertilization, in a female subject,
comprising administering to the mucosa of the reproductive tract,
e.g., the vaginal and/or cervical mucosa, of the subject a
crosslinker or composition of the invention in an amount effective
to inhibit sperm-egg fertilization, and thus prevent pregnancy. In
an additional embodiment, the method can be practiced on a male
subject to trap sperm in the epydidimal mucus. In certain
embodiments, the methods comprise additional steps such as one or
more of isolating/preparing the crosslinkers, preparing a
composition of the crosslinkers, determining the level of
antibodies in the mucus of the subject before administering the
crosslinkers, and determining the level of crosslinkers in the
mucus of the subject after administering the crosslinkers.
[0230] The crosslinkers and compositions of the present invention
according to the methods described herein are administered or
otherwise applied by delivering the composition, typically to the
reproductive tract. In some embodiments, the crosslinkers and
compositions may be topically delivered. In other embodiments, the
crosslinkers and compositions may be systemically delivered such
that the crosslinkers are secreted into the mucus of the subject.
Accordingly, the compositions as described above may be delivered
to a mucosal surface of the reproductive tract, e.g., to the vulva,
including the vaginal cavity. In the vaginal cavity, the
compositions may be applied to any portion of the uterus, including
inside the uterus and on the cervix, including the mucosa and/or
lining of the endo- and ecto-cervix.
[0231] An effective amount of the crosslinker can be administered.
As used herein, an "effective amount" of the crosslinker for
contraception refers to a dosage sufficient to inhibit sperm from
contacting an egg and fertilizing it. The effective amount can be
an amount sufficient to trap an amount of the sperm in mucus. As
will be recognized by one of skill in the art, the amount can vary
depending on the subject. The exact amount that is required will
vary from subject to subject, depending on the species, age, and
general condition of the subject, the particular carrier or
adjuvant being used, mode of administration, and the like. As such,
the effective amount will vary based on the particular
circumstances, and an appropriate effective amount can be
determined in a particular case by one of skill in the art using
only routine experimentation. In some instances, an effective
amount of crosslinker can be an amount that achieves a
concentration of crosslinker in the mucus of about 0.1 .mu.g/mL to
about 1000 .mu.g/mL, e.g., about 0.5 .mu.g/mL to about 100
.mu.g/mL, e.g., about 1 .mu.g/mL to about 50 .mu.g/mL or any range
therein.
[0232] As will be recognized by one of skill in the art, the term
"inhibiting" or "inhibition" does not refer to the ability to
completely eliminate the possibility of fertilization in all cases.
Rather, the skilled artisan will understand that the term
"inhibiting" refers to reducing the chances of sperm moving through
mucus beyond the mucus membrane such that fertilization of a
subject can occur, such as reducing chances of fertilization by a
sperm when such sperm is bound to trapping crosslinkers in mucus.
Such decrease in fertilization potential can be determined relative
to a control that has not been administered the crosslinkers of the
invention. In some embodiments, the decrease of fertilization
relative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, or 100% decrease.
[0233] In some embodiments providing contraception in a subject can
comprise trapping sperm in mucus. As such, in some embodiments a
method of trapping sperm in mucus is provided, which method
includes administering to a mucosa in the reproductive tract of the
subject a crosslinker or composition as described herein.
[0234] As used herein, the term "subject" refers to humans and
other animals. Thus, veterinary treatment is provided in accordance
with the presently-disclosed subject matter. As such, the
presently-disclosed subject matter provides for the treatment of
mammals such as humans, as well as those mammals of importance due
to being endangered, such as Siberian tigers; of economic
importance, such as animals raised on farms; and/or animals of
social importance to humans, such as animals kept as pets or in
zoos. Examples of such animals include but are not limited to:
carnivores such as cats and dogs; swine, including pigs, hogs, and
wild boars; ruminants and/or ungulates such as cattle, oxen, sheep,
giraffes, deer, goats, bison, and camels; and horses. Thus, also
provided is the treatment of livestock, including, but not limited
to, domesticated swine, ruminants, ungulates, horses (including
race horses), poultry, and the like.
[0235] In particular embodiments of the invention, more than one
administration (e.g., two, three, four, or more administrations) of
the crosslinker, composition, or device comprising the composition
can be employed over a variety of time intervals (e.g., hourly,
daily, weekly, monthly, etc.) to achieve prophylactic/contraceptive
effects. In some embodiments, the crosslinker, composition, or
device is administered regularly or constantly. In other
embodiments, the crosslinker, composition, or device is
administered on an as needed basis, e.g., prior to sexual activity
and/or following sexual activity.
[0236] In some embodiments, the method include administering an
crosslinker, and further administering an additional active agent,
e.g., prophylactic or therapeutic agent, e.g., a sperm-function
inhibitor and/or an antimicrobial, such as an antibiotic, an
antiviral, or other antimicrobial as would be known to one of skill
in the art. The additional active agents can be delivered
concurrently with the crosslinkers and compositions of the
invention. The additional active agents can be delivered in the
same composition as the crosslinker or in separate compositions. As
used herein, the word "concurrently" means sufficiently close in
time to produce a combined effect (that is, concurrently can be
simultaneously, or it can be two or more events occurring within a
short time period before or after each other).
VI. In Vitro Uses
[0237] Having demonstrated the usefulness of the crosslinkers of
the invention in trapping a target of interest on a substrate of
interest, another aspect of the invention relates to in vitro uses
of the crosslinkers to manipulate a target of interest. The
crosslinkers may be used in any method or technique where
noncovalent binding of a target to a substrate is desired. Examples
include, without limitation, methods of separating, purifying,
washing, filtering, or otherwise manipulating a target by using a
crosslinker to trap the target on a substrate, which may be, e.g.,
part of a chromatography, dialysis, or filtration medium. In some
embodiments, the substrate of interest may be contacted with the
crosslinker and then a composition comprising the target of
interest may be added to the substrate, e.g., as part of column
chromatography or patch chromatography.
[0238] In some embodiments, the crosslinker of the invention may be
used in a method or assay in which it is desirable to trap a target
of interest on a substrate of interest. Examples include, without
limitation, purification methods, filtration methods, separation
methods, immunoassays and other detection assays.
VII. Computer Programs and Systems
[0239] The present invention also provides a computer program
product comprising: a computer readable storage medium having
computer readable code embodied in the medium, the computer code
comprising: computer readable code to perform operations to carry
out the methods of this invention.
[0240] Further provided herein is a computer system, comprising: a
processor; and a memory coupled to the processor, the memory
comprising computer readable program code embodied therein that,
when executed by the processor, causes the processor to perform
operations to carry out the methods of this invention.
[0241] As noted above, a kit of this invention can comprise
electronic hardware components. In some embodiments of this
invention, the electronic hardware may perform and/or support
functionality that corresponding to various operations described
herein. For example, functions described and/or illustrated in
diagrams and/or flowchart illustrations of methods, apparatus
(systems) and/or computer program products according to some
embodiments may be performed by the electronic hardware. It is
understood that each block of the block diagrams and/or flowchart
illustrations, and combinations of blocks in the block diagrams
and/or flowchart illustrations, can be implemented by computer
program instructions. These computer program instructions may be
provided to a processor of a general purpose computer, special
purpose computer, and/or other programmable data processing
apparatus to produce a machine, such that the instructions, which
execute via the processor of the computer and/or other programmable
data processing apparatus, create means for implementing the
functions/acts specified in the block diagrams and/or flowchart
block or blocks.
[0242] These computer program instructions may also be stored in a
computer-readable memory that can direct a computer or other
programmable data processing apparatus to function in a particular
manner, such that the instructions stored in the computer-readable
memory produce an article of manufacture including instructions
which implement the function/act specified in the block diagrams
and/or flowchart block or blocks.
[0243] The computer program instructions may also be loaded onto a
computer or other programmable data processing apparatus to cause a
series of operational steps to be performed on the computer or
other programmable apparatus to produce a computer-implemented
process such that the instructions which execute on the computer or
other programmable apparatus provide steps for implementing the
functions/acts specified in the block diagrams and/or flowchart
block or blocks.
[0244] Accordingly, the present invention may be embodied in
hardware and/or in software (including firmware, resident software,
micro-code, etc.). Furthermore, embodiments of the present
invention may take the form of a computer program product on a
computer-usable or computer-readable non-transient storage medium
having computer-usable or computer-readable program code embodied
in the medium for use by or in connection with an instruction
execution system.
[0245] The computer-usable or computer-readable medium may be, for
example but not limited to, an electronic, optical,
electromagnetic, infrared, or semiconductor system, apparatus, or
device. More specific examples (a non-exhaustive list) of the
computer-readable medium would include the following: an electrical
connection having one or more wires, a portable computer diskette,
a random access memory (RAM), a read-only memory (ROM), an erasable
programmable read-only memory (EPROM or Flash memory), an optical
fiber, and a portable compact disc read-only memory (CD-ROM).
[0246] Computer program code for carrying out operations for
aspects of the present disclosure may be written in any combination
of one or more programming languages, including an object oriented
programming language such as Java, Scala, Smalltalk, Eiffel, JADE,
Emerald, C++, C#, VB.NET, Python or the like, conventional
procedural programming languages, such as the "C" programming
language, Visual Basic, Fortran 2003, Perl, COBOL 2002, PHP, ABAP,
dynamic programming languages such as Python, Ruby and Groovy, or
other programming languages. The program code may execute entirely
on the user's computer, partly on the user's computer, as a
stand-alone software package, partly on the user's computer and
partly on a remote computer or entirely on the remote computer or
server. In the latter scenario, the remote computer may be
connected to the user's computer through any type of network,
including a local area network (LAN) or a wide area network (WAN),
or the connection may be made to an external computer (for example,
through the Internet using an Internet Service Provider) or in a
cloud computer environment or offered as a service such as a
Software as a Service (SaaS).
[0247] Having described the present invention, the same will be
explained in greater detail in the following examples, which are
included herein for illustration purposes only, and which are not
intended to be limiting to the invention.
Example 1
Mathematical Modeling
Materials and Methods
[0248] Similar to previous studies (McKinley et al., PLoS One
9:e100598 (2014); Chen et al., Biophys. J. 106:2028 (2014);
Geonnotti et al., Biophys. J. 91:2121 (2006); Lai et al., Biophys.
J. 97:2379 (2009)), we modeled the diffusion of HIV from a
virion-rich (8.4.times.10.sup.5 virions, the average viremia in
semen of acutely infected males (McKinley et al., PLoS One
9:e100598 (2014); Gupta et al., J. Virol. 71:6271 (1997))) semen
layer (volume .about.3.0 mL (Rehan et al., Fertil. Steril. 26:492
(1975))) uniformly deposited on top of a cervicovaginal mucus layer
(CVM) that evenly covers the entire vaginal epithelial cell layer.
This results in a thickness of d=200 .mu.m and L=50 .mu.m for the
semen and CVM layers, respectively. Broadly neutralizing Ab
accumulate on HIV virions at rates depending on Ab-antigen
affinity, the number of available antigen sites on the virus
surface, and the local Ab concentration. For a model monoclonal
broadly neutralizing Ab against HIV, we focused on NIH45-46, which
binds to the CD4 binding site of gp120 and whose binding affinities
were previously described (Scheid et al., Science 333:1633 (2011)).
The number of Env spikes N* on individual HIV virions is variable,
and was estimated to follow a negative binomial distribution with
N*=14.+-.7 (range 4-35) based on cryo electron microscopy of HIV
virions (Zhu et al., Nature 441:847 (2006). We assume each Env
spike can bind up to 3 Ab without significant steric hindrance;
thus, individual Ab at concentration u(z,t) can bind and unbind
independently with rates k.sub.on and k.sub.off, and overall
binding/unbinding rates dependent on the number of unoccupied
binding sites 3N*-n. However, since the diffusivity for a
mucin-bound Ab u.sub.b(z,t) is reduced compared to free individual
Ab, the Smoluchowski encounter rate implies that the binding rate
for a mucin-bound Ab (k'.sub.on) to its antigen should be reduced
proportional to the difference in diffusivities of the Ab
(D.sub.Ab) and the virus (D.sub.v); hence,
k on ' = D v D Ab + D v k on .apprxeq. 1 2 5 k on .
##EQU00002##
The Ab-virion binding rate equations can be summarized as:
( Ab ) + ( Ab ) n Z ( t ) ( 3 N * - n ) ( k on f u f ( Z ( t ) , t
) + k on b u b ( Z ( t ) , t ) ) ( n + 1 ) k off ( Ab ) n + 1 Z ( t
) ( 1 ) ##EQU00003##
where (Ab) is an unbound Ab and (Ab).sup.nZ(t) denotes a virion at
Z(t) with n bound Ab. Individual Ab also bind and unbind to mucins
at rates m.sub.on[M] and m.sub.off, where [M] is the effective
concentration of Ab binding sites in the mucin network. In
addition, virion-bound Ab may also associate with mucins,
effectively immobilizing the entire Ab-virion complex for the
duration of the interaction.
[0249] We developed two different methods to simulate HIV
penetration across the vaginal mucosa. In the first method, a
stochastic particle simulation was used for virion diffusion,
virion-Ab binding, and Ab-mucin interactions. Virion diffusion for
a particle Z is given by the stochastic differential equation
(SDE)
dZ= {square root over (2D)}dW,
where W is Brownian motion and
D = { D HIV , free 0 , bound , ##EQU00004##
where "free" indicates that all virion-bound Ab are free from
mucin, and "bound" indicates that at least 1 virion-bound Ab is
associated to mucin. Ab binding and unbinding to virus is simulated
with a Poisson random variable with rates given by (1). Lastly,
Ab-mucin interactions were simulated with Poisson random variables
and rates dependent on the total number of virion-bound Ab and
those Ab currently interacting with mucins. Due to the great
computational expense involved in simulating Ab-mucin interactions
particularly when m.sub.on[M]>m.sub.off, we precomputed lookup
tables giving the distributions for the time that a virion spends
freely diffusing and the time that a virion spends interacting with
mucins. The (random) time that a virion spends freely diffusing is
given by the first time that at least one of its associated Ab
binds to mucin. Similarly, the time that a virion spends
interacting with mucins is given by the time that at least one of
the virion-bound Ab associates with mucin until the first time that
none of the surface bound Ab associates with mucin. We then sample
from these lookup tables whenever a virion's state changes (freely
diffusing or bound to mucin, binding or unbinding an Ab, crossing
the semen/CVM interface).
[0250] The second simulation method consists of a
reaction-diffusion partial differential equation (PDE) to capture
the average behavior of the virus population. The virus population
is represented by a vector {right arrow over (V)}(z,t), where the
component V.sub.n(z,t) represents the concentration of virus with n
bound Ab. We previously introduced a parameter
.alpha. = m off m off + [ M ] m on ##EQU00005##
to represent the fraction of time that Ab in CVM spend freely
diffusing [21]. Since Ab-mucin interactions likely occur at fast
time-scales
u f ( z , t ) = { .alpha. u ( z , t ) , 0 < d < 50 u ( z , t
) , 200 < d < 250 , and u b ( z , t ) = u ( z , t ) - u f ( z
, t ) . ##EQU00006##
[0251] In the limit m.sub.on,
m off .fwdarw. .infin. ( keeping = m off m off + [ M ] m on
##EQU00007##
fixed), the stochastic model can thus be approximated by the
reaction-diffusion system:
.differential. V .fwdarw. .differential. t = D .differential. 2 V
.fwdarw. .differential. z 2 - f .fwdarw. ( V .fwdarw. ( z , t ) ) +
g .fwdarw. ( V .fwdarw. ( z , t ) ) , .differential. u
.differential. t = D Ab 0 .differential. 2 u .differential. z 2 ,
##EQU00008##
where the diffusion tensor D, is a diagonal tensor with entries
D.sub.v.sub.0, .beta..sub.1D.sub.v.sub.0, . . . ,
.beta..sub.3N*D.sub.v.sub.0 along the diagonal (the diffusion
factors .alpha..sub.i are determined below) for 0<d<50 and
diagonal entries all D.sub.v.sub.0 for 50<d<250; and
D A b = { .alpha. D Ab 0 , 0 < d < 50 D Ab 0 , 50 < d <
250 . ##EQU00009##
[0252] We use a FTCS algorithm to model diffusion for both the
virus and Ab populations, with reflecting boundary conditions at
the semen/lumen interface; reflecting conditions for Ab and
absorbing conditions for virus at the CVM/cell interface; and
Fick's law for the discontinuous diffusion coefficients at the
semen/CVM interface. We assume that the number of Ab binding to
virions is negligible compared to the overall Ab population, so
there are no local depletion effects.
[0253] If each virion-bound IgG binds and unbinds to the mucin mesh
independently, then the virion spends approximately a fraction
.alpha..sup.n of time with all its bound Ab simultaneously free
from mucin. This yields a time-averaged diffusivity
D.sub.vn=.alpha..sup.nD.sub.v0 in the CVM. One adjustment is made,
however, to account for the lower diffusivity of a virion-Ab
complex compared with an individual Ab. Similar to the adjustments
on k.sub.on, define m.sub.first= 1/30, so that initial Ab-mucin
encounters occur at rate m.sup.firstm.sub.on[M]. For subsequent
interactions of virion-bound Ab with mucin, the rate may increase
because the Ab-virus complex is already located in close proximity
to a mucin molecule, or may be decreased due to the reduced
diffusivity of the Ab-virus complex associated to mucins relative
to a non-associated Ab-virus complex. Due to the lack of empirical
data in the literature, we assumed other Ab-bound to the same
virion with at least one Ab already associating with mucins will
associate with mucins with the same mucin-binding kinetics as a
free Ab molecule. In order to calculate the diffusion factors
.beta..sub.1, .beta..sub.2, . . . .beta..sub.3N, first neglect the
factor m.sup.first and consider the total time free.sub.i that a
virion with i bound Ab spends freely diffusing and the total time
bound that it spends bound to mucins up to a time T. Since the Ab
are assumed to bind and unbind to mucin independently,
lim T .fwdarw. .infin. free i free i + bound i = .alpha. i .
##EQU00010##
Now
[0254] .beta. i = m first free i m first free i + bound i = m first
free i / ( free i + bound i ) ( m first - 1 ) free i / ( free i +
bound i ) + 1 = m first .alpha. i ( m first - 1 ) .alpha. i + 1 .
##EQU00011##
[0255] The stochastic simulations and the reaction-diffusion
simulations show excellent agreement, particularly at rapid
m.sub.on[M] and m.sub.off (FIG. 7). Hence we utilized the
deterministic simulations except where noted.
[0256] Determining the number of Ab required to neutralize a given
HIV remains an active area of research, due to the difficulty in
simultaneously distinguishing the number of Ab necessary to
neutralize a particular Env spike and the minimum number of Ab-free
Env spike necessary for HIV to successfully infect (Magnus et al.,
PLoS Comput Biol 6:e1000713 (2010)). It was previously proposed
that the binding of a single Ab molecule to an Env spike appear to
be sufficient to inactivate the infectivity associated with that
spike (Yang et al., J. Virol. 79:3500 (2005)). The minimum number
of Ab-free Env spikes, and consequently the number of Env spikes
that must be inactivated to neutralize a virion, remain more
controversial. Estimates for minimum infectivity ranged from a
single Ab-free Env spike (Yang et al., J. Virol. 79:3500 (2005)) to
many (Schonning et al., J. Virol. 73:8364 (1999); McLain et al., J.
Gen. Virol. 75 (Pt 6):1457 (1994)). For our current model, we
assume that each additional Ab binding to a previously unoccupied
Env incrementally reduces the likelihood of infection, and we
measure the overall reduction in infectivity by the reduction in
number of unoccupied Env arriving at the vaginal epithelium over
the first two hours post ejaculation.
Results
[0257] Incorporating Mucin-Binding Kinetics into Previous Models
for HIV Penetration of Ab-Laden CVM:
[0258] We have previously modeled the diffusion of HIV through CVM
by combining a stochastic/deterministic hybrid model for the one
dimensional Brownian movement of individual HIV virions together
with a continuum model that describes the average local
concentration of broadly neutralizing monoclonal IgG in CVM (Chen
et al., Biophys. J. 106:2028 (2014)). That model allowed us to show
that a multitude of weak bonds between virion-bound IgG and mucins
alone, defined by the ratios of IgG diffusion in mucus vs. buffer
(a) in the range of 0.8-0.9, is sufficient to immobilize the vast
majority of HIV near the semenCVM interface. Nevertheless, to
further explore the IgG trapping potency across the full range of
IgG-nucin affinity, it was necessary to incorporate additional
complexity into the model. First, when modeling IgG that binds more
tightly to mucins, we made the assumption that the probability of a
successful bond between an IgG and the corresponding viral antigen
is directly proportional to the overall collision frequencies
between the two bodies, which can be described by the classical
Smoluchowski principle (Lai et al., Proc. Natl. Acad. Sci. USA
107:598 (2010)). Since an IgG bound to mucins will process far
reduced range of motion than a free, unbound IgG molecule, the
bound IgG should process a reduced k.sub.on rate, denoted as
k.sub.on', proportional to the reduction in collision frequency
with viral antigen, which in turn can be approximated by the ratios
of the diffusivity of IgG vs. mucins in CVM. While the diffusivity
of individual mucins in CVM remains unknown, we have previously
shown that CVM is composed of heavily bundled mucins that likely
reflect exceedingly limited range of motion for individual mucins
(Lai et al., Proc. Natl. Acad. Sci. USA 107:598 (2010)). We thus
made a very conservative estimate that an IgG bound to mucin will
possess a 25-fold reduced k.sub.on rate compared to individual free
IgG (i.e. k.sub.on'=k.sub.on/25), which roughly equates to assuming
the range of motion of mucins to match that for individual HIV
virions. While obviously an over-conservative assumption, further
reduction in k.sub.on' does not meaningfully impact estimates
generated by our model.
[0259] A second important detail we incorporated into our model is
the kinetics of IgG binding to and unbinding from mucins, which we
termed m.sub.on and m.sub.off, respectively. Experimentally,
m.sub.on and m.sub.off appears to be extremely transient and
difficult to measure individually (Saltzman et al., Biophys. J.
66:508 (1994); Olmsted et al., Biophys. J. 81:1930 (2001)).
Instead, IgG-mucin affinity is inherently reflected by the
diffusion coefficients of IgG in CVM vs. in buffer, which we denote
as .alpha.. .alpha. reflects the fraction of bound vs. unbound IgG
at any moment in time, and is equivalent to the ratio
m.sub.off/(m.sub.off+m.sub.on) at steady state. Assuming IgG
binding to its antigen does not increases its affinity to mucins,
the rates with which individual IgG can bind to mucins must be far
faster than the rate of virion-associated IgG binding to mucins. We
thus introduced a correction factor of .about.30 for the
mucin-association kinetics for virion-bound IgG, which is
equivalent to the difference in diffusivities of HIV vs. IgG in
CVM. This correction was necessary to ensure we do not overestimate
the trapping potency of viruses.
[0260] As a first step towards understanding how IgG-mucin affinity
can impact trapping potency, we modeled the probability and
duration of HIV-IgG complex associating to mucins in CVM containing
10 .mu.g/mL NIH45-46 with varying IgG-mucin affinity. Naturally,
HIV with surface IgG possessing no affinity to mucins, defined by
.alpha.=1, never binds to mucins, and the HIV-IgG complex undergoes
free diffusion the entire duration (FIG. 2A). When IgG-mucin
affinity is slightly increased such that individual IgG would
associate with mucins .about.5 and 10% of the time (i.e.
.alpha.=0.95 and 0.9, respectively), the fraction of time an
HIV-IgG complex spends freely diffusing in mucus begins to
decrease, with a corresponding increase in the fraction of time
spent associated with mucins (FIGS. 2B-2C). Interestingly, the
fraction of time the HIV-IgG complex associates with mucins appears
to peak between .alpha.=0.1 and 0.25 (FIGS. 2D-2F). This is
attributed to the fact that increased IgG-mucin affinity (i)
markedly reduces the fraction of NIH45-46 that can freely diffuse
and readily bind to HIV, especially in the semen layer far away
from the vaginal epithelium (FIG. 6), and (ii) that
mucin-associated IgG captures HIV with far lower efficiency (i.e.,
reduced k.sub.on' vs. k.sub.on). These two factors together
increases number of HIV with no bound IgG. With further increases
of mucin affinity to .alpha.=0.01, the amount of HIV free of bound
IgG dominates relative to HIV-IgG complexes, and most HT again
undergo Brownian motion in CVM (FIG. 2G).
[0261] Influence of IgG-Mucin Affinity on Maximizing Trapping
Potency and Vaginal Protection:
[0262] We next quantified how the probability of HIV-mucin
association impacts the fraction of HIV that can penetrate CVM and
reach the underlying vaginal epithelium, and the corresponding
reduction in infectivity based on the decrease in HIV-Env free of
bound IgG on those virions (McKinley et al., PLoS One 9:e100598
(2014)). In good agreement with the estimate of the fraction of
time spent associated with mucins, the maximum reduction in HIV
flux reaching the vaginal epithelium peaks at IgG-mucin affinities
corresponding to .alpha.=0.25 (FIG. 3A). At 5 and 10 .mu.g/mL of
NIH45-46 initially present in CVM and an IgG-mucin affinity
equivalent to .alpha.=0.25, only .about.3% and .about.0.3% of the
HIV viral load in semen is predicted to reach the vaginal
epithelium over the first 2 hrs post-ejaculation, equating to 10 to
100-fold reduced flux compared to the estimated .about.30% of HIV
load over the same duration in the absence of IgG-mucin affinity
(FIG. 3A). Under this scenario, each HIV virion on average
possesses .about.2 bound IgG (FIG. 3B), and the overall infectivity
is reduced by 86-94% (i.e. .about.7-16 fold) compared to IgG
without affinity to mucins (FIGS. 3C-3D). Note that the reduction
in infectivity is less than the reduction in flux of HIV viruses
since NIH45-46 without affinity to mucins can still neutralize the
virus.
[0263] When IgG-mucin affinity is further increased (i.e. lower a),
the fraction of HIV reaching the vaginal epithelium began to
increase. Furthermore, there is also substantially less number of
IgG bound to HIV-Env on virions that reach the vaginal epithelium.
Indeed, when a drops below 0.1, on average less than one IgG
molecule is bound on each virion over the entire population of HIV,
which implies that there must be HIV virions without any bound IgG
(FIG. 3B). As a result, infectivity of HIV may actually be greater
for hypothetical NIH45-46 that can bind tightly to mucins than if
NIH45-46 possessed no mucin-affinity at all (FIGS. 3C-3D).
[0264] To begin to understand how to engineer more potent
HIV-trapping IgG, we evaluated the relative impact of the rate of
IgG binding to the virus surface compared to IgG-mucin affinity.
The rate of IgG binding to HIV is the product of both the local IgG
concentration and k.sub.on, the binding kinetic constant: a
doubling of IgG k.sub.on has the same impact on IgG binding to HIV
as doubling the IgG concentration. The reduction of HIV flux
arriving at the vaginal epithelium, and the reduction in mean
number of NIH45-46-free Env proteins on HIV that reached the
vaginal epithelium, were sensitive to both IgG-antigen binding rate
and IgG-mucin affinity (FIGS. 4A-4D). When IgG-mucin affinity is
increased, the amount of initial NIH45-46 in CVM needed to reduce
the flux of viruses arriving the vaginal epithelium by 50% was
reduced from 5-35+.mu.g/mL for .alpha.=0.8-0.9 (in the range to
what was previously measured for IgG) to <1 .mu.g/mL when
.alpha.=0.25 (FIG. 4A). Similarly, the amount of NIH45-46 needed to
reduce the mean number of NIH45-46-free Env proteins on HIV by 80%
was reduced from 22-35+ .mu.g/mL for .alpha.=0.8-0.9 to 3 .mu.g/mL
for .alpha.=0.25 22-35+ .mu.g/mL to 3 .mu.g/mL (FIGS. 4C-4D).
Overall, the amount of NIH45-46 needed to reduce infectivity by 90%
was reduced from 5-6 .mu.g/mL when .alpha.=1 to 2 .mu.g/mL when
.alpha.=0.25 5-6 .mu.g/mL to 2 .mu.g/mL.
[0265] Influence of IgG-Antigen Binding Affinity on Maximizing
Trapping Potency and Vaginal Protection:
[0266] A longstanding assumption for neutralizing IgG against HIV
and other viruses is that higher binding affinity between IgG and
viral antigen would facilitate more potent protection. However, it
is important to note that high affinity IgGs are typically
identified and selected based on neutralization assays, typically
in the absence of mucus coatings and with some incubation time
between virus and IgG prior to exposure of the virus to target
cells. Thus, we sought to quantitatively evaluate whether high
antigen-IgG affinity that typically maximizes neutralization
potency in vitro would also be maximally protective against mucosal
HIV transmission in our model.
[0267] Interestingly, we found that the antigen-unbinding rate
k.sub.off generally possessed only a very minor effect on
increasing the fraction of HIV load that is trapped in mucus or
facilitate more effective protection, especially when the IgG
concentration or the k.sub.on rate is low (FIG. 5A). For example,
at 1 .mu.g/mL of IgG with k.sub.on of 1.5.times.10.sup.4
M.sup.-1s.sup.-1 (k.sub.on[Ab]=10.sup.-4 s.sup.-1), improvements of
k.sub.off from 10.sup.-3 s to 10.sup.-4 s.sup.-1 only reduced the
HIV flux arriving the vaginal epithelium by 1.6%, and a further
improvement from 10.sup.-4 s.sup.-1 to 10.sup.-5 s.sup.-1
essentially resulted in no appreciable difference in reduction of
flux (FIG. 5A). This is similarly reflected by the change in the
infectivity of the viruses relative to IgG with no mucin-affinity
from 100% with k.sub.off=10.sup.-3 s.sup.-1 to 96% with
k.sub.off=10.sup.-4 s.sup.-1 to finally 92% with
k.sub.off=10.sup.-5 s.sup.-1 (FIGS. 5C-5D). The lack of impact by
k.sub.off is directly attributed to the exceedingly limited number
of IgG that can accumulate on the surface of HIV when IgG is
present either at low to modest concentrations, or when IgG
possesses inadequate binding kinetics (FIG. 5B); slower unbinding
kinetics simply cannot enhance HIV trapping in mucus or
neutralization when few or no IgG is bound to HIV in the first
place.
[0268] A hallmark of HIV is its exceptionally high mutation rates,
which enables the virus to readily escape antibodies generated by
the immune system and prevent the host from mounting a protective
immune response. Comprehensive studies of elite controllers--the
rare individuals who can maintain undetectable viral load without
antiretroviral therapy--led to the discovery and cloning of
monoclonal antibodies that can broadly neutralize the vast majority
of HIV strains. These broadly neutralizing antibodies (bnAbs) were
thought to provide a template for the development of an HIV
vaccine. Unfortunately, HIV vaccine, including those that could
block vaginal transmission of HIV, remains elusive to date, likely
because of at least two reasons. First, bnAbs are typically highly
somatically mutated, and vaccines may not be able to elicit the
extent of somatic hypermutation needed in most individuals to
generate the desirable bnAbs. Second, many HIV vaccine candidates
are based on DNA or subunit proteins rather than attenuated virus;
the durability of antibody response from subunit vaccines are
generally shorter than vaccines based on attenuated virus, and the
level of antibody titers induced in the vagina may be inadequate to
block vaginal HIV transmission.
[0269] To overcome the challenges with HIV vaccines, a recently
emerged strategy is to enable passive immunization of the vagina
via sustained delivery of bnAbs locally (Whaley et al., J. Infect.
Dis. 210 Suppl 3:S674 (2014); Sherwood et al., Nat. Biotechnol.
14:468 (1996)). By dosing the bnAbs directly into the vagina, this
strategy not only bypasses the limitations of somatic
hypermutation, but also ensures protective levels of bnAbs are
present in the vagina to block HIV transmission. Despite these
important advantages, a critical shortcoming for passive
immunization is the relatively high costs of maintaining protective
levels of antibody in the body compared to vaccination. Much effort
has been spent on reducing the costs of antibody production, such
as the production of antibodies in plants (Ramessar et al., Proc.
Natl. Acad. Sci. USA 105:3727 (2008); Whaley et al., Hiatt A,
Zeitlin L (2011) Emerging antibody products and Nicotiana
manufacturing. Hum. Vaccin. 7:349 (2011)) as well as cheaper and
more efficient methods of purifying antibodies (Liu et al., MAbs
2:480 (2010); Zydney, Biotechnol. Bioeng. 113:465 (2016)). Here, we
introduce a novel and completely distinct approach based on tuning
IgG-mucin interactions--that could markedly reduce the dose of
bnAbs needed to block vaginal HIV transmission. The majority of
bnAbs against HIV appear to possess k.sub.on in the range of
10.sup.4 M.sup.s-1 that we previously estimated may require
concentrations in excess of 5-10 .mu.g/mL to facilitate effective
protection. Although we predict enhanced vaginal protection can be
accomplished with both increasing k.sub.on and optimizing IgG-mucin
affinity, the bnAbs generally bind to a very unique epitope on
HIV-Env that makes it unlikely to substantially improve the bnAbs
k.sub.on without compromising binding affinity (i.e. higher
k.sub.off). In contrast, simply by tuning the interactions between
IgG-Fc and mucins, we can potentially reduce dose of bnAb for
effective protection by 10-fold or more without jeopardizing the
broad antigen coverage of bnAb. Optimizing IgG-Fc interactions with
mucins thus offers a promising strategy to markedly reduce the
costs for effective passive immunization of the vagina. The
convergence of these various approaches may synergistically drive
down the costs and make passive immune protection against HIV cost
effective even in resource-poor settings.
[0270] Surprisingly, high affinity IgG-mucin interactions did not
appear to enhance protection. Instead, the ideal IgG-mucin affinity
that maximizes protection in our model (.alpha..about.0.25) is
comparable to the mucin-affinity previously measured for IgM
molecules (Saltzman et al., Biophys. J. 66:508 (1994); Olmsted et
al., Biophys. J. 81:1930 (2001)). It is also worth noting that IgM,
due to its pentameric structure, has 10 Fab arms compared to 2 Fab
arms for each IgG, and hence can bind to its antigenic target and
accumulate on the surface of virions with exceptional speed even if
each Fab possess relatively poor affinity compared to a fully
affinity-matured IgG. Thus, an IgM molecule appears to
simultaneously satisfy both design requirements we have identified
in this study--rapid k.sub.on and modest mucin affinity. IgM is the
first antibody isotype produced by our immune system and appears
early in the course of an infection. Virus-specific IgM also
usually reappear upon re-infection. While speculative, our study
raises the hypothesis that an evolved effector function of IgM is
to quickly begin purging a new pathogen from mucosal surfaces that
likely represents its first site of infection early in the course
of infection, and minimizing the viral titers that can enter the
systemic circulation.
[0271] As discussed above, it is unlikely that we can markedly
improve the k.sub.on for bnAbs without potentially compromising
their broad antigenic coverage. An alternative method to enhance
the overall rate of IgG accumulating on the viral surface at
mucosal secretions is to include IgGs targeting other viral
epitopes, including potentially non-neutralizing epitopes, since
trapping virions in mucus require only binding and not necessarily
neutralizing IgG. It is important to note that the immune system
typically generates a polyclonal Ab response against diverse
epitopes, rather than solely a neutralizing Ab response against a
single viral epitope. Indeed, many of the naturally produced IgG
against HI found in HIV patients associate with either the lipid
membrane of HIV virions, or other parts of the gp120 site on the
Env spike not directly involved in HIV infection of immune cells
(Santra et al., PLoS Pathog. 11:e1005042 (2015)). Likewise,
virtually all of the IgGs detected in the moderately successful
RV144 trial were non-neutralizing (Rerks-Ngarm et al., N. Engl. J
Med. 361:2209 (2009)). Such polyclonal response would likely result
in substantially faster rate of Ab accumulation than an individual
monoclonal IgG. Thus, co-delivery of multiple IgGs to enhance
passive immune protection of the vagina, or inclusion of multiple
immunogens (including non-neutralizing epitopes) in vaccine
formulations, would both be harnessing the same strategy our immune
system has evolved to fend off foreign pathogens.
[0272] Although often under-appreciated, CVM represents the first
line of defense against sexually transmitted infections in the
female reproductive tract. In addition to minimizing trauma to the
vaginal epithelium upon coital stirring, the presence of the CVM
layer also prevents virions in semen from immediately contacting
the vaginal epithelium upon ejaculation, and directly reduces the
virion flux and total viral load in semen that can reach target
cells over time. Reinforcing the CVM barrier against sexually
transmitted viral infections using virus-specific Ab that trap
viruses in mucus is likely an important mechanism of the vaginal
mucosal defense, but which is largely underappreciated and
continues to be underexplored. We expect the combination of
quantitative, predictive models with experimental validation will
enable development of improved passive immunization as well as
vaccination methods that harness the mucus barrier to reinforce the
mucosal defense against HIV and other sexually transmitted
infections.
Example 2
Anti-PEG Antibodies Alter the Mobility and Biodistribution of
Densely PEGylated Nanoparticles in Mucus
[0273] An emerging mechanism of mucosal immune protection,
pioneered by our group, is the immobilization of individual viruses
due to antibody-mucin interactions, which in turn prevents viral
translocation across mucus (Wang et al., Mucosal Immunol. 7:1036
(2014)). Previous work has shown that the interactions between
antibodies and mucus are low-affinity and transient; for example,
the diffusion of IgG and IgA molecules (diameter .about.10 nm) in
human mucus (pores .about.340.+-.50 nm (Lai et al., Proc. Nat.
Acad. Sci. USA 107:598 (2010))) is slowed only .about.5%-20%
compared to in buffer (Olmsted et al., Biophys. J. 81:1930 (2001);
Wang et al., Mucosal Immunol. 7:1036 (2014)). We found that this
seemingly negligible affinity is sufficient to trap HSV-1 virions
in human cervicovaginal mucus (CVM) with sub-neutralizing potency,
presumably because the array of virion-bound IgG ensures a
sufficient number of transient low-affinity bonds between the
virus/IgG complex and mucins at any given time (Wang et al.,
Mucosal Immunol. 7:1036 (2014)). Furthermore, a non-neutralizing
IgG facilitated effective protection against vaginal Herpes
infection in mice, but that protection was abolished when vaginal
mucus was removed. These results indicate that virus-binding
antibodies in mucus can directly alter the mobility of virions in
mucus, leading to a markedly reduced flux arriving at the
epithelium (Chen ei al., Biophys. J. 106:2028 (2014)). We thus
sought to evaluate here whether anti-PEG antibodies in mucus would
similarly trap PEGylated nanoparticles in mucus and impede
therapeutics delivery to mucosal surfaces.
Methods
[0274] Mouse Cervicovaginal Mucus Collection:
[0275] Mouse cervicovaginal mucus (mCVM) was obtained from 6-8 week
old CF-I mice (Harlan, Frederick, Md.), injected subcutaneously
with 2.5 mg of Depo-Provera.RTM. (DP; medroxyprogesterone acetate)
(Pharmacia & Upjohn Company, Kalamazoo, Mich.) seven days prior
to experiments. mCVM was collected by lavage with 20 .mu.L of
normal saline, and mCVM from 10-15 mice was pooled to collect
sufficient quantities for multiple particle tracking studies. The
collection procedure yields highly viscoelastic mucus, as observed
visually and through particle tracking experiments. Mucus was
stored at 4.degree. C. until used for microscopy within 48 hrs.
Mice were anesthetized prior to experimental procedures. All
experimental protocols were approved by the University of North
Carolina at Chapel Hill Institutional Animal Care and Use
Committee.
[0276] Nanoparticle Preparation and Characterization:
[0277] To produce PEGylated nanoparticles (PS-PEG), we covalently
modified 100 nm fluorescent, carboxyl-modified polystyrene beads
(PS; Molecular Probes, Eugene, Oreg.) with 2 kDa methoxy
poly(ethylene glycol) amine (PEG; Rapp Polymere, Tuebingen,
Germany) via a carboxyl-amine reaction, as published previously
(Lai et al., Proc. Natl. Acad. Sci. USA 104:1482 (2007); Yang et
al., Mol. Pharm. 11:1250 (2014)). Particle size and
.zeta.-potential were determined by dynamic light scattering and
laser Doppler anemometry, respectively, using a Zetasizer Nano ZS
(Malvern Instruments, Southborough, Mass.). Size measurements were
performed at 25.degree. C. at a scattering angle of 90.degree..
Samples were diluted in 10 mM NaCl solution, and measurements were
performed according to instrument instructions. High density
PEGylation was verified using the fluorogenic compound
1-pyrenyldiazomethane (PDAM) to quantify residual unmodified
carboxyl groups on the polystyrene beads (Yang ei al., Mol. Pharm.
11:1250 (2014)). PEG conjugation was also confirmed by a
near-neutral .zeta.-potential (Table 1) (Lai et al., Proc. Natl.
Acad. Sci. USA 104:1482 (2007)).
[0278] Antibody Binding to Nanoparticles:
[0279] Mouse anti-PEG IgG (3.3) and IgM (AGP-4) antibodies have
been previously described (Su et al., Bioconjug. Chem. 21:1264
(2010)). Anti-PEG IgG and IgM binding to PS-PEG nanoparticles was
confirmed by dot blot assay. Two microliters of PS or PS-PEG beads
were blotted onto nitrocellulose membranes. The membranes were
incubated with 1:2000 dilutions of anti-PEG IgM, control IgM
(anti-vancomycin; Santa Cruz Biotechnology, Dallas, Tex.), anti-PEG
IgG, or control IgG (anti-HIV-1 gp120; Santa Cruz Biotechnology,
Dallas, Tex.), followed by incubation with a 1:10000 dilution of
HRP-labeled goat anti-mouse IgM (Life Technologies, Carlsbad,
Calif.) or a 1:7000 dilution of HRP-labeled goat anti-mouse IgG
(Santa Cruz Biotechnology, Dallas, Tex.). Bound secondary antibody
was detected using an ECL kit (BioRad, Hercules, Calif.), and
imaged using a FluorChem E system (ProteinSimple, San Jose,
Calif.). The binding avidity for anti-PEG IgG (3.3) has been
reported to be 1.3.times.10.sup.-7 M at room temperature (Su et
al., MAbs 6:1069 (2014)), while that for anti-PEG IgM (AGP.4) is at
least 1.4.times.10.sup.-10 M (Ehrlich et al., J. Mol. Recognit.
22:99 (2009)).
[0280] Multiple Particle Tracking:
[0281] Dilute particle solutions (.about.10.sup.8-10.sup.9
particles/mL, 5% v/v) were added to 20 .mu.L of mCVM in custom-made
chambers, and samples were incubated 1 hr at 37.degree. C. before
microscopy. The trajectories of the fluorescent particles in mCVM
were recorded using an EMCCD camera (Evolve 512; Photometrics,
Tucson, Ariz.) mounted on an inverted epifluorescence microscope
(AxioObserver D1; Zeiss, Thornwood, N.Y.), equipped with an Alpha
Plan-Apo 100.times./1.46 NA objective, environmental (temperature
and CO.sub.2) control chamber and an LED light source (Lumencor
Light Engine DAPI/GFP/543/623/690). Videos (512.times.512, 16-bit
image depth) were captured with MetaMorph imaging software
(Molecular Devices, Sunnyvale, Calif.) at a temporal resolution of
66.7 ms and spatial resolution of 10 nm (nominal pixel resolution
0.156 .mu.m/pixel). The tracking resolution was determined by
tracking the displacements of particles immobilized with a strong
adhesive, following a previously described method (Apgar et al.,
Biophys. J. 79:1095 (2000)). Particle trajectories were analyzed
using MATLAB software as described previously (Wang et al., J.
Control. Release 220(Pt A):37 (2015)). Sub-pixel tracking
resolution was achieved by determining the precise location of the
particle centroid by light-intensity-weighted averaging of
neighboring pixels. Trajectories of n.gtoreq.40 particles per frame
on average (corresponding to n.gtoreq.100 total traces) were
analyzed for each experiment, and four independent experiments were
performed in mCVM collected from different mice. The coordinates of
particle centroids were transformed into time-averaged mean squared
displacements (MSD), calculated as
<.DELTA.r.sup.2(.tau.)>=[x(t+.tau.)-x(t)].sup.2+[y(r+)
y(t)].sup.2 (where .tau.=time scale or time lag), from which
distributions of MSDs and effective diffusivities (D.sub.eff) were
calculated, as previously demonstrated (Lai et al., Proc. Nat.
Acad. Sci. USA 104:1482 (2007); Dawson et al., J Biol. Chem.
278:50393 (2003); Suh et al., Adv. Drug Deliv. Rev. 57:63 (2005)).
MSD may also be expressed as MSD=4D.sub.0.tau..sup..alpha., where
.alpha., the slope of the curve on a log-log scale, is a measure of
the extent of impediment to particle diffusion (.alpha.=1 for pure
unobstructed Brownian diffusion; .alpha.<1 indicates increasing
impediment to particle movement as a decreases). Mobile particles
were defined as those with D.sub.eff.gtoreq.10.sup.-1.5
.mu.m.sup.2/s at .tau.=0.2667 s (this .tau. corresponds to a
minimum trajectory length of 5 frames), based on multiple datasets
of mobile and immobile nanoparticles (e.g., PS and PS-PEG
nanoparticles) in human mucus (Lai et al., Proc. Natl. Acad. Sci.
USA 107:598 (2010); Lai et al., Proc. Natl. Acad. Sci. USA 104:1482
(2007)). For particles .about.100 nm in size, a
D.sub.eff.gtoreq.10.sup.-1.5 .mu.m.sup.2/s effectively means that
the particles move a distance greater than their diameters within
0.2667 s.
[0282] Distribution of Nanoparticles in the Mouse Vagina:
[0283] To evaluate PEGylated nanoparticle trapping in vivo, we used
female 6-8 week old CF-1 mice (Harlan, Frederick, Md.) pretreated
with 100 mg of 17.beta.-estradiol benzoate (Sigma, St. Louis, Mo.)
injected subcutaneously two days before the experiments (Ensign et
al., Sci. Transl. Med. 4:138ra79 (2012)). Anti-PEG or control
(anti-vancomycin) IgM was first administered to the mouse vagina in
two doses: 10 .mu.L of 150 .mu.g/mL antibody in 0.5.times. normal
saline, and after a 10 min interval, another 10 .mu.L dose in
normal saline. The mildly hypotonic medium in the first dose
resulted in advective transport of antibody close to the epithelium
(Ensign et al., Biomaterials 34:6922 (2013)), which, combined with
the second dose in isotonic medium, ensured antibody was well
distributed throughout the luminal layer. After another 10 min
interval, 20 .mu.L of either PS-PEG or control PS (0.025% w/v) was
administered in a slightly hypotonic medium (0.75.times. normal
saline). Mice were sacrificed after 10 min, and the entire vagina
was then gently removed and frozen in Tissue-Tek O.C.T..TM.
Compound (Sakura Finetek U.S.A., Inc., Torrance, Calif.).
Transverse sections were obtained at various points along the
length of the tissue (between the introitus and the cervix) with a
Microm HM 500 M Cryostat (Microm International). The thickness of
the sections was set to 6 .mu.m to achieve single-cell layer
thickness. The sections were then stained with ProLong Gold
antifade reagent (Invitrogen, Grand Island, N.Y.) with
4',6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei and
retain particle fluorescence. Fluorescence images of the sections
were obtained with an inverted fluorescence microscope at 10.times.
magnification. The same procedures were performed for at least n=4
mice per condition. Mice were anesthetized for the duration of
experiments. All experimental protocols were approved by the Johns
Hopkins Institutional Animal Care and Use Committee.
[0284] Statistical Analysis:
[0285] Data averages are presented as means with standard error of
the mean (SEM) indicated. Statistical significance was determined
by a one-tailed, Student's t-test (.alpha.=0.05).
Results
[0286] Due to both passive transudation and active transport by the
MHC class I-related neonatal Fc receptor (Li et al., Proc. Natl.
Acad. Sci. USA 108:4388 (2011)), IgG, rather than IgA, is the
predominant immunoglobulin inhuman CVM secretions (>10-fold more
IgG than IgA) (Usala et al., J. Reprod. Med. 34:292 (1989)).
However, the routes of PEG exposure that may lead to gradual
induction of anti-PEG IgG in humans are not yet known and would be
difficult to produce in mice. Therefore, we first tested whether
exogenous murine anti-PEG IgG added to ex vivo mouse CVM (mCVM) at
concentrations typical of pathogen-specific antibodies in humans
(Wang et al., Mucosal Immunol. 7:1036 (2014)) could alter the
mobility of polystyrene (PS) nanoparticles with PEG grafting at
densities well into the dense brush regime (PS-PEG; diameter
.about.100 nm) (Table 1). Both anti-PEG IgG and IgM (previously
shown to bind the PEG backbone) bound specifically to PS-PEG but
not uncoated PS nanoparticles (FIGS. 12A-12D), presumably because
the degree of curvature on the nanoparticles would cause PEG chains
to increasingly assume a more diffuse mushroom conformation that
exposes the polymer backbone at distances far from the particle
core.
TABLE-US-00001 TABLE 1 Characterization of PEG-modified
nanoparticles, and ratios of the ensemble average diffusion
coefficients in water (D.sub.w) compared to in mCVM (D.sub.m). Size
and .xi.-potential values for carboxyl- modified beads are provided
for comparison. PEG .xi.- Density Size Surface Diameter potential
(PEG/ D.sub.w/ (nm)* Chemistry (nm) (mV) nm.sup.2).dagger.
D.sub.m.dagger-dbl. 100 COOH 109 .+-. 4 -55 .+-. 5 N/A N/A 100 PEG
132 .+-. 3 -7 .+-. 3 2.3 .+-. 0.1 4.9 *Provided by the
manufacturer. .dagger.Calculated from % COOH substitution measured
by PDAM assay. .dagger-dbl.Effective diffusivity values are
calculated at a time scale of 0.2667 s. D.sub.w is calculated from
the Stokes-Einstein equation.
[0287] In mCVM without exogenously added anti-PEG IgG or treated
with control mouse IgG, PS-PEG exhibited largely unhindered
Brownian motion, with particles capable of diffusing many microns
on the order of seconds (FIG. 8A). However, in mCVM first treated
with anti-PEG IgG to a final concentration of 10 g/mL prior to the
addition of nanoparticles, a large fraction of PS-PEG became
immobilized. We quantified the speeds of PS-PEG in mucus treated
with different antibodies using multiple particle tracking, a
technique that allows quantitative measurements of hundreds of
individual particles. PS-PEG particles in control mCVM were only
slowed 5-fold compared to their theoretical speeds in water (Table
1), in good agreement with previous reports (Lai et al., Proc.
Natl. Acad. Sci. USA 107:598 (2010); Ensign et al., Mol. Pharm.
10:2176 (2013)). The addition of anti-PEG IgG reduced the
geometrically averaged ensemble mean squared displacement
(<MSD>) by .about.30-fold compared to control IgG (FIG. 8B;
at a time scale of 0.2667 s). The impediment to free Brownian
diffusion caused by anti-PEG IgG was also reflected by the slope a
from the log-log <MSD> vs. time scale plots (.alpha.=1 for
pure unobstructed Brownian diffusion, e.g., particles in water, and
.alpha. becomes smaller and approaches zero as obstruction to
Brownian diffusion increases): the average a value was 0.86 for
PS-PEG in control CVM, but 0.52 for PS-PEG in mCVM treated with
anti-PEG IgG (p<0.05 vs. control). Importantly, the mobile
PS-PEG fraction (see Methods) was reduced from 96% in control mCVM
to only 34% in anti-PEG treated mCVM. These results demonstrate the
ability for anti-PEG IgG to alter the mobility of PEGylated
nanoparticles in mucus secretions.
[0288] We next sought to test whether an agglutinating antibody may
trap PEGylated particles even more extensively. Secretory IgA
(sIgA) is another common immunoglobulin found in mucus. The
polymeric nature of sIgA, as well as IgM, has long been suggested
to facilitate "immune exclusion", the agglutination of
microorganisms by polymeric immunoglobulins into clusters too large
to diffuse through mucus (Roche et al., Mucosal Immunol. 8:176
(2015)). The relevance of sIgA- and IgM-mediated immune exclusion
in the female reproductive tract is vividly illustrated by
agglutination of otherwise vigorously motile sperm, which make
little to no forward progress after agglutination and cannot `swim`
through mucus (Cone, Mucus, in Handbook of Mucosal Immunology, P.L.
Ogra, et al., Editors. 1999, Academic Press: San Diego. p. 43-64).
Unfortunately, anti-PEG sIgA is not commercially available and
cannot be readily generated in the lab. Therefore, we evaluated the
effects of anti-PEG IgM on the diffusion behavior of PEGylated
nanoparticles in mucus. Addition of anti-PEG IgM immobilized an
even greater fraction of PS-PEG than did anti-PEG IgG and at a
lower antibody concentration (5 pug/mL vs. 10 .mu.g/mL; FIG. 9A).
In contrast, addition of control IgM did not alter the diffusion of
PS-PEG. The <MSD> of PS-PEG in mCVM with anti-PEG IgM was
150-fold lower than for the same particles in mCVM treated with
control IgM (FIG. 9B), and reflects a drop in the mobile fraction
from 97% to 7% (FIG. 9C). Anti-PEG IgM also reduced the average a
value of PS-PEG from 0.79 to 0.45 (p<0.05 vs. control),
underscoring the extent of antibody-mediated immobilization.
Interestingly, we did not observe any nanoparticle agglutination in
mCVM with anti-PEG IgM; agglutination was only observed when
nanoparticles were pre-mixed with anti-PEG IgM prior to addition to
mucus (FIGS. 13A-13C).
[0289] Lastly, we wanted to evaluate whether the observed changes
to the diffusion of PEGylated nanoparticles in physiological mucus
specimens ex vivo would also alter nanoparticle distribution at
mucosal surfaces in vivo. We therefore topically administered
anti-PEG or control IgM to the mouse vagina, followed by addition
of PS-PEG in hypotonic medium. We observed that a substantial
fraction of PS-PEG was drawn advectively through luminal mucus and
accumulate immediately adjacent to the epithelium in mice dosed
with control IgM, with a significant fraction penetrating deep into
the rugae (FIG. 10A). In contrast, PS-PEG nanoparticles were
largely trapped in luminal mucus in mice that received anti-PEG
IgM; far fewer particles reached the vaginal epithelium or
penetrated into the rugae (FIG. 10B), similar to mucoadhesive
carboxylated latex beads (Ensign et al., Sci. Transl. Med.
4:138ra79 (2012)). These results are consistent with previous
observations that nanoparticles that interact with and bind to the
mucin mesh fibers overlaying the epithelium are unable to penetrate
the mucus layer and reach the epithelium (Ensign et al., Sci.
Transl. Med. 4:138ra79 (2012)).
[0290] The emergence of anti-PEG antibodies represents a potential
Achilles' heel to the increasingly common use of PEG in
nanomedicine. Rodent studies have clearly and unequivocally
illustrated that anti-PEG immunity directly abrogates the extended
circulation times that PEGylation generally affords to
therapeutics. Anti-PEG immunity may also result in serious
complications beyond poor pharmacokinetics. Indeed, anti-PEG
response as a result of repeated weekly injections of PEGylated
liposomes containing synthetic oligodeoxynucleotides led to
significant morbidity and mortality in mice (Semple et al., J.
Pharmacol. Exp. Ther. 312:1020(2005)). Inhuman clinical studies of
PEG-uricase for the treatment of gout, the presence of anti-PEG
antibodies was associated with early elimination and poor outcomes
(Ganson et al., Arthritis Res. Ther. 8:R12 (2006); Sundy et al.,
Arthritis Rheum. 56:1021 (2007)). While these studies all highlight
the systemic effects associated with anti-PEG response, potential
mucosal anti-PEG response has not been evaluated to date. Our
finding that anti-PEG IgG or IgM can directly impede the mobility
and consequently alter the biodistribution of PEGylated
nanoparticles at mucosal surfaces adds yet another pitfall of
anti-PEG immunity in vivo (FIG. 11).
[0291] More antibodies are secreted into mucus than into blood or
lymph; thus, a major part of the physiological immune response is
likely intended to occur in the mucus secretions coating exposed
surfaces of the respiratory, gastrointestinal and urogenital
tracts. However, the notion that secreted antibodies can work
together with mucus to impede the mobility of pathogens and foreign
particulates and reduce the flux arriving at the epithelium remains
largely unrecognized. This is in part a consequence of the
exceedingly weak affinity between antibodies and mucus, which would
suggest that individual antibodies are incapable of crosslinking a
pathogen or particle to mucus. It was not until very recently that
virus-binding antibodies were shown to be capable of immobilizing
viruses in human mucus secretions (Wang et al., Mucosal Immunol.
7:1036 (2014)). Our finding that anti-PEG antibodies can immobilize
PEGylated nanoparticles in mCVM offers further evidence that
trapping by antibody-nucin crosslinking may be a universal
mechanism of mucosal immunity across different animal species.
[0292] We found that anti-PEG IgM was substantially more potent in
trapping individual PEGylated nanoparticles in mucus than anti-PEG
IgG. This may be due to a number of reasons. First, anti-PEG IgM
has a higher binding avidity to PEG than anti-PEG IgG (see
Methods), which likely translates to greater antibody accumulation
on the surface of PEGylated nanoparticles. Second, IgM likely
possesses greater affinity to mucins than IgG. This can be inferred
from the diffusivity of individual IgM molecules in mucus, which is
slowed nearly 50% compared to in buffer, whereas individual IgG
molecules are slowed only .about.5-20% in mucus compared to in
buffer (Olmsted et al., Biophys. J. 81:1930 (2001)). Because both
IgG and IgM are far too small relative to the mucin mesh spacing
(Lai et al., Proc. Natl. Acad. Sci. USA 107:598 (2010)) to be
slowed substantially by steric hindrance, IgM must be slowed to a
greater extent than IgG due to more or stronger adhesive
interactions with mucus constituents. The greater mucin-of IgM
implies fewer particle-bound IgM would be needed to immobilize a
PEGylated nanoparticle compared to IgG; indeed, greater trapping of
PS-PEG was achieved with IgM than IgG even at a lower
concentration.
[0293] Interestingly, we observed negligible levels of
agglutination in all of our microscopy studies. This is likely
because even a single particle-bound IgM can substantially impede
the Brownian motion of the particle (assuming the binding of IgM to
PEG does not alter IgM affinity to mucins, a single IgM bound to
the nanoparticle may slow it down by .about.50%), and lower
particle mobility directly reduces the frequency of colliding with
other particles, a necessary first-step to agglutination. Thus,
IgM-induced agglutination is unlikely to be prevalent when the
kinetics of IgM accumulating onto the nanoparticle surface is
substantially faster than the rate of collision between
nanoparticles. This directly challenges the general dogma that
antibody-mediated agglutination--often supported by micrographs
showing the formation of large immune complexes upon incubation of
antibodies and pathogens in buffer in the absence of mucus--is a
dominant mechanism of mucosal immunity by polymeric
immunoglobulins.
[0294] The vast majority of the literature on anti-PEG immunity
focuses on the acute induction of anti-PEG immune response by
specific PEGylated liposomes or nanoparticle therapeutics. The
possibility of pre-existing anti-PEG response is rarely addressed.
Because most of the population has yet to receive PEGylated
therapeutics, the presence of PEG-specific antibodies in bodily
secretions such as mucus most likely results from induction by
alternative routes of exposure. Since PEG and other PEG-containing
polymers are often found in soaps and other detergents, a
hypothetical route of exposure could be via open cuts and wounds
cleaned with PEG-containing soaps, or bleeding gums at the oral
mucosa exposed to PEG-containing toothpaste. The combination of PEG
and soap surfactants that are toxic to cells may provide a danger
signal that inadvertently induces immune cells to generate
antibodies against PEG. Alternatively, PEG is also frequently found
in numerous foods as well as oral and topical products. Repeated
exposure to PEG at the oral, gastrointestinal and vaginal mucosa,
including exposure accompanied by surfactants, could potentially
induce systemic and/or mucosal anti-PEG immunity over time, with
significant implications for the delivery of PEGylated therapeutics
via relevant routes of administration.
[0295] PEG is widely used in nanomedicine as a stealth coating
polymer to improve circulation time and therapeutic efficacy.
However, a growing body of evidence generated over the past two
decades shows that systemic exposure to PEG can induce anti-PEG
antibodies that not only significantly alter the systemic
circulation kinetics of PEG-modified nanotherapeutics, but may also
directly result in adverse outcomes. Our finding that anti-PEG
antibodies can also trap PEG-coated nanoparticles in mucus
secretions ex vivo and alter particle biodistribution at mucosal
surfaces in vivo further suggests that mucosal anti-PEG immunity
may be an under-recognized challenge. Mucosal exposure to PEG and
the prevalence of anti-PEG antibodies in human mucus is not yet
well understood. In light of the large arsenal of PEGylated
therapeutics that are FDA-approved or in clinical development for
both systemic and mucosal applications, we believe further
investigations into the origin and characteristics of pre-existing
anti-PEG immunity are of critical importance to support ongoing
efforts in translational nanomedicine development.
Example 3
A Blueprint for Robust Crosslinking of Mobile Species in Biogels
Using Third Party Crosslinking Anchors with Short-Lived
Anchor-Matrix Bonds
[0296] Biopolymeric matrices are ubiquitous in living systems,
generically composed of a highly entangled and cross-linked mesh of
macromolecules in buffer. Within cells, cytoskeletal networks of
actin and microtubules control cell migration, maintain cell shape
and polarity, and facilitate proper routing and sorting of
intracellular cargo (Huber et al., Adv. Phys. 62:1 (2013)). At the
extracellular scale, networks of fibronectin and collagen not only
provide scaffolds for mechanical support and tissue organization,
but also regulate the dynamic behavior of cells through variations
in local microstructure and stiffness (Theocharis et al., Adv. Drug
Deliv. Rev. 97:4 (2016)). At the tissue scale, secreted mucins
create a viscoelastic gel that serves both as a lubricant and as a
transport barrier to prevent pathogens and particulates from
reaching the underlying epithelium (Lai et al., Adv. Drug Deliv.
Rev. 61:86 (2009)).
[0297] A major function of biogels is to regulate transport. Gels
can impede the passive diffusion of particulates and active motion
of bacteria and cells by steric obstruction from as well as
adhesive interactions to the matrix constituents. Given that the
majority of nanoparticles and viruses are smaller than the mesh
spacings, their diffusion across a gel can only be hindered by
adhesive interactions. However, due to evolutionary pressure, it is
exceedingly unlikely that direct adhesive interactions with
matrices comprised of relatively homogeneous constituents, such as
mucins or laminins, can alone effectively block the transport of
the full diversity of nanoparticulates typically encountered in
nature. For example, viruses must penetrate the dense mucin mesh to
infect underlying cells; thus, it is hardly surprising that the
vast majority of viruses that transmit at mucosal surfaces (HIV,
Herpes, HPV, Norwalk, etc.) are able to evade binding to mucins and
diffuse rapidly through the low viscosity interstitial fluids
within pores of mucus gels (Olmsted et al., Biophys. J. 81:1930
(2001)).
[0298] An alternative strategy is to utilize "third party"
molecular anchors to crosslink nano-scale species (henceforth
referred to as nanoparticles) to the matrix, such as antibodies
(Ab) that can specifically recognize and bind invading pathogens.
The diffusion coefficients of IgG and IgA Ab in human mucus are
.about.5-10% lower compared to buffer, whereas 10-fold larger
viruses can diffuse in mucus unhindered. The slightly retarded
diffusion of both Ab implies they must be slowed by weak and
transient interactions with the mucus matrix. Surprisingly, despite
this seemingly negligible affinity, herpes-binding IgG can
specifically and effectively immobilize Herpes Simplex Viruses
(HSV-1) in human cervicovaginal mucus even at sub-neutralizing IgG
concentrations, and trapping HSV-I in mucus directly prevented
vaginal herpes transmission in mice (Wang et al., IgG in
cervicovaginal mucus traps HSV and prevents vaginal Herpes
infections. Mucosal Immunol. 7:1 (2014)). It remains unclear
whether the trapping potency of IgG can be further enhanced by
tuning its affinity with matrix constituents. To potentially
develop more potent anchors, we sought to examine the
characteristics of IgG that could maximize net adhesive
interactions between nanoparticles and biopolyner matrices. The
widely held and intuitively reasonable assumption is that anchors
with long-lived, high-affinity bonds to both the nanoparticle and
matrix would confer superior trapping efficiency. Surprisingly, we
herein show theoretically and experimentally that anchor-matrix
bonds that are rapid and short-lived relative to
anchor-nanoparticle bonds greatly enhances trapping potency of
molecular anchors.
Methods
[0299] Preparation of PEG-Coated Nanoparticles:
[0300] To produce PEGylated nanoparticles (PS-PEG), we covalently
modified 200 nm fluorescent, carboxyl-modified polystyrene beads
(PS-COOH; Invitrogen) with 2 kDa methoxy poly(ethylene glycol)
amine (PEG; Sigma) via a carboxyl-amine reaction, as published
previously (Lai et al., Proc. Natl. Acad. Sci. USA 104:1482 (2007);
Yang et al., Mol. Pharm. 11:1250 (2014)). Particle size and
.zeta.-potential were determined by dynamic light scattering and
laser Doppler anemometry, respectively, using a Zetasizer Nano ZS
(Malvern Instruments, Southborough, Mass.). Size measurements were
performed at 25.degree. C. at a scattering angle of 900. Samples
were diluted in 10 mM NaCl solution, and measurements were
performed according to instrument instructions. High density
PEGylation (>1 PEG/nm.sup.2) was verified using the fluorogenic
compound 1-pyrenyldiazomethane (PDAM) to quantify residual
unmodified carboxyl groups on the polystyrene beads (Yang et al.,
Mol. Pharm. 11:1250 (2014)). PEG conjugation was also confirmed by
a near-neutral .zeta.-potential (Lai et al., Proc. Natl. Acad. Sci.
USA 104:1482 (2007)).
[0301] Preparation of BM, Laminin/Entactin, and Collagen Gels:
[0302] Growth-factor reduced Matrigel (Corning) was dialyzed
against PBS for a minimum of 24 hours, then biotinylated with
20-fold molar concentration NHS-PEG4-biotin (ThermoFisher). This
biotinylated Matrigel (final concentration 2.2 mg/mL) was mixed
with neutravidin (ThermoFisher; final concentration 0 or 4 g/mL),
BSA (Sigma, final concentration 1 mg/mL), Eagle's Minimum Essential
Medium (EMEM, Lonza BioWhittacker), on ice for 15 minutes.
Fluorescent PS-COOH (Ex: 505 nm, Em515 nm; final concentration
4.5*10.sup.8 beads/mL) and PS-PEG (Ex: 625 nm, Em645 nm; final
concentration 0.3*10.sup.8 beads/mL) nanoparticles and anti-PEG
IgG.sub.1 (CH2076 or CH2076B, Silver Lake Research, final
concentration 10 .mu.g/mL) was combined on ice. The mixture was
added to a custom-made micro-volume glass chamber slide, incubated
at 37.degree. C. for 45 minutes in a custom hydration chamber, then
sealed and incubated for another 30 minutes prior to microscopy.
Trapping of PS-COOH beads in BM was used as an internal control in
all microscopy experiments as a measure of complete polymerization
of BM constituents.
[0303] High Resolution Multiple Particle Tracking:
[0304] The trajectories of the fluorescent particles were recorded
using an EMCCD camera (Evolve 512; Photometrics, Tucson, Ariz.)
mounted on an inverted epifluorescence microscope (AxioObserver D1;
Zeiss, Thornwood, N.Y.), equipped with an Alpha Plan-Apo
100.times./1.46 NA objective, environmental (temperature and
CO.sub.2) control chamber and an LED light source (Lumencor Light
Engine DAPI/GFP/543/623/690). 20s videos (512.times.512, 16-bit
image depth) were captured with MetaMorph imaging software
(Molecular Devices, Sunnyvale, Calif.) at a temporal resolution of
66.7 ms and spatial resolution of 10 nm (nominal pixel resolution
0.156 m/pixel). The tracking resolution was determined by tracking
the displacements of particles immobilized with a strong adhesive,
following a previously described method (Apgar et al., Biophys. J.
79:1095 (2000)). Particle trajectories were analyzed using MATLAB
software as described previously (Wang et al., J. Control. Release
220:37 (2015)). Sub-pixel tracking resolution was achieved by
determining the precise location of the particle centroid by
light-intensity-weighted averaging of neighboring pixels.
Trajectories of n.gtoreq.40 particles per frame on average
(corresponding to n.gtoreq.100 total traces per specimen/condition)
were analyzed for each experiment, and 3-4 independent experiments
were performed for each condition. The coordinates of particle
centroids were transformed into time-averaged mean squared
displacements (MSD), calculated as
<.DELTA.r.sup.2(.tau.)>=[x(t+.tau.)-x(t)].sup.2+[y(t+t)-y(t)].sup.2
(where, .tau.=time scale or time lag), from which distributions of
MSDs and effective diffusivities (D.sub.eff) were calculated, as
previously demonstrated (Lai et al., Proc. Natl. Acad. Sci. USA
104:1482 (2007); Dawson et al., J. Biol. Chem. 278:50393 (2003);
Suh et al., Adv. Drug Deliv. Rev. 57:63 (2005)). MSD may also be
expressed as MSD=4D.sub.0.tau..sup..alpha., where .alpha., the
slope of the curve on a log-log scale, is a measure of the extent
of impediment to particle diffusion (.alpha.=1 for pure
unobstructed Brownian diffusion; .alpha.<1 indicates
sub-diffusive motion due to interactions with the elastic as well
as viscous properties of the polymeric gel). Mobile particles were
defined as those with D.sub.eff>10.sup.-1.5 m.sup.2/s at
.tau.=0.2667 s (this .tau. corresponds to a minimum trajectory
length of 5 frames), based on multiple datasets of mobile and
immobile nanoparticles (e.g., PS and PS-PEG nanoparticles) in human
mucus Lai et al., Proc. Nat. Acad. Sci. USA 104:1482 (2007); Lai et
al., Proc. Nat. Acad. Sci. USA 107:598 (2010)).
[0305] Biolayer Interferometry Experiments:
[0306] On an Octet QK instrument (ForteBio), streptavidin
biosensors (ForteBio) were loaded with biotinylated Matrigel and
blocked with free biotin. Antibody (biotinylated and native) at
different concentrations was associated with these customized
biosensors and dissociated into running buffer. Data were adjusted
for reference sensors and baseline values and aligned to
dissociation, then processed with Savitzky-Golay filtering.
Analysis was performed with ForteBio software using a 1:1 global
curve fit model to obtain values for k.sub.on, k.sub.off, and
K.sub.D.
[0307] Mathematical Model:
[0308] Instead of developing a reaction-diffusion model for a
concentration of nanoparticle species P, we take a stochastic
approach and focus on the motion of a single nanoparticle P. The
main goal is to maximize the fraction of time that P spends bound
to the polymer network.
[0309] Let N be the total number of anchor binding sites on the
nanoparticle P. The crosslink enhancement effect requires the
cooperative action of multiple anchors; it requires N>1 anchor
binding sites (e.g., antigenic epitopes) on the nanoparticle. The
reactions [eq:1] and [eq:2] describe the case of a single binding
site (i.e., N=1). The first crosslink bond forms at a diffusion
limited reaction rate according to [eq:2]. If N>1, additional
anchors might be bound to the nanoparticle. Once the first
crosslink forms many additional binding sites M are very close by,
allowing additional anchor-matrix bonds to form. The intra-complex
reaction is given by
M + AP ( AM ) n - 1 Ca on .fwdarw. .rarw. na off P ( AM ) n , n
> 1 , [ eq : 3 ] ##EQU00012##
where C is a nondimensional parameter that scales the intra-complex
binding rate and assumed to be 1 in the current work. Molecules
within a large complex may not react with each other at the same
rate as they do when they are freely diffusing. When nanoparticles,
anchors, and matrix elements are bound within the same complex,
they are mechanically linked. Mechanical forces imposed by
surrounding elements of the complex confine random molecular
motion. Similar biomechanical reactions are common in biology
(e.g., molecular motor transport (Goychuk et al, PLoS One 9:1
(2014)) and DNA transcription (Matsuda et al., Biophys. J. 106:1801
(2014))). For our present situation, it is a reasonable first
approximation to assume that the intra-complex dissociation rates
remain the same as the bimolecular dissociation rates (i.e.,
a.sub.off and k.sub.off). However, intra-complex binding rates are
different from the Smoluchowski bimolecular reaction rates for
diffusing molecules. The binding rate between two molecules within
the complex depends on their relative distance and effective random
mobility. Molecules within a single nanocomplex are quite close so
that they do not have to move far in order to bind. On the other
hand, they have lower relative mobility when mechanically confined
within the complex.
[0310] Let n be the number of occupied binding sites, and let s be
the number of anchors crosslinking the nanoparticle to the polymer
network. The chemical system can be modeled as a Markov process
with state transitions given by
n ( N - n ) k on .fwdarw. .rarw. ( n - s + 1 ) k off n + 1 , ( s ,
n ) .delta. s , 0 ( N - n ) k on ' .fwdarw. .rarw. ( s + 1 ) k off
( s + 1 , n + 1 ) , [ eq : 4 ] s g ( s ) ( n - s ) a on .fwdarw.
.rarw. ( s + 1 ) a off s + 1 , [ eq : 5 ] where g ( s ) = { D P / D
A , s = 0 C , s .gtoreq. 1 . ##EQU00013##
[0311] The process is described by its probability density p(n, s,
x, t). The probability that at time t, the nanoparticle is bound to
n anchors, s of which are bound to elements of the polymer matrix,
and located within a small distance dx of position x is
Prob[n(t)=n,s(t)=s,x<x(t)<x+dx].apprxeq.p(n,s,x,t)dx+o(dx).
[0312] Since the model is a continuous time Markov process, the
probability density function satisfies the differential
Chapman-Kolmogorov equation,
.differential. .differential. t p ( n , s , x , t ) = .delta. s , 0
D P .gradient. 2 p + [ s + n , s ] p , [ eq : 6 ] ##EQU00014##
where, .sub.s the N.times.N transition rate matrix for [eq:5] and
.sub.n,s is the N.times.N transition rate matrix for [eq:4].
[0313] Multiple Timescale Analysis:
[0314] Consider the case where .tau..sub.M<<.tau..sub.P.
Notice that the fast reaction [eq:5] conserves n. While n changes
slowly, transitions in s are at quasi-steady-state. Since we are
primarily concerned with the motion of the nanoparticle, and not
necessarily the state of any bound anchors, our goal is to obtain a
good approximation to the marginal probability
u ( x , t ) = n = 0 N s = 0 N p ( n , s , x , t ) .
##EQU00015##
[0315] Using the rule of conditional probability, we can rewrite
the full probability density for the process as
p(n,s,x,t)=.rho..sub.t(s,t|n,x).rho..sub.t(n,t|x)u(x,t).
[0316] Since s changes rapidly compared to n, which changes rapidly
compared to x, the conditional probabilities .rho..sub.t rapidly
equilibrate, which means that
.rho..sub.t.apprxeq..rho..sub..infin.=.rho.. Since the transition
rates are independent of position x, it follows that .rho.(s|n,
x)=.rho.(s|n) and .rho.(n|x)=.rho.(n). The two probability
distributions p(sIn) and p(n) are called quasi steady state
distributions, and they satisfy
s = 0 N s .rho. ( s | n ) = 0 , s = 0 N n = 0 N n , s .rho. ( s | n
) .rho. ( n ) = 0. [ eq : 7 ] ##EQU00016##
[0317] We can take advantage of the separation of time scales with
an asymptotic approximation, namely
p(n,s,x,t).about..rho.(s|n).rho.(n)u(x,t). [eq:8]
[0318] First we average out the fastest reaction, the transition in
s. Let p(n,x,t)=.rho.(n)u(x,t). Substituting [eq:8] into [eq:6],
summing over s, and using [eq:7] yields
.differential. .differential. t p ( n , x , t ) = D P .rho. ( 0 | n
) .gradient. 2 p + n p , [ eq : 9 ] ##EQU00017##
where .sub.n is the transition rate matrix for the averaged slow
reaction:
( N - n ) .kappa. ( n ) ##EQU00018## n .rarw. .fwdarw. n + 1 , ( n
+ 1 ) k off ##EQU00018.2##
where .kappa.(n)=.rho.(0|n)k.sub.on+k'.sub.on. Given n, the
stationary distribution for the number of anchors s<n on the
nanoparticle that are bound to the matrix is
.rho. ( s | n ) = { D A C .alpha. n ( D A C - D P ) .alpha. n + D P
, s = 0 D P ( n s ) ( 1 - .alpha. ) s .alpha. n - s ( D A C - D P )
.alpha. n + D P , s > 0 , ##EQU00019##
where .alpha.=a.sub.off/(Ca.sub.on+a.sub.off).
[0319] We can apply the same procedure to average out n as follows.
The quasi-steady-state distribution for n is given by
.rho. ( n ) = k off n ( N n ) j = 0 n - 1 ( .rho. ( 0 | j ) k on +
k on ' ) , ##EQU00020##
where is a normalization factor. Substituting
p(n,x,t)=.rho.(n)u(x,t) into [eq:9], summing over n, and using
[eq:7] yields
.differential. .differential. r u ( x , t ) = D eff .gradient. 2 u
, D eff = D P n = 0 N .rho. ( 0 | n ) .rho. ( n ) . [ eq : 10 ]
##EQU00021##
[0320] Monte-Carlo Simulations:
[0321] To determine the accuracy of the above approximation, we use
Monte-Carlo simulations. Using the Gillespie algorithm, we simulate
the Markov chain [eq:4] and [eq:5], (which is independent of x). A
single realization is generated through m state transitions. The
total elapsed time t.sub.m and the total time spend with s=0 (the
free diffusing state) t.sub.m.sup.(0) are updated with each
transition. It is easy to show that
P [ s = 0 ] = lim m .fwdarw. .infin. t m ( 0 ) t m .
##EQU00022##
[0322] Because all increments from free diffusion are independent,
an estimator for the effective diffusivity is
D m eff .ident. D P t m ( 0 ) t m . ##EQU00023##
[0323] Saturated Regime:
[0324] The reaction rate for any individual free anchor is
substantially reduced by the saturation of matrix binding sites.
Because [A.sub.T]>>[M], the fraction of unoccupied matrix
binding sites is equivalent to the fraction of time an individual
matrix binding site is unoccupied. Hence
.xi. = a off [ A T ] [ M ] a o n + a off .apprxeq. a off [ M ] a on
[ A T ] . ##EQU00024##
[0325] It follows that the binding rate for an individual
freely-diffusing anchor is
a on ' = .xi. a on .apprxeq. a off [ M ] [ A T ] . [ eq : 11 ]
##EQU00025##
[0326] Similarly, the intra-complex binding rate (see Eqn. [eq:3])
is a''.sub.on=Ca.sub.off[M]/[A.sub.T]. Based on the modified
binding rate [eq:11], the anchor-matrix kinetic timescale
becomes
.tau. AM = 1 ( 1 + [ M ] [ A T ] ) a off .apprxeq. 1 / a off .
##EQU00026##
[0327] The effective diffusivity in the saturated regime is
obtained by substituting
[ A ] = [ A * ] , .PHI. .apprxeq. 1 - [ M ] [ A T ] , and .alpha.
.apprxeq. 1 - C [ M ] [ A T ] into [ eq : 10 ] ##EQU00027##
Results and Discussion
[0328] Anchor-Mediated Trapping of Nanoparticles is Far More
Efficient with Rapid, Short-Lived Anchor-Matrix Bonds:
[0329] The highly viscoelastic nature of physiological mucus gels
makes it exceedingly difficult to chemically modify and
subsequently remove crosslinkers without irreversibly perturbing
its rheological properties. Instead, we took advantage of the
thermo-gelling properties of Matrigel.RTM., which enables us to
biotinylate the matrix as a low viscosity fluid at 4.degree. C. yet
study its diffusional barrier properties as a viscoelastic gel at
37.degree. C. Similar to with mucins, IgG possess exceedingly weak
affinity with Matrigel.RTM., as reflected by the high recovery rate
of IgG from Matrigel.RTM. by simple centrifugation. This allowed us
to investigate, using anti-PEG IgG as molecular anchors, whether
the mobility of polyethylene glycol-modified polystyrene
nanoparticles (PS-PEG; diameter .about.200 nm) that exhibits rapid
diffusion in the biotinylated Matrigel.RTM. can be altered by
tuning the affinity of anchor-matrix bonds.
[0330] We mixed neutravidin and biotinylated IgG that specifically
bind PEG into biotinylated Matrigel.RTM. to create high affinity
IgG-matrix bonds prior to temperature-induced gelation of the
matrix; we verified that both molecules were able to bind with high
affinity to biotinylated Matrigel using biolayer interferometry. In
biotinylated Matrigel.RTM. mixed with neutravidin, either lacking
exogenous IgG altogether or treated with control IgG, PS-PEG
exhibited rapid diffusion, with a geometrically averaged ensemble
effective diffusivity (<D.sub.eff>; 0.27 .mu.m.sup.2/s) only
8.7-fold reduced compared to their theoretical diffusivity in
buffer (FIGS. 14A-14B). The conjugation of biotinylated IgG to
Matrigel.RTM. did not reduce gel formation or the barrier
properties of Matrigel.RTM. against 200 nm uncoated
carboxyl-modified nanoparticles, which were immobilized to a
similar extent as unmodified Matrigel.RTM. (FIGS. 15A-15B).
Surprisingly, despite anchoring 10 g/mL anti-PEG IgG to
Matrigel.RTM. with long-lived, high affinity biotin-neutravidin
bonds, the matrix largely failed to immobilize PS-PEG. Indeed, the
<D.sub.eff> (0.14 .mu.m.sup.2/s) of PS-PEG at .tau.=0.2667s
was not statistically significantly different than in the same
Matrigel without anti-PEG IgG, and over 80% of particles remained
mobile (defined as nanoparticles with
<D.sub.eff>.gtoreq.10.sup.-1 .mu.m.sup.2/s at .tau.=0.2667s;
FIGS. 14A-14C). Modest trapping of PS-PEG by matrix-bound anti-PEG
IgG was observed only with prolonged incubation, e.g., 24 hrs
(FIGS. 16A-16B).
[0331] In contrast, despite the seemingly negligible affinity
between native unmodified IgG and biotinylated Matrigel, the
addition of 10 .mu.g/mL of anti-PEG IgG in biotinylated
Matrigel.RTM. reduced the <D.sub.eff> of PS-PEG by nearly
40-fold, with nanoparticles slowed on average almost 600-fold
compared to their mobility in water. The fraction of mobile PS-PEG
was reduced from 81% to 14% with the addition of anti-PEG IgG. The
immobilization was not due to agglutination of PS-PEG, since
trapped nanoparticles appeared identical to non-agglutinated
nanoparticles in Matrigel.RTM. treated with control IgG. PS-PEG
were also unlikely to be immobilized due to marked increase in the
nanoparticle hydrodynamic diameter; a complete coating of IgG on
200 nm nanoparticles would add no more than 10 nm to the
hydrodynamic diameter, and larger nanoparticles remained largely
diffusive in Matrigel. These results directly demonstrate that
short-lived anchor-matrix bonds is far more efficient in
facilitating immobilization of nanoparticles than long-lived
anchor-matrix bonds.
[0332] Proposed Theoretical Framework and Assumptions:
[0333] Our observations motivated us to develop a model to
recapitulate the observations and examine the features of molecular
anchors and matrix that could maximize trapping potency of
nanoparticles by the matrix. The model assumes three reactive
species: molecular anchors A, nanoparticles 1, and matrix
constituents M. Assuming that anchors must simultaneously possess
some affinity to both the matrix and the nanoparticle, our model
reveals that the most robust crosslinking of nanoparticles to the
matrix, as reflected by a minimum particle diffusivity D.sub.eff,
is achieved when the following six conditions are met:
[0334] 1. Markedly faster anchor-matrix binding-unbinding kinetics
than anchor-nanoparticle kinetics. In other words, lifetime of
anchor-matrix bonds (.tau..sub.AM) is short relative to the
lifetime of anchor-particle bonds (.tau..sub.AP) i.e.
.tau..sub.AM<<.tau..sub.AP
[0335] 2. Nanoparticles possess multiple, i.e., N>1 independent
binding sites such that multiple anchors can simultaneously
crosslink the same nanoparticle to the matrix
[0336] 3. Anchors are much smaller than the nanoparticle, and
consequently, the anchor diffusivity D.sub.A is much larger than
the free nanoparticle diffusivity D.sub.P; i.e.,
D.sub.A>>D.sub.P
[0337] 4. The anchor-nanoparticle binding is fast enough that many
anchors are likely to bind to the nanoparticle before it diffuses
out of the polymer matrix of thickness L; i.e.,
.tau..sub.AP<.tau..sub.L
[0338] 5. .tau..sub.AM is sufficiently short such that anchors do
not saturate the binding sites in the matrix
[0339] 6. Anchor concentration [A] is modest, such that on average
a single nanoparticle will not be simultaneously bound by two
anchors that are immobilized to the matrix. In other words, average
[A] does not exceed one anchor per volume of the nanoparticle (i.e.
[A]<<1/V.sub.P).
[0340] In general, with the proposed components M, A and P, there
are two reaction sequences that form the desired complex (MAP),
corresponding to a trapped nanoparticle. In particular, the MAP
complex is formed either by a matrix-bound anchor capturing a free
nanoparticle:
M + A .rarw. .fwdarw. a on a off MA , MA + P .rarw. .fwdarw. k on '
k off MAP , [ eq : 1 ] ##EQU00028##
or by a nanoparticle-anchor complex (formed when free anchors
accumulate on a diffusing nanoparticle) interacting with and
binding to the matrix:
A + P .rarw. .fwdarw. k on k off AP , M + AP .rarw. .fwdarw. D P D
A a on a off MAP , [ eq : 2 ] ##EQU00029##
[0341] The anchor-nanoparticle binding rates for free anchors
(k.sub.on) and matrix-bound anchors (k.sub.on') are given by the
Smoluchowski encounter relation, namely
k.sub.on=(D.sub.P+D.sub.A).phi.[A]R.sub.0,
k'.sub.on=(D.sub.P+D.sub.M)(1-.phi.)[A]R.sub.0,
respectively, where Ro is the effective binding distance at which
two molecules react. Note that the diffusivity of the polymer
matrix is effectively zero, i.e., D.sub.M.apprxeq.0. The fraction
of free A at steady state is related to the binding (a.sub.on) and
unbinding (a.sub.off) rates of anchors to the matrix, given by
.PHI. = a off a on + a off . ##EQU00030##
Note that .phi.=0 and .phi.=1 represent extremes where all anchors
and no anchors are bound to the matrix, respectively.
[0342] Why Short-Lived Anchor-Matrix Bonds Maximize Trapping
Potency:
[0343] Instinctively, one may expect that .phi.=0 maximizes the
fraction of trapped nanoparticles and that trapping potency is
reduced as .phi. rises until it is eliminated altogether when
.phi.=1. Nevertheless, this was not supported by our experiments
where IgG, anchored to the matrix with long-lived biotin-avidin
bonds, failed to trap nanoparticles with the same potency as IgG
that exhibit only weak and short-lived interactions with the
matrix. To begin to understand why long-lived anchor-matrix bonds
may compromise nanoparticle trapping, it is important to note that
a nanoparticle is unlikely to simultaneously encounter multiple
matrix-bound (immobilized) anchors unless the anchor concentration
is very high (i.e., anchors are generically spaced at distances
much greater than the dimensions of the nanoparticles). For
example, we have previously observed trapping of .about.100-200 nm
nanoparticles and viruses at IgG concentrations of 1-3 .mu.g/mL;
the average distance between each IgG at these concentrations is
roughly 440-630 nm. At these concentrations, if anchors are
permanently bound to the matrix, the average number of anchors on
each 100-200 nm nanoparticle that has been cross-linked to the
matrix must be at most one. Conversely, to achieve an average
distance of .ltoreq.100 nm between each IgG would require IgG
concentrations in excess of 250 .mu.g/mL, an exceedingly high
concentration for a single anchor species.
[0344] Recall from the Smoluchowski encounter relation that when
anchors are immobilized, the rate of a nanoparticle binding to an
anchor is proportional to particle diffusivity D.sub.P, whereas the
binding rate of free anchors to the nanoparticle is proportional to
D.sub.P+D.sub.A. Since we postulated that D.sub.A>>D.sub.P,
nanoparticles must encounter freely diffusing anchors much more
frequently and quickly than matrix-bound anchors. Consequently,
when .phi.>0, multiple anchors will begin to accumulate on the
surface of the nanoparticle, and multiple bonds can form (i.e.,
PA.sub.n.fwdarw.P(AM).sub.n) when a freely diffusing
nanoparticle-anchor complex encounters matrix constituents. While a
single anchor might rapidly unbind from the matrix, resulting in a
very short association lifetime of the complex, a
nanoparticle-anchor complex with multiple nanoparticle-bound
anchors, i.e., PA.sub.n, can increase the collective crosslink
lifetime because only one MAP bond is necessary to keep the
nanoparticle immobilized at any given time. So long as the anchors
stay bound to the nanoparticle, they do not diffuse away as quickly
after the nanoparticle-anchor complex unbinds from the matrix as
would individual free anchors, and thus can more rapidly rebind to
the matrix. Assuming each anchor-matrix bond is independent, the
PA.sub.n complex crosslink lifetime increases exponentially with
the number of anchors n bound to the same nanoparticle, and it
becomes exceedingly rare for all anchors to simultaneously unbind
from the matrix. We therefore reach the seemingly counterintuitive
conclusion that short-lived anchor-matrix bonds can actually
facilitate more complete crosslinking of nanoparticles to the
matrix.
[0345] Of course, some fraction of A must bind to the polymer
network with some probability or frequency; if .phi.=1, then
anchors never bind to the matrix. It follows that the crosslink
lifetime of a nanoparticle-anchor complex to the matrix must
eventually begin to decrease as the anchor-matrix binding affinity
is reduced below some optimal fraction of free anchors,
0<.phi.<1, corresponding to the most robust crosslinking of
nanoparticles to matrix.
[0346] We seek to precisely identify the optimal affinity .phi. and
timescale of anchor-matrix interactions for minimizing nanoparticle
flux through a gel layer. To do so, we first define a
characteristic length scale L of interest in the system (e.g., the
height of a mucus layer lining the surface of the lung or GI
tract). There are three timescales to consider: diffusion
(.tau..sub.L=L.sup.2/(2D.sub.P)), anchor-matrix interactions
(.tau..sub.AM=1/(a.sub.on+a.sub.off)), and anchor-nanoparticle
interactions (.tau..sub.AP=1/(D.sub.A[A]R.sub.0+k.sub.off)). The
diffusion time scale determines the average amount of time needed
to diffuse through a matrix layer. The two kinetic timescales
characterize the average duration of consecutive bind-unbind
events. We assume that L is large enough that many anchor-matrix
and anchor-nanoparticle interactions can occur before the
nanoparticles diffuse out of the system, i.e., .tau..sub.AP,
.tau..sub.AM<<T.sub.L, which allows us to derive an effective
diffusivity for the nanoparticle D.sub.eff.ltoreq.D.sub.P (see
Methods section for the derivation) that characterizes the
effective trapping potency of the anchors. Naturally, the smaller
D.sub.eff is, the more immobilized the nanoparticle is:
D.sub.eff=D.sub.P indicates anchors that have no effect on the
native diffusivity of the nanoparticle, whereas
D.sub.eff<D.sub.P reflects anchors that can at least transiently
immobilize the nanoparticle to the matrix. We now seek to explore
how anchor-matrix affinity influences nanoparticle trapping under
two important regimes: rapid yet short-lived anchor-matrix
kinetics, where .tau..sub.AM<.tau..sub.AP, and slow long-lived
anchor-matrix kinetics, where .tau..sub.AM>.tau..sub.AP.
[0347] If .tau..sub.AM>>.tau..sub.AP, then on the timescale
of A/P kinetics, anchors do not bind to or unbind from the matrix.
Because we assume that [A] is not unrealistically high (see above
and Assumption 6), nanoparticles can effectively only bind to a
single matrix-bound anchor at a time. In this regime, D.sub.eff is
minimized when .phi.=0, i.e., very high anchor-matrix affinity
(FIG. 2A, .tau..sub.AP/.tau..sub.AM.about.0.01). Assuming
D.sub.A/D.sub.P=20 and only 15 antigens on each 100 nm
nanoparticle, this results in a D.sub.eff/D.sub.P that is reduced
.about.55% on average compared to if anchors have no affinity to
matrix (FIG. 17A), which translates to only a .about.5% reduction
in the fraction of nanoparticles that can penetrate a 50 .mu.m
thick layer over 2 hours (FIG. 17B).
[0348] A much different result is obtained with rapid and
short-lived anchor-matrix bonds relative to anchor-nanoparticle
bonds, i.e., .tau..sub.AM<<.tau..sub.AP. D.sub.eff/D.sub.P
for the same nanoparticle drops significantly as
.tau..sub.AP/.tau..sub.AM increases, with a nontrivial optimal
.phi. that minimizes D.sub.eff (FIG. 17A). Indeed, when
.tau..sub.AP/.tau..sub.AM approaches 20 and with the steady state
free fraction of anchors in the .about.20-40% range (i.e.,
.phi..about.0.2-0.4), D.sub.eff/D.sub.P is reduced by over 90%,
effectively restricting transport of the nanoparticles. This drop
in D.sub.eff directly correlates to >50% reduction in the
fraction of nanoparticles that can penetrate across a 50 .mu.m
thick matrix layer over 2 hrs, a greater than 10-fold increase in
trapping potency compared to the long-lived anchor-matrix bonds
scenario (FIG. 17B). Increases in .tau..sub.AP/.tau..sub.AM also
directly reduces the amount of anchors needed to reduce the flux of
nanoparticles penetrating and exiting the matrix layer (FIG. 17C).
These results confirm Condition 1 of our proposed model, namely
that short-lived anchor-matrix bonds (.tau..sub.AM<.tau..sub.AP)
maximizes trapping potency.
[0349] Efficient Crosslinking of Nanoparticles to Matrix Also
Requires Multiple Antigens and Rapid Anchor Diffusivity:
[0350] Hypothetically, if there is only one epitope available per
nanoparticle, then at most only one anchor can bind to the
nanoparticle (i.e., N=1). Naturally, in this scenario,
D.sub.eff/D.sub.P would decrease monotonically as .phi..fwdarw.0,
since maximum trapping is achieved when the nanoparticle-bound
anchor never dissociates from the matrix, as shown in FIG. 18A. In
contrast, for N>1, D.sub.eff/D.sub.P is an exponentially
decreasing function of N: the more antigen sites available on a
nanoparticle, the more likely and quickly the nanoparticle will
accumulate anchors on its surface and become trapped in the matrix
(FIG. 18A). With even a modest number of anchor binding sites on
each nanoparticle (N.about.20), nanoparticle D.sub.eff can be
reduced by over 90% when combined with rapid (i.e.
.tau..sub.P/.tau..sub.M=20) and weak (i.e. .phi..about.0.2-0.4)
anchor-matrix interactions. To place this in perspective, Influenza
and Herpes Simplex Virus have hundreds of hemaglutinin and gD
glycoprotein epitopes per viral particle, respectively. These
results confirm Condition 2 of our proposed model.
[0351] In addition to the number of binding sites, the rate of
anchor accumulation depends on the frequency with which anchors can
collide with the nanoparticle. The latter is in turn proportional
to the diffusivity of the anchor as predicted by the Smoluchowski
encounter relation. Although greater nanoparticle diffusivity
D.sub.P can theoretically increase the encounter and anchor
accumulation rate on the nanoparticle, this also reduces the time
.tau..sub.L available for sufficient quantities of anchor to
accumulate on the nanoparticle before the nanoparticle diffuses
through the barrier fluid. As shown in FIG. 18B, nanoparticle
D.sub.eff/D.sub.P drops as D.sub.A increases; thus, anchors that
are smaller and more mobile than the nanoparticle are preferred for
nanoparticle trapping. These results confirm Condition 3 of our
proposed model.
[0352] Balancing Thickness of Polymeric Matrices and Anchor
Concentrations to Maximize Trapping Potency:
[0353] The barrier properties of biogels are naturally dependent on
both the thickness of the gel layer as well as the concentration of
the molecular anchors. This is particularly relevant for
diffusional barriers such as mucus and basement membranes, where
minimizing the fraction of viruses that can penetrate through the
gel layer can directly reduce the probability of transmission or
spread of the infection systemically. To address the balance
between these two parameters, we assert that the most effective
balance of timescales to immobilize nanoparticles is
.tau..sub.AM<<.tau..sub.AP<<T.sub.L (i.e., imposing
Conditions 1 and 4 of our model). We have already shown above that
maximal trapping occurs with rapid anchor-matrix binding kinetics
i.e. .tau..sub.AM<<.tau..sub.AP. In addition, as explained
above, in order for nanoparticles to become trapped in the matrix,
the nanoparticle must be captured by at least one anchor before it
diffuses through the matrix. Recall that the average time the
nanoparticle, unhindered by anchors, needs to diffuse through a
matrix layer is .tau..sub.L=L.sup.2/(2D.sub.P). Hence, the matrix
layer thickness must be L> {square root over
(2.sub.DP.tau..sub.P)} for anchors to have sufficient time to
accumulate on the nanoparticle. To illustrate the effect of the
timescale .tau..sub.D, we use a numerical approximation of the
solution to equation [eq:9] (see Methods Section), and compute the
probability that a nanoparticle can diffuse across a polymer matrix
layer of thickness L. We term this the absorption probability; a
low absorption probability indicates effective trapping by anchors.
Not surprisingly, D.sub.eff/D.sub.P (FIG. 19A) and the absorption
probability (FIG. 19B) both decrease with increasing anchor
concentration (FIG. 19A), in both cases approaching a minimum
D.sub.eff/D.sub.P when .phi..about.0.35. Interestingly, when we
compare the relative importance of L vs. anchor concentrations, we
found that exponentially higher anchor concentrations are required
when L is smaller than .about.40-50 .mu.m thick in order to
maintain a comparably effective diffusional barrier (FIG. 19C),
implying that effective diffusional barriers in vivo should be at
least 40-50 .mu.m thick. These estimates agree remarkably well with
both (i) the thickness of mucus coatings lining the respiratory,
gastrointestinal and cervicovaginal tracts (typically
.about.50-100+ .mu.m), as well as (ii) the Ab concentrations
present in mucus (typically .about.0.1-10 .mu.g/mL) (Wang et al.,
Mucosal Immunol. 7:1 (2014)). This analysis confirms Condition 4 of
our proposed model.
[0354] Short-Lived Anchor-Matrix Bonds Enable Robust Trapping of
Multiple Nanoparticle Species by Minimizing Potential Saturation of
Anchor-Binding Sites within the Matrix:
[0355] To selectively control transport against multiple species of
nanoparticles in the same polymeric matrix, such as trapping a
diverse array of pathogens that impinge on mucus coating the
airways and gastrointestinal epithelium, many corresponding anchor
species must coexist without impeding each other's trapping
potency. In other words, even when anchors that bind any given
species represent only a tiny fraction of all anchors present, the
specific anchor-matrix affinity must remain unaltered in order to
maintain comparable trapping potency. We introduce the term
trapping robustness to describe the ability to immobilize multiple
nanoparticle species.
[0356] Since the concentration of matrix constituents are finite,
the number of anchor binding sites in a polymeric gel must by
definition be finite. Thus, if anchor-matrix bonds are long lived,
a matrix-bound anchor prevents other anchors from binding to the
same binding site on the matrix. Thus, at concentrations sufficient
for trapping (e.g., 1-5 .mu.g/ml IgG), the system could accommodate
only a relatively limited number of anchor species (<103 for a
2% w/v gel, assuming 10 anchor binding sites per matrix molecule
and an average MW of ZZ MDa) before additional anchors become
unable to effectively reduce D.sub.eff/D.sub.P (FIG. 20). However,
when .tau..sub.AM<.tau..sub.AP, the short duration of
anchor-matrix bonds would greatly increase the number of unoccupied
anchor binding sites available on the matrix at any moment in time.
This in turn enables the anchor-matrix system to both immobilize a
far greater number of particle species simultaneously as well as
reduce the minimum D.sub.eff D.sub.P that could be achieved with
each anchor (FIG. 20). Indeed, when
.tau..sub.AP/.tau..sub.AM.gtoreq.20, a biogel reinforced with
appropriate molecular anchors can effectively immobilize at least
30-fold more (i.e. .about.3.times.10.sup.4) distinct nanoparticle
species without appreciable loss in trapping potency (i.e., similar
minimum D.sub.eff/D.sub.P for all species), underscoring the
potential trapping robustness of the system. Altogether, these
results confirm Condition 5 of our proposed model.
[0357] Finally, we examined the relative importance for each of the
parameters described above by evaluating the partial derivatives of
log(D.sub.eff/D.sub.P) under a range of parameter values. We
observed the greatest impact with .tau..sub.AP/.tau..sub.AM (in
particular at low .tau..sub.AP/.tau..sub.AM values) and the rates
of anchor accumulation k.sub.on[A], with more modest impact with
changes in antigenic epitope density (i.e., maximum number of bound
anchor) N and the diffusivity of anchors relative to the
nanoparticle species D.sub.A/D.sub.P(FIG. 21). These results
underscore short-lived anchor-matrix bonds relative to
anchor-nanoparticle bonds as a crucial separation of timescales for
enabling molecular anchors that can substantially enhance the
barrier properties of either biological or synthetic polymeric
matrices to multiple nanoparticle species.
Summary
[0358] A critical function of polymeric matrices in biological
systems is to exert selective control over the transport of
thousands of nanoparticulate species. By eliminating the need for
matrix constituents to recognize diverse antigenic species, an
anchor-matrix system can enable an effective diffusional barrier
against many nanoparticle species while maintaining relatively
static biochemistry and microstructure of the matrix. This suggests
that anchors, such as IgG and other antibodies produced by the
immune system that can adapt and bind diverse molecular entities,
represent an ideal platform to control nanoparticle transport.
Here, we demonstrate both experimentally and theoretically that
short-lived anchor-matrix interactions represents the critical
attribute enabling potent and robust control over nanoparticle
transport in biogels. Given the weak and short-lived interactions
between individual IgG antibodies and Matrigel constituents, it was
reasonable to conclude that IgG exerts only limited selective
control over nanoparticle transport in Matrigel.RTM.. In contrast,
the observed trapping potency against pathogens and particulates is
a cooperative effect of multiple IgG. Combined with our earlier
observations that IgG can potently immobilize viruses and
nanoparticles in different mucus secretions, it is likely that the
proposed strategy, whereby the barrier properties are tuned by
modest concentration of highly mobile molecular anchors with
exceedingly short anchor-matrix bond times relative to
anchor-nanoparticle bond times, is a universal feature of biogels
in living systems. Our findings provide a blueprint for engineering
of molecular anchors with optimal short-lived anchor-matrix bonds
to selectively tune the barrier properties of polymeric gels.
Example 4
Selective Tuning of the Barrier Properties of Hydrogels Using IgG
and IgM Antibodies
[0359] Biopolymeric matrices are ubiquitously present in living
systems, and provide structural support and mechanical function
essential to health (Kruegel et al., Cell. Mol. Life Sci 67:2879
(2010)). For example, extracellular matrices known as basement
membranes (BM) provide a scaffold for epithelial cells to anchor to
underlying loose connective tissue (Kruegel et al., Cell. Mol. Life
Sci 67:2879 (2010)). Similarly, mucus gels lubricate mucosal
surfaces such as the gastrointestinal and female reproductive
tracts to allow for passage of food or copulation (Lai et al., Adv.
Drug Deliv. Rev. 61:86 (2009)), and the vitreous humor maintains
the spherical shape of the eye critical for proper vision (Kokavec
et al., Clin. Exp. Ophthalmol. 44:597 (2016)).
[0360] To attain the necessary viscous and elastic modulus for
suitable biological functions, these biopolymers typically undergo
extensive crosslinking or entanglement to create a viscoelastic
gel. Such supramolecular structure of network matrices in turn
enables a second important function: selectively permeable
membranes that determine which substances will be able to enter the
epithelium. Naturally, the matrices can restrict diffusion by
steric obstruction when objects are larger than the pores present,
such as preventing malignant cells from invading the deeper tissues
(Arends et al., PLoS One 10:1 (2015)) or aerosolized particles from
reaching the epithelium along conducting airways (Cheng et al.,
AAPS PharmSciTech 15:630 (2014)), In order to restrict diffusion of
entities not adequately trapped by steric obstruction, biopolymeric
matrices must rely on adhesive interactions (Lieleg et al.,
Biophys. J. 97:1569 (2009)). It is highly unlikely that matrices
with relatively static biochemical structures can adhesively
innobilize, on their own, the full diversity of pathogens and
macromolecules present in any living system. Indeed, the vast
majority of viruses that transmit at mucosal surfaces (HIV, HSV,
HPV, Norwalk, etc.) evade adhesion to mucins and diffuse rapidly
through the low viscosity interstitial fluids that fill the pores
of mucus gels (Olmsted et al., Biophys. J 81:1930 (2001)).sup.7.
Thus, to selectively immobilize a diverse array of entities smaller
than the pores of the matrices, it is necessary to utilize highly
adaptive third-party crosslinkers that can evolve and crosslink
viruses and nanoparticles to matrix constituents.
[0361] Antibodies (Ab) represent a potential platform of
crosslinkers that can be readily generated by living systems.
Specifically, due to the process of somatic hypermutation and
affinity maturation, antibodies possessing high specificity against
a diverse array of pathogens can be readily induced and secreted.
Here, we investigated whether Ab can facilitate trapping of
pathogens in extracellular matrices of the basement membrane (BM).
Our results demonstrate that Ab-matrix interaction is likely a long
overlooked molecular mechanism enabling selective permeability of
BM and presumptively other biopolymeric matrices.
Materials and Methods
[0362] Preparation of PEG-Coated Nanoparticles:
[0363] To produce PEGylated nanoparticles (PS-PEG), we covalently
modified 200 nm fluorescent, carboxyl-modified polystyrene beads
(PS-COOH; Invitrogen) with 2 kDa methoxy polyethylene glycol amine
(PEG; Sigma) via a carboxyl-amine reaction, as published previously
(Lai et al., Proc. Natl. Acad. Sci. USA 104:1482 (2007); Yang et
al., Mol. Pharm. 11:1250 (2014)). Particle size and
.zeta.-potential were determined by dynamic light scattering and
laser Doppler anemometry, respectively, using a Zetasizer Nano ZS
(Malvern Instruments, Southborough, Mass.). Size measurements were
performed at 25.degree. C. at a scattering angle of 90.degree..
Samples were diluted in 10 mM NaCl solution, and measurements were
performed according to instrument instructions. PEG conjugation was
also confirmed by a near-neutral .zeta.-potential (Table 2) (Lai et
al., Proc. Natl. Acad. Sci. USA 104:1482 (2007)). Dense PEG
grafting (>1 PEG/nm) was further verified using the fluorogenic
compound 1-pyrenyldiazomethane (PDAM) to quantify residual
unmodified carboxyl groups on the polystyrene beads (Yang et al.,
Mol. Pharm. 11:1250 (2014)).
[0364] Preparation of BM, LAM, and Collagen Gels:
[0365] A mixture of the following was added to a custom-made
micro-volume (.about.20 .mu.L) glass chamber slide: (i)
growth-factor reduced Matrigel (Corning), high-concentration
laminin/entactin (Corning), or collagen; (ii) BSA (Sigma); (iii)
Eagle's Minimum Essential Medium (EMEM, Lonza BioWhittacker); (iv)
fluorescent PS-COOH (Ex: 505 nm, Em515 nm) and PS-PEG (Ex: 625 nm,
Em645 nm) nanoparticles; and (v) different Ab. The mixture was
incubated at 37.degree. C. for 45 minutes in a custom hydration
chamber, then sealed and incubated for another 30 minutes prior to
microscopy. Final concentrations of reagents in slides were as
follows: 2.2 mg/mL Matrigel, 1 mg/mL BSA, .about.4.3*10.sup.8
beads/mL for both PEG- and COOH-modified nanoparticles, antibody as
listed, EMEM, q.s. In LAM experiments, the final concentration of
LAM complex (Corning) was 1.5 mg/mL (comparable to the
concentration of laminin in 2.2 mg/mL Matrigel). In COL
experiments, the final concentration of COL was as high as 3 mg/mL.
Trapping of PS-COOH beads in BM was used as an internal control in
all microscopy experiments as a measure of complete polymerization
of BM constituents.
[0366] Native Anti-PEG IgG and IgM and Deglycosylation:
[0367] Anti-PEG IgG.sub.1 (CH2074, Silver Lake Research) and
anti-PEG IgM (AGP4, IBMS) were used as test Ab and anti-Biotin IgG
(33, Santa Cruz) and anti-Vancomycin IgM (2F10, Santa Cruz) were
used as control Ab. All native Ab were used as is provided by the
manufacturer. To prepare deglycosylated Ab, N-glycans on anti-PEG
IgG and IgM were removed with rapid non-reducing PNGase F enzyme
(New England Biolabs) according to manufacturer's protocol. The
deglycosylation reaction was confirmed with two NuPAGE 4-12%
Bis-Tris gels (Novex) in MOPS buffer. The first gel was transferred
to a nitrocellulose membrane (Novex) with a Semi-Dry Blotter
(Novex), blocked with carbo-free buffer (Vector Labs), labeled
overnight at 4.degree. C. in 2 g/mL biotinylated concavalin A
(Vector), and probed with anti-biotin peroxidase (Vector) before
imaging with Clarity Western Blot ECL Substrate (BioRad) in a
FluorChemE unit (Cell Biosciences). The second gel was silver
stained with Pierce Silver Stain Kit (Thermo Scientific) and imaged
with the same unit (FIG. 22). The remaining deglycosylated IgG and
IgM was buffer-exchanged into PBS using 50 MWCO spin-x columns
(Corning), quantified based on A.sub.28, and their binding affinity
to PEG was measured using ELISA.
[0368] High Resolution Multiple Particle Tracking:
[0369] The trajectories of the fluorescent particles were recorded
using an EMCCD camera (Evolve 512; Photometrics, Tucson, Ariz.)
mounted on an inverted epifluorescence microscope (AxioObserver DI;
Zeiss, Thornwood, N.Y.), equipped with an Alpha Plan-Apo
100.times./1.46 NA objective, environmental (temperature and
CO.sub.2) control chamber and an LED light source (Lumencor Light
Engine DAPI/GFP/543/623/690). 20s videos (512.times.512, 16-bit
image depth) were captured with MetaMorph imaging software
(Molecular Devices, Sunnyvale, Calif.) at a temporal resolution of
66.7 ms and spatial resolution of 10 nm (nominal pixel resolution
0.156 .mu.m/pixel). The tracking resolution was determined by
tracking the displacements of particles immobilized with a strong
adhesive, following a previously described method (Apgar et al.,
Biophys. J. 79:1095 (2000)). Particle trajectories were analyzed
using MATLAB software as described previously (Wang et al., J.
Control. Release 220:37 (2015)). Sub-pixel tracking resolution was
achieved by determining the precise location of the particle
centroid by light-intensity-weighted averaging of neighboring
pixels. Trajectories of n.gtoreq.40 particles per frame on average
(corresponding to n.gtoreq.100 total traces) were analyzed for each
experiment, and 3-4 independent experiments were performed for each
condition. The coordinates of particle centroids were transformed
into time-averaged mean squared displacements (MSD), calculated as
<.DELTA.r.sup.f(r)>=[x(t+.tau.)-x(t)]2+[y(t+.tau.)-y(t)].sup.2
(where .tau.=time scale or time lag), from which distributions of
MSDs and effective diffusivities (D.sub.eff) were calculated, as
previously demonstrated (Lai et al., Proc. Natl. Acad. Sci. USA
104:1482 (2007); Dawson et al., J. Biol. Chem. 278:50393 (2003);
Suh et al., Adv. Drug Deliv. Rev. 57:63 (2005)). MSD may also be
expressed as MSD=4D.sub.0.tau..sup..alpha., where .alpha., the
slope of the curve on a log-log scale, is a measure of the extent
of impediment to particle diffusion (.alpha.=1 for pure
unobstructed Brownian diffusion; .alpha.<1 indicates increasing
impediment to particle movement as a decreases). Mobile particles
were defined as those with D.sub.eff.gtoreq.10.sup.-1 .mu.m.sup.2/s
at .tau.=0.2667 s (this .tau. corresponds to a minimum trajectory
length of 5 frames), based on multiple datasets of mobile and
immobile nanoparticles (e.g., PS and PS-PEG nanoparticles) in human
mucus (Lai et al., Proc. Natl. Acad. Sci. USA 104:1482 (2007); Lai
et al, Proc. Natl. Acad. Sci. USA 107:598 (2010)).
[0370] Biolayer Interferometry Experiments:
[0371] On an Octet QK instrument (ForteBio), streptavidin
biosensors (ForteBio) hydrated in running buffer (PBS) were loaded
with biotinylated biopolymer or biotinylated PEG at 5 .mu.g/mL for
600s. Sensors were washed in running buffer for 180s and a baseline
set in running buffer for 300s. Antibody (IgG or IgM) at different
concentrations was associated with these customized biosensors for
600s and dissociated for 600s. Loading ligands and analytes were
diluted in running buffer. Data were adjusted for reference sensors
and baseline values and aligned to dissociation, then processed
with Savitzky-Golay filtering. Analysis was performed with ForteBio
software using a 1:1 global curve fit model to obtain values for
k.sub.on, k.sub.off, and K.sub.D.
[0372] Salmonella Invasion Across BM:
[0373] BM (final concentration 2.2 mg/mL) or LAM (final
concentration 1.5 mg/mL) with Luria Broth (LB, BD Falcon), BSA, and
varying concentrations of anti-Salmonella typhimurium IgG.sub.1
(6331; WHERE) were mixed and incubated for 2 hours at 37.degree. C.
in the upper chamber of a HTS FluoroBlok MultiWell System with 3.0
.mu.m pores (BD Falcon) in a custom hydration chamber. After
confirming mobility in the above-mentioned epifluorescence
microscope, GFP-labeled S. typhimurium in 10 .mu.L LB was then
added to the top of each well, and 200 .mu.L LB to each well in the
bottom chamber. After 2 hours of incubation at 37.degree. C., the
top chamber was removed and OD600 in the lower chamber was measured
with a SpectraMax M2 (Molecular Devices). Experiment was performed
in triplicate, n=3.
[0374] Statistical Analysis:
[0375] MSD data were log-transformed and compared within groups
using a repeated-measures two-way ANOVA and post hoc Tukey test.
Log-transformed average D.sub.eff and % mobile were compared with
ANOVA and subsequent Tukey's HSD tests. Binding curve K.sub.D,
k.sub.on, and k.sub.off were compared with two-way ANOVA and post
hoc Tukey test. Salmonella invasion data was nonnalized within each
replicate by controlling for background (defined as OD600 of LB
only) and maximal invasion of Salmonella in a given matrix. Data
were compared within groups using a repeated-measures two-way ANOVA
and post hoc Neuman-Keuls test. In all analyses, global
.alpha.=0.05. Error bars and .+-.represent SEM.
Results
[0376] PEG-Binding IgG and IgM Immobilizes PEG-Coated Nanoparticles
in BM:
[0377] To begin to investigate whether the barrier properties of BM
can be selectively tuned, we first mixed fluorescent polystyrene
nanoparticles modified with either carboxyl groups (PS-COOH) or a
dense layer of polyethylene glycol (PS-PEG) into reconstituted BM
from Engelbreth-Holm-Swarm sarcoma (commercially available as
Matrigel.RTM.) and performed high resolution multiple particle
tracking to quantify the diffusion of hundreds of individual
nanoparticles in each specimen. In good agreement with previous
reports (Lieleg et al., Biophys. J. 97:1569 (2009)), nearly all
PS-PEG nanoparticles exhibited largely unhindered Brownian motion,
capable of diffusing many microns on the order of seconds, hindered
only 1.6-fold compared to its movement in water (FIGS. 23A-23E). In
contrast, PS-COOH beads in the same BM specimen were extensively
immobilized, exhibiting a geometrically averaged ensemble effective
diffusivity (<D.sub.eff>).about.4000-fold reduced compared to
PS-PEG (FIGS. 23A-23E; p<0.0018). Only 0.2%.+-.0.09% of PS-COOH
beads were classified as mobile (possessing <D.sub.eff> in
excess of 10.sup.-6 mm.sup.2/s) vs. 96%.+-.2% for PS-PEG beads. The
effective immobilization of PS-COOH but not PS-PEG nanoparticles
confirms BM affords a sufficiently rigid matrix that can immobilize
virus-sized nanoparticles by adhesive interactions, and that PS-PEG
nanoparticles are adequately modified with PEG to evade adhesive
interactions with the matrix constituents.
[0378] We next tested whether exogenously introduced Ab can
specifically immobilize the otherwise readily diffusing PS-PEG
beads in BM. The addition of control IgG did not appreciably alter
the diffusive motion of PS-PEG; the <D.sub.eff> of PS-PEG
were slowed only .about.1.6 fold compared to their theoretical
speeds in water (Table Si). However, in BM treated with anti-PEG
IgG to a final Ab concentration of 10 .mu.g/mL, the majority of
PS-PEG became effectively immobilized, moving less than the
particle diameter over the course of 20 s movies, similar to
PS-COOH nanoparticles (FIG. 24A). Indeed, the <D.sub.eff> of
PS-PEG was reduced by .about.17-fold compared to control IgG (FIG.
24B), and the mobile fraction was reduced from 96.+-.1% in control
IgG to 21.+-.9% (p=0.018). The impediment to free Brownian
diffusion caused by anti-PEG IgG was also reflected by the slope a
from the log-log plot of geometrically averaged ensemble mean
squared displacements <MSD> vs. time scale plots (.alpha.=1
for pure unobstructed Brownian diffusion, e.g., particles in water,
and .alpha. becomes smaller and approaches zero as obstruction to
Brownian diffusion increases): the average a value was 0.88 and
0.55 for PS-PEG in control IgG- and anti-PEG IgG-treated BM,
respectively (p=0.0014 vs. control). We further reduced the
anti-PEG IgG concentrations in BM and observed comparable trapping
potency at 5 .mu.g/mL (FIG. 1), but not at 1 .mu.g/mL. These
results underscore the ability for antigen-specific IgG to
immobilize virus-sized nanoparticles in BM.
TABLE-US-00002 TABLE 2 Effective diffusivity of PEG beads with IgG
and IgM antibodies in BM and LAM as compared to the theoretical
diffusivity of PEG beads in water at 37.degree. C. D.sub.water/
Antibody Matrix D.sub.matrix None (PS-COOH) BM 5943.9 None (PS-PEG)
BM 1.53 Control IgG BM 1.6 IgG 5 .mu.g/mL BM 26.0 IgG 10 .mu.g/mL
BM 26.7 Control IgM BM 0.6 IgM 1 .mu.g/mL BM 61.8 IgM 3 .mu.g/mL BM
136.5 IgM 5 .mu.g/mL BM 240.5 Deg. IgG 10 .mu.g/mL BM 11.6 Deg IgM
5 .mu.g/mL BM 21.8 Control IgG LAM 8.6 IgG 10 .mu.g/mL LAM 187.2
Control IgM LAM 6.2 IgM 5 .mu.g/mL LAM 242.6
[0379] In addition to IgG, another major class of antibody is IgM,
the pentameric antibody that occurs early in the adaptive immune
response against foreign pathogens. We therefore also investigated
whether IgM can also immobilize virus-sized nanoparticles in BM.
Addition of control IgM did not alter the diffusion of PS-PEG; the
<D.sub.eff> of PS-PEG were slowed to only .about.0.6 fold
compared to their theoretical speeds in water (Table 2), with
97.+-.2% of the nanoparticles exhibited <D.sub.eff> in excess
of 10.sup.-6 m.sup.2/s. In contrast, anti-PEG IgM immobilized an
even greater fraction of PS-PEG than anti-PEG IgG, even at lower
concentrations (FIGS. 25A and 25B). Anti-PEG IgM at 5 .mu.g/mL
reduced the <MSD> of PS-PEG in BM by .about.90-fold compared
to control IgM (FIG. 25C), reflecting a drop in the mobile fraction
to 26.+-.13% (FIG. 25E). Anti-PEG IgM also reduced the average a
value of PS-PEG from 0.86 to 0.61 (p=0.0014 vs. control),
underscoring the extent of antibody-mediated immobilization.
Anti-PEG IgM mediated potent trapping of PS-PEG beads even at 1 and
3 .mu.g/mL, reducing the <D.sub.eff> by 62- and 137-fold,
respectively. Surprisingly, despite the common assumption that IgM
can mediate efficient agglutination, we did not observe appreciable
nanoparticle agglutination in BM with anti-PEG IgM.
[0380] Elucidating the Molecular Basis of Ab-BM Interactions:
[0381] We next sought to determine the molecular basis of Ab-BM
bonds that facilitate crosslinking of nanoparticles to BM
constituents. Since nanoparticle trapping in BM require F.sub.ab
specificity, we hypothesized that it is the F.sub.c-domain, in
particular N-glycans on IgG-F.sub.c as well as on the heavy chains
of IgM, that mediate binding interactions with BM constituents. To
test this hypothesis, we treated anti-PEG IgG and IgM with PNGase F
and confirmed that removal of N-glycans did not appreciably alter
the binding affinity of either Ab to PEG (FIGS. 22A-22B). In good
agreement with our hypothesis, removal of N-glycans from anti-PEG
IgG substantially reduced its trapping potency. The fraction of
mobile PS-PEG nanoparticles in BM treated with deglycosylated
anti-PEG IgG was markedly increased (52.+-.6%) compared to native
anti-PEG IgG (21.+-.9%) (p=0.032), consistent with the
.about.10-fold increase in <MSD> (p<0.0001; FIGS.
26A-276). Similarly, the fraction of mobile PS-PEG beads and their
corresponding <MSD> in BM treated with deglycosylated
anti-PEG IgM was markedly increased. These results suggest
N-glycans on Ab contribute to the association of both IgG and IgM
to BM constituents.
[0382] Laminin/entactin associate strongly with each other and
together represent the largest constituent of BM by mass. To
determine the components of BM involved in Ab-mediated trapping, we
first studied matrices composed of only laminin/entactin (LAM).
Similar to native BM, LAM was able to effectively immobilize
PS-COOH but not PS-PEG nanoparticles, confirming the presence of a
rigid matrix. We found that anti-PEG IgG and IgM also exhibited
comparable trapping potency in LAM. The <D.sub.eff> of PS-PEG
was reduced .about.20-fold and .about.40-fold in anti-PEG IgG- and
IgM-treated LAM compared to corresponding controls, respectively.
Similarly, the fraction of mobile PS-PEG nanoparticles was reduced
from 70.+-.7% and 75.+-.10% in LAM mixed with control IgG and IgM
to 21.+-.2% and 5.+-.2.5% in LAM mixed with anti-PEG IgG- and IgM,
respectively (FIGS. 27A-27E). Since collagen represents another
major constituent in BM, we also sought to investigate matrices
consisting of purely collagen. However, the collagen-only gel never
formed an adequately intact and rigid matrix to immobilize PS-COOH
beads, and thus trapping potency was not measured.
[0383] Ab-BM interactions can prevent bacterial translocation:
Finally, as a proof of concept that we can harness Ab to reinforce
the barrier properties of BM, as well as demonstrate that Ab-BM
bonds are sufficient to immobilize even highly motile bacterial
pathogens, we decided to evaluate whether IgG that binds Salmonella
typhimurium can prevent the invasion of the bacteria across BM gel.
We first added BM and Ab to the upper chamber of transwell system
and allowed the gel to form, followed by the addition of
fluorescent Salmonella bacteria, and monitored the amount of
bacteria found in the bottom chamber. Anti-LPS IgG markedly reduced
the flux of Salmonella in native BM in a dose-dependent manner,
with >90% reduction in the amount of Salmonella that could
penetrate across the BM layer at IgG doses .gtoreq.10 .mu.g/mL
(FIG. 28). We further investigated matrix composed of LAM only, and
found no appreciable difference in the abilities of BM or LAM to
prevent Salmonella flux in conjunction with Abs.
[0384] We next assessed whether we can selectively tune the barrier
properties of Matrigel.RTM. simply by introducing specific
antibodies. We mixed in different concentrations of IgG that
specifically bind PEG; the antibody possesses weak affinity to
collagen and laminin as measured by biolayer interferometry (Table
3). In Matrigel.RTM. that contains a final concentration of 10
.mu.g/mL anti-PEG IgG, the <D.sub.eff> for PS-PEG was reduced
.about.167-fold compared to control anti-biotin IgG (FIG. 24D), and
the mobile nanoparticle fraction was reduced from 96.+-.0.7% in
control IgG to 13.+-.4.2% (p=0.018; FIG. 24E). Anti-PEG IgG enabled
substantial trapping of PS-PEG at 5 .mu.g/mL (FIG. 24), but not at
1 .mu.g/mL. The appearance of the PS-PEG nanoparticles remained
identical between control IgG and anti-PEG IgG conditions,
indicating that the impeded motion of the nanoparticles were not
attributed to anti-PEG IgG crosslinking multiple nanoparticles
together.
TABLE-US-00003 TABLE 3 Binding affinities of anti-PEG IgG and IgM
to BLI probes coated with biotinylated collagen or laminin Collagen
Laminin kon Koff KD kon Koff KD [1/Ms] [1/s] [M] [1/Ms] [1/s] [M]
IgG 4.05 .times. 9.49 .times. 2.34 .times. 4.23 .times. 10.sup.4
2.98 .times. 10.sup.-4 7.05 .times. 10.sup.3 10.sup.-4 10.sup.-7
10.sup.-9 IgM 5.54 .times. 8.79 .times. 1.59 .times. 4.71 .times.
10.sup.4 1.77 .times. 10.sup.-4 3.76 .times. 10.sup.4 10.sup.-4
10.sup.-8 10.sup.-9
[0385] To further confirm the antigen specificity and ability to
reinforce barrier properties against different antigens, we mixed
in nanoparticles conjugated with biotin-PEG, and found they were
effectively immobilized in Matrigel.RTM. treated with anti-biotin
IgG (FIGS. 29A-29B). To demonstrate the ability of antigen-specific
IgG to mediate trapping even in the presence of large quantities of
other crosslinkers, we further evaluated the mobility of PS-PEG in
BM simultaneously treated with both 10 .mu.g/mL anti-PEG IgG and
100-fold excess anti-biotin IgG (i.e., 1 mg/mL). We found no
appreciable reduction in the trapping potency of anti-PEG IgG
despite the excess levels of other antigen-specific IgG (FIGS.
30A-30B). These results underscore antigen-specific IgG can
robustly immobilize virus-sized nanoparticles in Matrigel.RTM., and
that this strategy can accommodate trapping of a large number of
diverse nanoparticle species without compromising the ability to
trap any individual foreign species.
[0386] Since many antibody-effector functions are influenced by
N-glycans on IgG-Fc, not being bound by the theory, it is possible
that N-glycans on Fc may also mediate interactions with BM
constituents. We deglycosylated anti-PEG IgG and IgM with PNGase F
and found that the removal of N-glycans substantially abrogated the
trapping potency of IgG and IgM, with the fraction of mobile
nanoparticles increasing from 13.+-.4.2% to 57.+-.5.3% for IgG
(p=0.0006) and from 13.+-.6.7% to 67.+-.9.4% for IgM (0<0.0001;
FIG. 31). Both IgG and IgM antibodies appear to anchor
nanoparticles to laminin/entactin (LAM), since we observe
comparable trapping with a hydrogel composed of LAM alone: the
<D.sub.eff> of PS-PEG was reduced .about.20-fold and
.about.40-fold in anti-PEG IgG- and IgM-treated LAM compared to
corresponding controls, respectively (FIG. 27).
[0387] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
* * * * *